Ultrafast Header Processing in All-Optical Packet Switched-Network

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Thermo Fisher Scientific UltraPlex 1-Step ToughMix

UltraPlex ® 1-Step ToughMix ® Low ROX™ (4X)DescriptionUltraPlex 1-Step ToughMix Low ROX is a ready-to-use, 4X-concentrated master mix for reverse transcription quantitative PCR (RT-qPCR) of RNA templates using hybridization probe detection chemistries such as TaqMan ® 5’-hydrolysis probes on real-time PCR systems that utilize ROX passive reference with 585 nM excitation. First-strand cDNA synthesis and PCR amplification are carried out in the same tube without opening between procedures. It is ideal for highly sensitive quantification of RNA viruses or low abundance RNA targets in uni- or multiplexed RT-qPCR applications as well as high throughput gene-expression studies. The system has been optimized to deliver maximum RT-qPCR efficiency, sensitivity, and specificity in reduced reaction volumes and fast cycle times. UltraPlex 1-Step ToughMix Low ROX contains all required components for RT-qPCR except RNA template and probe. It is compatible with all dual-labeled probe chemistries.qScript ® XLT is an engineered M-MLV reverse transcriptase (RT) with reduced RNase H activity for improved activity and stability at higher temperatures. The use of higher temperatures (50 to 54°C) for the first-strand step of one-step RT-qPCR provides higher specificity for primer annealing and disruption of RNA secondary structure that can interfere with cDNA synthesis. These beneficial properties of qScript XLT RT are further enhanced by a hot-start mechanism for the reverse transcription step. Minimizing off-target extension by the RT during reaction assembly provides highly reproducible low copy quantification as well as extended room temperature stability of fully assembled reactions for high throughput operations.UltraPlex 1-Step ToughMix Low ROX is highly resistant to PCR inhibitors. A key component of the ToughMix is an ultra pure, highly processive thermostable DNA polymerase that is combined with high avidity monoclonal antibodies. This provides an extremely stringent automatic hot-start that minimizes the potential for primer-dimer and other non-specific PCR artifacts.Instrument CompatibilityDifferent real-time PCR systems employ different strategies for the normalization of fluorescent signals and correction of well-to-well optical variations. It is critical to match the appropriate qPCR reagent to your specific instrument. UltraPlex 1-Step ToughMix Low ROX provides seamless integration on the Applied Biosystems 7500, 7500 Fast, ViiA 7, QuantStudio or Stratagene MX series of real-time PCR systems. Please visit our web site at to find an optimized kit for your instrument platform(s).ComponentsReagent DescriptionUltraPlex 1-Step ToughMix Low ROX (4X) 4X reaction buffer containing dATP, dCTP, dGTP, dTTP, magnesium, qScript XLT reverse transcriptase, RNase inhibitor protein, hot-start DNA polymerase, stabilizers, and ROX Reference DyeStorage and Stability Store components in a constant temperature freezer at -25°C to -15°C protected from light upon receipt. For lot specific expiry date, refer to package label, Certificate of Analysis or Product Specification Form.Guidelines for One-Step RT-qPCRThe design of highly specific primers and probes is a critical parameter for successful one-step RT-qPCR. The use of computer aided primerdesign programs is encouraged in order to minimize the potential for internal secondary structure and complementation at 3’-ends within each primer, the primer pair, and primer/probe combinations. Regions of strong RNA secondary structure should be avoided as this can interfere with primer hybridization and/or impede procession of the reverse transcriptase. For best results, amplicon size should be between 70 and 150 bp. Optimal results may require titration of primer concentration between 400 and 900 nM. A final concentration of 450 nM each primer and 100 to 150 nM probe is effective for most applications. The efficacy and efficiency of any primer/probe set should be validated under fast cycling and/or rapid ramp rate protocols before use in qPCR studies.Cat No. 95168-100Size: 100 x 20 µL reactions (500 µL) Store at -25ºC to -15°C protected from light95168-500 95168-01K 95168-10K 500 x 20 µL reactions (5 x 500 µL) 1000 x 20 µL reactions (1 x 5 mL) 10000 x 20 µL reactions (1 x 50 mL)Guidelines for One-Step RT-qPCR continued:▪If frozen, thaw UltraPlex 1-Step ToughMix Low ROX on ice. Thoroughly mix by vortexing, and then centrifuge to collect contents to the bottom of the tube.Retain on ice before use.▪First-strand synthesis can be carried out between 42°C and 60°C. Optimal results are generally obtained with a 10-minute incubation at 50°C. Longer incubation times for first-strand synthesis (up to 20 min) may be used. Incubation at temperatures over 54°C may result in delayed Cqs for assays that are optimal at 48 - 50°C.▪We recommend a minimum of 30s incubation at 95°C to inactivate the RT and activate the hot-start polymerase prior to PCR cycling. Longer activation times (2 – 10 minutes) will not adversely affect product performance and may reduce early cycle background noise experienced with some hydrolysis probe chemistries.▪The kit is compatible with both fast or standard qPCR cycling protocols. Annealing and or extension temperatures may need to be optimized for a given primer/probe design or fluorogenic probe chemistry. Use the suggested protocol as a starting point. Multiplexed RT-qPCR may benefit from a slightly longer extension time (60 to 90s). Use of a slower ramp rate (~2.5°C/s) between the denaturation step and annealing/extension step may improve performance for some assays.▪To maximize specificity, reactions should be assembled and retained on ice before transfer to the qPCR instrument.▪Preparation of a reaction cocktail is recommended to reduce pipetting errors and maximize assay precision. Assemble the reaction cocktail with all required components except RNA template and mix thoroughly by vortexing. Then, dispense equal aliquots into each reaction tube. Add RNA to each reaction as the final step. Addition of sample as 2 to 5-µL volumes will improve assay precision.▪Suggested input quantities of template are: 1 pg to 100 ng total RNA; 10 fg to 10 ng poly A(+) RNA; 10 to 1x108 copies viral RNA.▪After sealing each reaction, mix contents by vortexing, and then centrifuge briefly to collect components at the bottom of the reaction tube.Reaction AssemblyComponent Volume for 20-μL rxn. Final ConcentrationUltraPlex 1-Step ToughMix Low ROX (4X) 5 µL 1XForward primer(s) variable 300 – 900 nMReverse primer(s) variable 300 – 900 nMProbe(s) variable 50-200 nMNuclease-free water variableRNA template 2 to 10 µL variableFinal Volume (μL) 20 µLNote: For smaller, or larger, reaction volumes scale all components proportionally.RT-qPCR Cycling ProtocolIncubate complete reaction mix in a real-time PCR detection system as follows:cDNA Synthesis 50°C, 10 minInitial denaturation 95°C, 3 minPCR cycling (30 - 45 cycles) 95°C, 3s to 10s60°C, 30s to 90s (data collection step)‡‡Note: The use of longer extension times (90s at 60°C), or a 3-step cycling protocol with an extension step of 60s at 72°C can help mitigate suppression of a low copy amplicon when co-amplified with a high copy target sequence.Quality ControlKit components are free of contaminating DNase and RNase. UltraPlex 1-Step ToughMix Low ROX is functionally tested in duplexed RT-qPCR. Kinetic analysis must demonstrate linear resolution over six orders of dynamic range (r2≥ 0.990) and a PCR efficiency ≥ 90% for the primary GOI with constant detection for the limiting exogenous positive control assay.Limited Label LicensesUse of this product signifies the agreement of any purchaser or user of the product to the following terms:1.The product may be used solely in accordance with the protocols provided with the product and this manual and for use with components contained in the kitonly. Quantabio, LLC. grants no license under any of its intellectual property to use or incorporate the enclosed components of this kit with any components not included within this kit except as described in the protocols provided with the product, this manual, and additional protocols available at . Some of these additional protocols have been provided by Quantabio product users. These protocols have not been thoroughly tested or optimized by Quantabio, LLC. Quantabio, LLC. neither guarantees them nor warrants that they do not infringe the rights of third-parties.2.Other than expressly stated licenses, Quantabio, LLC. makes no warranty that this kit and/or its use(s) do not infringe the rights of third-parties.3.This kit and its components are licensed for one-time use and may not be reused, refurbished, or resold.4.Quantabio, LLC. specifically disclaims any other licenses, expressed or implied other than those expressly stated.5.The purchaser and user of the kit agree not to take or permit anyone else to take any steps that could lead to or facilitate any acts prohibited above. Quantabio, LLC.may enforce the prohibitions of this Limited License Agreement in any Court, and shall recover all its investigative and Court costs, including attorney fees, in any action to enforce this Limited License Agreement or any of its intellectual property rights relating to the kit and/or its components.©2021 Quantabio, LLC. 100 Cummings Center Suite 407J Beverly, MA 01915; Telephone number: 1-888-959-5165.Quantabio products are manufactured in Beverly, Massachusetts, Frederick, Maryland and Hilden, Germany.Intended for molecular biology applications. This product is not intended for the diagnosis, prevention or treatment of a disease.UltraPlex, ToughMix and qScript are registered trademarks of Quantabio, LLC. TaqMan is a registered trademark of Roche Molecular Systems, Inc. ROX is a trademark of Life Technologies Corporation.。

高斯错误修改总结

A list of error messages and possible solutions -Gaussian calculations can fail with various error messages. Some error messages from .out and .log files - and possible solutions - have been compiled here to facilitate problem solving.-These are divided into:-Syntax and similar errors-语法类错误Memory and similar errors-内存类错误Convergence problems -不收敛错误Errors in solvent calculations -溶剂中的计算错误Errors in log files-错误文件-ERROR MESSAGES IN OUTPUT FILES-Syntax and similar errors:End of file in ZSymb.-Error termination via Lnk1e in /global/apps/gaussian/g03.e01/g03/l101.exe Solution: The blank line after the coordinate section in the .inp file is missing. (输入文件空行丢失)Unrecognized layer "X".-（不识别层X）Error termination via Lnk1e in /global/apps/gaussian/g03.e01/g03/l101.exeSolution: Error due to syntax error(s) in coordinate section (check carefully). If error is "^M", it is caused by DOS end-of-line characters (e.g. if coordinates were written under Windows). Remove ^M from line ends using e.g. emacs. To process .inp files from command line, use sed -i 's/^M//' File.inp (Important: command does not work if ^M is written as characters - generate ^M on command line using ctrl-V ctrl-M).-QPERR --- A SYNTAX ERROR WAS DETECTED IN THE INPUT LINE.-Solution: Check .inp carefully for syntax errors in keywords -RdChkP: Unable to locate IRWF=0 Number= 522.-Error termination via Lnk1e in /global/apps/gaussian/g03.e01/g03/l401.exe or-FileIO operation on non-existent file.-[...] Error termination in NtrErr:-NtrErr Called from FileIO.Solution: Operation on .chk file was specified (e.g.geom=check, opt=restart), but .chk was not found. Check that:-%chk= was specifed in .inp-.chk has the same name as .inp-.chk is in the same directory as .inp -run script transports .chk to temporary folder upon job start. Run scripts downloaded here should do this. -The combination of multiplicity N and M electrons is impossible.-（多重性）Error termination via Lnk1e in /global/apps/gaussian/g03.e01/g03/l301.exeSolution: Either the charge or the multiplicity of the molecule was not specified correctlyin .inp.-（电荷和多重性指定错误）Memory and similar errors: Out-of-memory error in routine RdGeom-1 (IEnd= 1200001 MxCore= 2500)-Use %mem=N MW to provide the minimum amount of memory required to complete this step-Error termination via Lnk1e in /global/apps/gaussian/g03.e01/g03/l101.exe or-Not enough memory to run CalDSu, short by 1000000 words.-Error termination via Lnk1e in /global/apps/gaussian/g03.e01/g03/l401.exe or-[...] allocation failure: -（表示配分失败）Error termination via Lnk1e in /global/apps/gaussian/g03.e01/g03/l1502.exe Solution: Specify more memory in .inp (%mem=Nmb). Possibly, also increase pvmem value in run script. Especially solvent calculations can exhibit allocation failures and explicit amounts of memory should be specified.-galloc: could not allocate memory.-（无法分配内存）Solution: The %mem value in .inp is higher than pvmem value in run script. Increase pvmem or decrease %mem. -Probably out of disk space(磁盘空间). Write error in NtrExt1 Solution: /scratch space is most likely full. Delete old files in temporary folder. -Convergence problems: Density matrix is not changing but DIIS error= 1.32D-06 CofLast= 1.18D-02.-（收敛问题）The SCF is confused. Error termination via Lnk1e in/global/apps/gaussian/g03.e01/g03/linda-exe/l502.exel Solution: Problem with DIIS. Turn it off completely, e.g. using SCF=qc, or partly by usingSCF=(maxconventionalcycles=N,xqc), where N is the number of steps DIIS should be used (see SCF keyword). -Convergence criterion not met. SCF Done: E(RHF) = NNNNNNN A.U. after 129 cycles -[...] Convergence failure -- run terminated. Error termination via Lnk1e in/global/apps/gaussian/g03.e01/g03/linda-exe/l502.exe Solution: One SCF cycle has a default of maximum 128 steps, and this was exceeded without convergence achieved. Possible solution: In the route section of input file, specify SCF=(MaxCycle=N), where N is the number of steps per SCF cycles. Alternatively, turn of DIIS (e.g. by SCF=qc) (see SCF keyword).--Problem with the distance matrix.-（距离矩阵）Error termination via Lnk1e in /pkg/gaussian/g03/l202.exe Solution: Try to restart optimization from a different input geometry. -（重新不同几何异构体的输入优化）New curvilinear step not converged（新曲线步骤不收敛）. Error imposing constraints-Error termination via Lnk1e in /pkg/gaussian/g03/l103.exe-Solution: Problem with constrained coordinates (e.g. in OPT=modredun calculation). Try to restart optimization from a slightly different input geometry. -（一种稍微不同的输入几何）-Optimization stopped. -- Number of steps exceeded, NStep= N-[..] Error termination request processed by link 9999.-Error termination via Lnk1e in /global/apps/gaussian/g03.e01/g03/l9999.exe Solution: Maximum number of optimization steps is twice the number of variables to be optimized. Try increasing the value by specifying OPT=(MaxCycle=N) in .inp file, where N is the number of optimization steps (see OPT keyword). Alternatively, try to start optimization from different geometry.--Errors in solvent calculations: AdVTs1: ISph= 2543 is engulfed by JSph= 2544 but Ae( 2543) is not yet zero!-Error termination via Lnk1e in /global/apps/gaussian/g03.e01/g03/l301.exe Solution: Problem is related to building of the cavity in solvent calculations（溶剂效应优化计算错误）. One possible solution is to change the cavity（腔） model (default in g03 is UAO, can be changed by adding RADII keyword in section below coordinates in the .inp file, e.g. RADII=UFF, see SCRF keyword).--Hydrogen X has 2 bounds. Keep it explicit at all point on the-potential energy surface to get meaningful results.Solution: In UAO cavity model, spheres are placed on groups of atoms, with hydrogens assigned to the heavy atom, they are bound to. If assignment fails (e.g. because heavy atom-H bond is elongated), cavity building fails. Possible solutions: a) use cavity model that also assigns spheres to hydrogens (e.g. RADII=UFF) or b) Assign a sphere explicity on problematic H atom (use SPHEREONH=N, see SCRF keyword)--ERROR MESSAGES IN LOGFILES =>> PBS: job killed: wall time N exceeded limit M-signal number 15 received. Solution: Job did not finish within specified wall time. Retrieve .out and .chk files from temporary folder /global/work/$USER/$JOB (or $PBS_JOBID) and restart calculation if possible (using e.g. opt=restart or scf=restart). -cp: cannot stat $JOB.inp: No such file or directory Solution: The .inp file is not in the directory from where the job was submitted (or its name was misspelled during submission. If error reads: cp: cannot stat $JOB .inp .inp, the .inp file was submitted with extension).-ntsnet: unable to schedule the minimum N workers Solution: The value of %N proc Linda=N in the .inp file is higher than the number of nodes asked for during submission. Make sure these values match.Connection refused [...] died without ever signing in-Sign in timed out after 0 worker connections. Did not reach minimum (N), shutting downSolution: Error appears if you run parallel calculations but did not add this file to your $HOME directory: .tsnet.config containing only the line: Tsnet.Node.lindarsharg: ssh (see also guidelines for submission). -Density matrix is not changing but DIIS error - Suggested solutions1/- SCF=qc will probably solve the problem, albeit at a cost- Change the SCF converger to either SD, Quadratic or Fermi2/- lower the symmetry of optimize with and optimizewith the "nosymm" keywordI solved the problem using a variation on the first suggestion. Normally the scf took less than 80 cycles to converge. So i used scf=(Maxconventionalcycles=100,xqc) which resulted in a good compromise between using scf=qc and optimisation speed.In the case of the DIIS error the scf always took more than 100 cycles before the error,so by adding scf=(Maxconventionalcycles=100,xqc) the scf switched to qc after 100 cycles in the standard DIIS mode.l9999错误是优化圈数不够，把out文件保存成gjf，修改后接着优化。

镍柱纯化 HisTrap HP

GE HealthcareInstructions 71-5027-68 AH HisTrap affinity columns HisTrap HP, 1 ml and 5 mlHisTrap™ HP is a ready to use column, prepacked with precharged Ni Sepharose™ High Performance. This prepacked column is ideal for preparative purification of Histidine-tagged recombinant proteins by immobilized metal ion affinity chromatography (IMAC).The special design of the column, together with the high-performance matrix of the Ni Sepharose medium, provides fast, simple, and easy separations in a convenient format.Ni Sepharose High Performance has low nickel (Ni2+) ion leakage and is compatible with a wide range of additives used in protein purification. HP columns can be operated with a syringe, peristaltic pump, or liquid chromatography system such as ÄKTA™ design chromatography systems.CAUTION! Contains nickel. May produce an allergic reaction..1Pack size available by special order.Connector kit1Union 1/16” female/M6 male is also needed.2Union M6 female/1/16” male is also needed.Table of contents1.Description..............................................................................32.General considerations.....................................................63.Operation.................................................................................74.Optimization........................................................................115.Stripping and recharging..............................................126.Cleaning-in-place.............................................................137.Scaling-up............................................................................138.Storage..................................................................................149.Troubleshooting................................................................1410.Intended use.......................................................................1711.Ordering Information (18)Code No. Product No. supplied 17-5247-01 HisTrap HP 5 × 1 ml 17-5247-05 HisTrap HP 100 × 1 ml 117-5248-01 HisTrap HP 1 × 5 ml 17-5248-02 HisTrap HP 5 × 5 ml 17-5248-05HisTrap HP 100 × 5 ml 1Connectors supplied Usage No. supplied 1/16” male/luer femaleConnection of syringe to top of HiTrap column1Tubing connector flangeless/M6 femaleConnection of tubing (e.g. Peristaltic Pump P1) to bottom of HiTrap column 11Tubing connector flangeless/M6 male Connection of tubing (e.g. Peristaltic Pump P1) to top of HiTrap column 21Union 1/16” female/M6 male Connection to original FPLC™ System through bottom of HiTrap column1Union M6 female/1/16” maleConnection to original FPLC System through top of HiTrap column 1Stop plug female, 1/16”Sealing bottom of HiTrap column 2, 5 or 71DescriptionMedium propertiesHisTrap HP 1 ml and 5 ml columns are prepacked with NiSepharose High Performance, which consists of 34 µm highlycross-linked agarose beads with an immobilized chelating group.The medium has then been charged with Ni2+-ions.Several amino acids, for example histidine, form complexes with many metal ions. Ni Sepharose High Performance selectively binds proteins if suitable complex-forming amino acid residues areexposed on the protein surface.Additional histidines, such as in the case of (histidine)6 -tag,increase affinity for Ni2+ and generally make the histidine-tagged protein the strongest binder among other proteins in for example an E. coli extract.Column propertiesHisTrap HP columns are made of biocompatible polypropylenethat does not interact with biomolecules. Columns are deliveredwith a stopper on the inlet and a snap-off end on the outlet. Thecolumns have porous top and bottom frits that allow high flowrates. They cannot be opened or refilled.Columns can be operated with either a syringe and the supplied luer connector, a peristaltic pump, or a chromatography systemsuch as ÄKTA design.Note:To prevent leakage, ensure that the connector is tight.Table 1. HisTrap HP characteristics.1Dynamic binding capacity conditions:Note: Dynamic binding capacity is protein-dependent.2H 2O at room temperature 3Ni 2+-stripped mediumMatrixHighly cross-linked spherical agarose, 6%Average particle size 34 µmMetal ion capacity ~ 15 µmol Ni 2+/ml mediumDynamic binding capacity 1 At least 40 mg (histidine)6-tagged protein/ml medium Column volumes 1 ml or 5 ml Column dimensions i.d. × H:0.7 × 2.5 cm (1 ml) 1.6 × 2.5 cm (5 ml)Recommended flow rate 1 and 5 ml/min for 1 and 5 ml column respectivelyMax. flow rates 4 and 20 ml/min for 1 and 5 ml column respectively Max back pressure 20.3 MPa, 3 barCompatibility during use Stable in all commonly used buffers, reducingagents, denaturants, and detergents (s ee Table 2)Chemical stability 30.01 M HCl, 0.1 M NaOH. Tested for 1 week at 40°C.1 M NaOH, 70% acetic acid. Tested for 12 hours.2% SDS. Tested for 1 hour.30% 2-propanol. Tested for 30 min.Avoid in buffers Chelating agents, e.g. EDTA, EGTA, citrate (see Table 2)pH stability 3short term (at least 2 hours): 2 to 14 long term (≤ 1 week): 3 to 12Storage 20% ethanol Storage temperature4°C to 30°CSample:1 mg/ml (histidine)6-tagged pure protein (M r 28 000 or 43 000) inbinding buffer (Q B, 10% determination) or (histidine)6-tagged protein bound from E. coli extractColumn volume: 0.25 ml or 1 ml Flow rate: 0.25 ml/min or 1 ml/min Binding buffer: 20 mM sodium phosphate, 0.5 M NaCl, 5 mM imidazole, pH 7.4Elution buffer: 20 mM sodium phosphate, 0.5 M NaCl, 0.5 M imidazole, pH 7.4The Ni 2+-charged medium is compatible with all commonly used aqueous buffers, reducing agents, denaturants such as 6 M Gua-HCl and 8 M urea, and a range of other additives (see Table 2).Table 2. Ni Sepharose High Performance is compatible with the following compounds, at least at the concentrations given.1See Notes and blank run, p. 10–11.2Tested for 1 week at 40°C.3The strong chelator EDTA has been used successfully in some cases, at 1 mM.Generally, chelating agents should be used with caution (and only in the sample, not the buffers). Any metal-ion stripping may be counteracted by addition of a small excess of MgCl 2 before centrifugation/filtration of the sample. Note that stripping effects may vary with applied sample volume.Reducing agents 15 mM DTE 5 mM DTT20 mM ß-mercaptoethanol 5 mM TCEP10 mM reduced glutathione Denaturing agents 8 M urea 26 M guanidine hydrochloride 2Detergents2% Triton™ X-100 (nonionic)2% Tween™ 20 (nonionic)2% NP-40 (nonionic)2% cholate (anionic)1% CHAPS (zwitterionic)Other additives500 mM imidazole 20% ethanol 50% glycerol 100 mM Na 2SO 41.5 M NaCl 1 mM EDTA 360 mM citrate 3Buffer substances50 mM sodium phosphate, pH 7.4100 mM Tris-HCl, pH 7.4100 mM Tris-acetate, pH 7.4100 mM HEPES, pH 7.4100 mM MOPS, pH 7.4100 mM sodium acetate, pH 422General considerationsHisTrap HP is supplied precharged with Ni2+ ions. In general, Ni2+ is the preferred metal ion for purification of recombinant histidine-tagged proteins. Note, however, that in some cases it may be wise to test other metal ions, for example Zn2+ and Co2+, as the strength of binding depends on the nature of the histidine-tagged protein as well as the metal ion (see Optimization).We recommend binding at neutral to slightly alkaline pH (pH 7–8) in the presence of 0.5–1.0 M NaCl. Sodium phosphate buffers are often used. Tris-HCl can generally be used, but should be avoided in cases where the metal-protein affinity is very weak, since it may reduce binding strength. Avoid chelating agents such as EDTA or citrate in buffers, see Table2.Including salt, for example 0.5–1.0 M NaCl in the buffers andsamples, eliminates ion-exchange effects but can also have amarginal effect on the retention of proteins.Imidazole at low concentrations is commonly used in the binding and the wash buffers to minimize binding of host cell proteins. For the same reason, it is important to also include imidazole in thesample (generally, at the same concentration as in the washbuffer). At somewhat higher concentrations, imidazole may alsodecrease the binding of histidine-tagged proteins. The imidazole concentration must therefore be optimized to ensure the bestbalance of high purity (low binding of host cell proteins) and high yield (binding of histidine-tagged target protein). This optimalconcentration is different for different histidine-tagged proteins,and is usually slightly higher for Ni Sepharose High Performance than for similar IMAC media on the market (see Data File18-1174-40). Use highly pure imidazole; such imidazole givesessentially no absorbance at 280 nm.As alternatives to imidazole elution, histidine-tagged proteins can be eluted from the medium by several other methods orcombinations of methods – a lowering of pH within the range of2.5–7.5 can be used, for example. At pH values below 4, metal ionswill be stripped off the medium.Note:If the proteins are sensitive to low pH, we recommendcollection of the eluted fractions in tubes containing1 M Tris-HCl, pH 9.0 (60–200 µl/ml fraction) to restore thepH to neutral.Chelating agents such as EGTA or EDTA will also strip metal ionsfrom the medium and thereby cause protein elution, but the target protein pool will then contain Ni2+ ions. In this case, Ni2+ ions can be removed by desalting on a HiTrap™ Desalting, a PD-10 Desalting Column, or HiPrep™ 26/10 Desalting, (see Table3).Leakage of Ni2+ from Ni Sepharose High Performance is very low under all normal conditions, lower than for other IMAC mediatested. For applications where extremely low leakage duringpurification is critical, leakage can be even further reduced byperforming a blank run (see page 11). Likewise, a blank run should also be performed before applying buffers/ samples containingreducing agents (see page 11).Whatever conditions are chosen, HisTrap HP columns can beoperated with a syringe, peristaltic pump, or chromatographysystem.Note:If Peristaltic Pump P-1 is used, the maximum flow rate that can be run on a HisTrap HP 1 ml column is 3 ml/min.3OperationBuffer preparationWater and chemicals used for buffer preparation should be of high purity. Filter buffers through a 0.22 µm or a 0.45 µm filter beforeuse.Use high purity imidazole as this will give very low or noabsorbance at 280 nm.If the recombinant histidine-tagged protein is expressed asinclusion bodies, include 6 M Gua-HCl or 8 M urea in all buffers and sample. On-column refolding of the denatured protein may bepossible.Recommended conditionsSample preparationFor optimal growth, induction, and cell lysis conditions for your recombinant histidine-tagged clones, please refer to established protocols.Adjust the sample to the composition and pH of the binding buffer by: Adding buffer, NaCl, imidazole, and additives fromconcentrated stock solutions; by diluting the sample with binding buffer; or by buffer exchange, (see Table 3). Do not use strong bases or acids for pH-adjustment (precipitation risk). Filter the sample through a 0.22 µm or a 0.45 µm filter and/or centrifuge it immediately before applying it to the column.To prevent the binding of host cell proteins with exposed histidine, it is essential to include imidazole at a low concentration in the sample and binding buffer (see Optimization).Binding buffer: 20 mM sodium phosphate, 0.5 M NaCl,20–40 mM imidazole, pH 7.4 (The optimal imidazole concentration is protein-dependent; 20–40 mM is suitable for many proteins.)Elution buffer:20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole, pH 7.4 (The imidazole concentration required for elution is protein-dependent).Table 3. Prepacked columns for desalting and buffer exchangePurification1Fill the syringe or pump tubing with distilled water. Remove the stopper and connect the column to the syringe (use the luerconnector provided), laboratory pump or chromatographysystem tubing “drop-to-drop” to avoid introducing air into thesystem.2Remove the snap-off end at the column outlet.3Wash the column with 3–5 column volumes of distilled water.4Equilibrate the column with at least 5 column volumes of binding buffer. Recommended flow rates are 1 ml/min or5 ml/min for the 1 and 5 ml columns respectively.In some cases a blank run is recommended before finalequilibration/ sample application (see page 11).5Apply the pretreated sample using a syringe or a pump.6Wash with binding buffer until the absorbance reaches a steady baseline (generally, at least 10–15 column volumes).Note:Purification results are improved by using imidazole insample and binding buffer (see Optimization).7Elute with elution buffer using a one-step or linear gradient.Five column volumes are usually sufficient if the protein ofinterest is eluted by a one-step gradient. A shallow gradient, forexample a linear gradient over 20 column volumes or more,may separate proteins with similar binding strengths.Note:If imidazole needs to be removed from the protein, use HiTrap Desalting, a PD-10 Desalting Column, or HiPrep26/10 Desalting depending on the sample volume (seeTable3).Note:Ni Sepharose High Performance is compatible withreducing agents. However, removal of any weakly boundNi2+ ions by performing a blank run without reducingagents (as described on page 11) before applying buffer/sample including reducing agents is recommended. Do notleave HisTrap HP columns with buffers including reducingagents when not in use.Note:Leakage of Ni2+ from Ni Sepharose High Performance is low under all normal conditions. The leakage is lower than forother IMAC media tested (see Data File Ni Sepharose HighPerformance, 18-1174-40). For very critical applications,leakage during purification can be even further diminishedby performing a blank run (as described below) beforeloading sample.Blank run:Use binding buffer and elution buffer without reducing agents.1Wash the column with 5 column volumes of distilled water (to remove the 20% ethanol).2Wash with 5 column volumes of elution buffer.3Equilibrate with 10 column volumes of binding buffer.4OptimizationImidazole at low concentrations is commonly used in the binding and the wash buffers to minimize binding of host cell proteins. For the same reason, it is important to also include imidazole in thesample (generally, at the same concentration as in the washbuffer). At somewhat higher concentrations, imidazole may alsodecrease the binding of histidine-tagged proteins. The imidazole concentration must therefore be optimized to ensure the bestbalance of high purity (low binding of host cell proteins), and high yield (binding of histidine-tagged target protein). This optimalconcentration is different for different histidine-tagged proteins,and is usually slightly higher for Ni Sepharose High Performance than for similar IMAC media on the market (see Data File NiSepharose High Performance, 18-1174-40). Finding the optimalimidazole concentration for a specific histidine-tagged protein is a trial-and-error effort, but 20–40 mM in the binding and washbuffers is a good starting point for many proteins. Use a high purity imidazole, such imidazole gives essentially no absorbance at280 nm.Ni2+ is usually the first choice metal ion for purifying most(histidine)6-tagged recombinant proteins from nontagged host cell proteins, and also the ion most generally used. Nevertheless, it isnot always possible to predict which metal ion will be best for agiven protein. The strength of binding between a protein and ametal ion is affected by several factors, including the length,position, and exposure of the affinity tag on the protein, the type of ion used, and the pH of buffers, so some proteins may be easier to purify with ions other than Ni2+.A quick and efficient way to test this possibility and optimizeseparation conditions is to use HiTrap IMAC HP 1 ml columns,which are packed with IMAC Sepharose High Performance (notcharged with metal ions). Each column can be charged withdifferent metal ions, for example Cu2+, Co2+, Zn2+, Ca2+, or Fe2+.Instructions are included with each column.A study to compare the purification of six (histidine)6-taggedrecombinant proteins, including three variants of maltose-binding protein, with different metal ions has indicated that Ni2+ generally gives best selectivity between (histidine)-tagged and nontaggedhost-cell proteins (see Application Note 18-1145-18).5Stripping and rechargingNote:The column does not have to be stripped and recharged between each purification if the same protein is going to bepurified; it is sufficient to strip and recharge it after 5–7purifications, depending on the cell extract, extract volume,target protein, etc.Recommended stripping buffer: 20 mM sodium phosphate,0.5 M NaCl, 50 mM EDTA, pH 7.4Strip the column by washing with at least 5–10 column volumes of stripping buffer. Wash with at least 5–10 column volumes ofbinding buffer and 5–10 column volumes of distilled water before recharging the column.Recharge the water-washed column by loading 0.5 ml or 2.5 ml of0.1 M NiSO4 in distilled water on HisTrap HP 1 ml and 5 ml column,respectively. Salts of other metals, chlorides, or sulfates, may also be used (see“Optimization”). Wash with 5 column volumes distilled water, and 5 column volumes binding buffer (to adjust pH) before storage in 20% ethanol.6Cleaning-in-placeWhen an increase in back-pressure is seen, the column can becleaned. Before cleaning, strip off Ni2+ ions using therecommended procedure described above.After cleaning, store in 20% ethanol (wash with 5 column volumes) or recharge with Ni2+ prior to storage in ethanol.The Ni2+-stripped column can be cleaned by the followingmethods;•Remove ionically bound proteins by washing the column with several column volumes of 1.5 M NaCl; then wash the columnwith approximately 10 column volumes of distilled water.•Remove precipitated proteins, hydrophobically bound proteins, and lipoproteins by washing the column with 1 MNaOH, contact time usually 1–2 hours (12 hours or more forendotoxin removal). Then wash the column withapproximately 10 column volumes of binding buffer, followedby 5–10 column volumes of distilled water.•Remove hydrophobically bound proteins, lipoproteins, and lipids by washing the column with 5–10 column volumes 30%isopropanol for about 15–20 minutes. Then wash the columnwith approximately 10 column volumes of distilled water.Alternatively, wash the column with 2 column volumes ofdetergent in a basic or acidic solution. Use, for example,0.1–0.5% nonionic detergent in 0.1 M acetic acid, contact time1–2 hours. After treatment, always remove residual detergentby washing with at least 5 column volumes of 70% ethanol.Then wash the column with approximately 10 column volumesof distilled water.7Scaling-upTwo or three HisTrap HP 1 ml or 5 ml columns can be connected in series for quick scale-up (note that back-pressure will increase).Ni Sepharose High Performance, the medium prepacked inHisTrap HP columns, is supplied preswollen in 25 and 100 ml labpacks (see Ordering Information). An alternative scale-up strategy is thus to pack the medium in empty columns – Tricorn™ and XK columns are suitable for this purpose.8StorageStore HisTrap HP columns in 20% ethanol at 4°C to 30°C.9TroubleshootingThe following tips may be of assistance. If you have any furtherquestions about your HisTrap HP column, please visit/hitrap, contact our technical support, or your local representative.Note:When using high concentrations of urea or Gua-HCl,protein unfolding generally takes place. Refolding on-column (or after elution) is protein-dependent.Tips:To minimize dilution of the sample, solid urea or Gua-HCl can be added.Tips:Samples containing urea can be analyzed directly by SDS-PAGE whereas samples containing Gua-HCl must be buffer-exchangedto a buffer with urea before SDS-PAGE.Column has clogged:•Cell debris in the sample may clog the column. Clean the column according to the section Cleaning-in-place.•It is important to centrifuge and/or filter the sample through a0.22 µm or a 0.45 µm filter, see Sample preparation. Sample is too viscous:•If the lysate is very viscous due to a high concentration of host nucleic acid, continue sonication until the viscosity is reduced,and/or add DNase I to 5 µg/ml, Mg2+ to 1 mM, and incubate onice for 10–15 min. Alternatively, draw the lysate through asyringe needle several times.Protein is difficult to dissolve or precipitates during purification:•The following additives may be used: 2% Triton X-100, 2% Tween 20, 2% NP-40, 2% cholate, 1% CHAPS, 1.5 M NaCl,50% glycerol, 20 mM ß-mercaptoethanol, 1–3 mM DTT or DTE(up to 5 mM is possible but depends on the sample and thesample volume), 5 mM TCEP, 10 mM reduced glutathione, 8 Murea, or 6 M Gua-HCl. Mix gently for 30 min to aid solubilizationof the tagged protein (inclusion bodies may require longermixing). Note that Triton X-100 and NP-40 (but not Tween)have a high absorbance at 280 nm. Furthermore, detergentscannot be easily removed by buffer exchange.No histidine-tagged protein in the purified fractions:•Elution conditions are too mild (histidine-tagged protein still bound: Elute with an increasing imidazole gradient ordecreasing pH to determine the optimal elution conditions.•Protein has precipitated in the column: For the nextexperiment, decrease amount of sample, or decrease proteinconcentration by eluting with linear imidazole gradient insteadof imidazole steps. Try detergents or changed NaClconcentration, or elute under denaturing (unfolding)conditions (add 4–8 M urea or 4–6 M Gua-HCl).•Nonspecific hydrophobic or other interaction: Add a nonionic detergent to the elution buffer (e.g. 0.2% Triton X-100)or increase the NaCl concentration.•Concentration of imidazole in the sample and/or binding buffer is too high: The protein is found in the flow-throughmaterial. Decrease the imidazole concentration.•Histidine-tag may be insufficiently exposed: The protein is found in the flowthrough material; perform purification ofunfolded protein in urea or Gua-HCl as for inclusion bodies.•Buffer/sample composition is incorrect: The protein is found in the flowthrough material. Check pH and composition ofsample and binding buffer. Ensure that chelating or strongreducing agents are not present in the sample at too highconcentration, and that the concentration of imidazole is nottoo high.SDS-PAGE of samples collected during the preparation of thebacterial lysate may indicate that most of histidine-tagged protein is located in the centrifugation pellet. Possible causes andsolutions are:•Sonication may be insufficient: Cell disruption may be checked by microscopic examination or monitored bymeasuring the release of nucleic acids at A260. Addition oflysozyme (up to 0.1 volume of a 10 mg/ml lysozyme solution in25 mM Tris-HCl, pH 8.0) prior to sonication may improveresults. Avoid frothing and overheating as this may denaturethe target protein. Over-sonication can also lead tocopurification of host proteins with the target protein.•The protein may be insoluble (inclusion bodies): The protein can usually be solubilized (and unfolded) from inclusion bodiesusing common denaturants such as 4–6 M Gua-HCl, 4–8 Murea, or strong detergents. Prepare buffers containing 20 mMsodium phosphate, 8 M urea, or 6 M Gua-HCl, and suitableimidazole concentrations, pH 7.4–7.6. Buffers with urea shouldalso include 500 mM NaCl. Use these buffers for samplepreparation, as binding buffer and as elution buffer. Forsample preparation and binding buffer, use 10–20 mMimidazole or the concentration selected during optimizationtrials (including urea or Gua-HCl).The protein is collected but is not pure (multiple bands on SDS polyacrylamide gel):•Partial degradation of tagged protein by proteases: Add protease inhibitors (use EDTA with caution, see Table2).•Contaminants have high affinity for nickel ions: Elute with a stepwise or linear imidazole gradient to determine optimalimidazole concentrations to use for binding and for wash; addimidazole to the sample in the same concentration as thebinding buffer. Wash before elution with binding buffercontaining as high concentration of imidazole as possible,without causing elution of the tagged protein. A shallowimidazole gradient (20 column volumes or more), mayseparate proteins with similar binding strengths. If optimizedconditions do not remove contaminants, further purificationby ion exchange chromotography (HiTrap Q HP orHiTrap SP HP) and/or gel filtration (Superdex™ Peptide,Superdex 75 or Superdex 200) may be necessary.•Contaminants are associated with tagged proteins: Add detergent and/or reducing agents before sonicating cells.Increase detergent levels (e.g. up to 2% Triton X-100 or2% Tween 20), or add glycerol (up to 50%) to the wash bufferto disrupt nonspecific interactions.Histidine-tagged protein is eluted during sample loading/wash:•Buffer/sample composition is incorrect: Check pH and composition of sample and binding buffer. Ensure thatchelating or strong reducing agents are not present in thesample at a too high concentration, and that theconcentration of imidazole is not too high.•Histidine-tag is partially obstructed: Purify under denaturing conditions (use 4–8 M urea or 4–6 M Gua-HCl).•Column capacity is exceeded: Join two or three HisTrap HP1 ml columns together or change to a HisTrap HP 5 ml column. 10Intended useThe HisTrap HP is intended for research use only, and shall not be used in any clinical or in vitro procedures for diagnostic purposes.11Ordering InformationProduct No. supplied Code No.HisTrap HP 5 × 1 ml 17-5247-01100 × 1 ml1 17-5247-051 × 5 ml 17-5248-015 × 5 ml 17-5248-02100 × 5 ml1 17-5248-05 Related products No. supplied Code No.100 ml 17-5268-02 HiTrap Desalting 5 × 5 ml 17-1408-01100 × 5 ml1 11-0003-29 PD-10 Desalting Column 30 17-0851-01 HiPrep 26/10 Desalting 1 × 53 ml 17-5087-014 × 53 ml 17-5087-02HisTrap FF 5 × 1 ml 17-5319-01100 × 1 ml1 17-5319-025 × 5 ml 17-5255-01100 × 5 ml1 17-5255-02 HisTrap FF crude 5 × 1 ml 11-0004-58100 × 1 ml1 11-0004-595 × 5 ml 17-5286-01100 × 5 ml1 17-5286-02 HisTrap FF crude Kit 1 kit 28-4014-77 HisPrep™ FF 16/10 1 × 20 ml 28-9365-511Pack size available by special order.1 One connector included in each HiTrap package.2 Two, five, or seven stop plugs female included in HiTrap packages depending on the product.3One fingertight stop plug is connected to the top of each HiTrap column at delivery.AccessoriesNo. Supplied Code No.1Tubing connector flangeless/M6 female 12 18-1003-68Tubing connector flangeless/M6 male 1 2 18-1017-98Union 1/16” female/M6 male 1 6 18-1112-57Union M6 female /1/16” male 1 5 18-3858-01Union luerlock female/M6 female 218-1027-12HiTrap/HiPrep, 1/16” male connector for ÄKTA design8 28-4010-81Stop plug female, 1/16”2 5 11-0004-64Fingertight stop plug, 1/16”35 11-0003-55Related literatureCode No.Recombinant Protein Purification Handbook, Principles and Methods18-1142-75Affinity Chromatography Handbook, Principles and Methods18-1022-29Affinity Chromatography, Columns and Media Selection Guide18-1121-86Ni Sepharose and IMAC Sepharose, Selection Guide 28-4070-92HiTrap Column Guide18-1129-81。

OpticsExpress

#189766 - $15.00 USD (C) 20132013; revised 3 Jun 2013; accepted 3 Jun 2013; published 18 Jul 2013 29 July 2013 | Vol. 21, No. 15 | DOI:10.1364/OE.21.017769 | OPTICS EXPRESS 17769
©2013 Optical Society of America
OCIS codes: (190.4370) Nonlinear optics, fibers; (320.7110) Ultrafast nonlinear optics; (320.5540) Pulse shaping; (060.5295) Photonic crystal fibers.
Abstract: Formation of parabolic pulses at femtosecond time scale by means of passive nonlinear reshaping in normally dispersive optical fibers is analyzed. Two approaches are examined and compared: the parabolic waveform formation in transient propagation regime and parabolic waveform formation in the steady-state propagation regime. It is found that both approaches could produce parabolic pulses as short as few hundred femtoseconds applying commercially available fibers, specially designed all-normal dispersion photonic crystal fiber and modern femtosecond lasers for pumping. The ranges of parameters providing parabolic pulse formation at the femtosecond time scale are found depending on the initial pulse duration, chirp and energy. Applicability of different fibers for femtosecond pulse shaping is analyzed. Recommendation for shortest parabolic pulse formation is made based on the analysis presented.

超图电子HV9861ADB1高光LED驱动板说明书

Supertex inc.HV9861ADB1General DescriptionThe HV9861ADB1 demoboard is a high-brightness LED driver employing the patented average-mode, constant current control scheme by Supertex Inc. The power conversion stage of the HV9861ADB1 consists of a diode bridge rectifier followed by a buck converter operating with fixed off-time of 20µs.The HV9861ADB1 LED driver features tight regulation of the LED current within a few milliamps over the entire range of the input AC line and the output LED string voltage. The LEDcurrent accuracy is almost insensitive to the passive component tolerances, such as the output filter inductance or the timing resistor. The accuracy of the LED current is mainly determined by the internal 270mV ± 3% reference voltage of the HV9861A control IC and by the external current sense resistor tolerance.(Note, that the linear dimming input of the HV9861A disables switching, when its voltage falls below a 200mV threshold.Expect the LED driver to shut off when the LED current falls below 50 ~ 55mA.)PWM dimming can be achieved by applying a pulse-width-modulated square wave signal between the PWMD and GND pins.The HV9861ADB1 features protection from an output short circuit condition. Open LED protection is inherent, since theoutput filter capacitor can accept the full rated rectified AC line voltage. Please, note that the demoboard is not CISPR15 compliant.An additional input EMI filter circuit is required to make the board meet conducted electro-magnetic emission limits. Also, note that:NO SAFETY ISOLATION OF THE LED LOAD FROM THE AC MAINS VOLTAGE IS PROVIDED!21-Watt, Universal, AC LED Driver Demoboard with Accurate Average-Mode, Constant Current ControlConnection Diagram WARNINGDo not connect earth-grounded test instruments. Doing so will short the AC line, resulting in damage to the instrument and/or the HV9861ADB1. Use floating high voltage differ -ential probes or isolate the demoboard by using an isolat-ing transformer.No galvanic isolation. Dangerous voltages are present when connected to the AC line.1. Connect the input AC voltage between the AC INterminals as shown in the connection diagram.2. Connect the LED string between LED+ (anode ofLED string) and LED- (cathode of LED string).3. Connect the PWMD terminal to the VDD terminalusing the jumper provided to enable the LED driver.4. The current level can be adjusted by applying 0 -1.5V between LD and GND. Leave LD floating to use the internal current setting.Connections90-265VAC50/60Hz0 -1.5V for LD function or leave openSilk ScreenPWM DimmingThe HV9861ADB1 is capable of being PWM dimmed by apply-ing a square wave TTL compatible signal between PWMD and GND terminals. However, since there is no galvanic isolation on the board, care must be taken to prevent damage to the PWM dimming source and/or the HV9861ADB1. One simple way is to isolate the LED driver from the AC line using an isola-tion transformer. Another approach is to use an opto-isolator to drive the PWMD pin as shown in the following figure.5.0Vsquarewavesignal(<1.0kHz)PWMDVDD(e.g. LTV-814 from Lite-On) Schematic60L E D C u r r e n t (A )AC Line Voltage (VAC)AC Line Voltage (VAC)L E D C u r r e n t (A )PWM Dimming Response Short Circuit CurrentI LEDPWMDV IN = 240VACTypical CharacteristicsSupertex inc. does not recommend the use of its products in life support applications, and will not knowingly sell them for use in such applications unless it receives an adequate “product liability indemnification insurance agreement.” Supertex inc. does not assume responsibility for use of devices described, and limits its liability to the replacement of the devices determined defective due to workmanship. No responsibility is assumed for possible omissions and inaccuracies. Circuitry and specifications are subject to change without notice. For the latest product specifications refer to the Supertex inc. (website: http//)©2013 Supertex inc.All rights reserved. Unauthorized use or reproduction is prohibited.Supertex inc.。

Ultrafast transient absorption spectroscopy principles and application to photosynthetic systems

REVIEWUltrafast transient absorption spectroscopy:principles and application to photosynthetic systemsRudi Berera ÆRienk van Grondelle ÆJohn T.M.KennisReceived:18February 2009/Accepted:5June 2009/Published online:4July 2009ÓThe Author(s)2009.This article is published with open access at Abstract The photophysical and photochemical reac-tions,after light absorption by a photosynthetic pigment–protein complex,are among the fastest events in biology,taking place on timescales ranging from tens of femto-seconds to a few nanoseconds.The advent of ultrafast laser systems that produce pulses with femtosecond duration opened up a new area of research and enabled investigation of these photophysical and photochemical reactions in real time.Here,we provide a basic description of the ultrafast transient absorption technique,the laser and wavelength-conversion equipment,the transient absorption setup,and the collection of transient absorption data.Recent appli-cations of ultrafast transient absorption spectroscopy on systems with increasing degree of complexity,from bio-mimetic light-harvesting systems to natural light-harvest-ing antennas,are presented.In particular,we will discuss,in this educational review,how a molecular understanding of the light-harvesting and photoprotective functions of carotenoids in photosynthesis is accomplished through the application of ultrafast transient absorption spectroscopy.Keywords Ultrafast spectroscopy ÁPhotosynthesis ÁLight-harvesting antennasAbbreviations(B)Chl (Bacterio)Chlorophyll BPheo Bacteriopheophtin EADS Evolution-associated difference spectra ESA Excited-state absorption FWHM Full-width at half maximum LHC Light-harvesting complex PSII Photosystem II RC Reaction center SADS Species-associated difference spectra SE Stimulated emissionIntroductionThe process of photosynthesis relies upon the efﬁcient absorption and conversion of the radiant energy from the Sun.Chlorophylls and carotenoids are the main players in the process.While the former are involved in light-har-vesting and charge separation process,the latter also play vital photoprotective roles.Photosynthetic pigments are typically arranged in a highly organized fashion to con-stitute antennas and reaction centers,supramolecular devices where light harvesting and charge separation take place.The very early steps in the photosynthetic process take place after the absorption of a photon by an antenna sys-tem,which harvests light and eventually delivers it to the reaction center (Van Grondelle et al.1994).Despite the enormous variety of photosynthetic organisms,the primary events leading to photosynthetic energy storage areremarkably similar (Sundstro¨m 2008).In order to compete with internal conversion,intersystem crossing,and ﬂuo-rescence,which inevitably lead to energy loss,the energyR.Berera ÁR.van Grondelle ÁJ.T.M.Kennis (&)Department of Physics and Astronomy,Faculty of Sciences,VU University Amsterdam,De Boelelaan 1081,1081HV Amsterdam,The Netherlands e-mail:john@nat.vu.nlPresent Address:R.BereraInstitute of Biology and Technology of Saclay,CEA (Commissariat a l’Energie Atomique),URA 2096CNRS (Centre National de la Recherche Scientiﬁque),91191Gif/Yvette,FrancePhotosynth Res (2009)101:105–118DOI 10.1007/s11120-009-9454-yand electron transfer processes thatﬁx the excited-state energy in photosynthesis must be extremely fast.In order to investigate these events,ultrafast techniques down to a sub-100fs resolution must be used.In this way,energy migration within the system as well as the formation of new chemical species such as charge-separated states can be tracked in real time.This can be achieved by making use of ultrafast transient absorption spectroscopy.The basic principles of this technique,instrumentation,and some recent applications to photosynthetic systems that involve the light-harvesting and photoprotective functions of carotenoids are described in this educational review.For earlier reviews on ultrafast spectroscopy,see e.g.,Jimenez and Fleming(1996),Groot and Van Grondelle(2008),and Zigmantas et al.(2008).Ultrafast transient absorption spectroscopyThe principle of ultrafast transient absorption spectroscopyThe process of energy transfer in a photosynthetic mem-brane typically takes place on a time scale from less than 100fs to hundreds of ps(Sundstro¨m et al.1999;Van Amerongen and Van Grondelle2001;Van Grondelle et al. 1994).The advent of ultrashort tunable laser systems in the early1990s has opened up a new and extremely fascinating area of research.Nowadays,the high(sub50fs)time resolution has made it possible to investigate the very early events taking place within a light-harvesting antenna in real time(Sundstro¨m2008).In transient absorption spectros-copy,a fraction of the molecules is promoted to an elec-tronically excited state by means of an excitation(or pump) pulse.Depending on the type of experiment,this fraction typically ranges from0.1%to tens of percents.A weak probe pulse(i.e.,a pulse that has such a low intensity that multiphoton/multistep processes are avoided during prob-ing)is sent through the sample with a delay s with respect to the pump pulse(Fig.1).A difference absorption spec-trum is then calculated,i.e.,the absorption spectrum of the excited sample minus the absorption spectrum of the sample in the ground state(D A).By changing the time delay s between the pump and the probe and recording a D A spectrum at each time delay,a D A proﬁle as a function of s and wavelength k,i.e.,a D A(k,s)is obtained.D A(k,s) contains information on the dynamic processes that occur in the photosynthetic system under study,such as excited-state energy migration,electron and/or proton transfer processes,isomerization,and intersystem crossing.In order to extract this information,global analysis procedures may be applied(see below).One advantage of time-resolved absorption spectroscopy over time-resolvedﬂuorescence is that with the former,the evolution of non-emissive states and dark states can be investigated.This is of particular importance in photosynthesis where carotenoid dark(non-emissive)states play a number of vital roles.In general,a D A spectrum contains contributions from various processes:(1)Theﬁrst contribution is by ground-state bleach.As afraction of the molecules has been promoted to the excited state through the action of the pump pulse,the number of molecules in the ground state has been decreased.Hence,the ground-state absorption in the excited sample is less than that in the non-excited sample.Consequently,a negative signal in the D A spectrum is observed in the wavelength region of ground state absorption,as schematically indicated in Fig.1(dashed line).(2)The second contribution is by stimulated emission.For a two-level system,the Einstein coefﬁcients for absorption from the ground to the excited state(A12)and stimulated emission from the excited to the ground state(A21)are identical.Thus,upon popula-tion of the excited state,stimulated emission to the ground state will occur when the probe pulse passes through the excited volume.Stimulated emission will occur only for optically allowed transitions and will have a spectral proﬁle that(broadly speaking)follows theﬂuorescence spectrum of the excited chromo-phore,i.e.,it is Stokes shifted with respect to the ground-state bleach.During the physical process of stimulated emission,a photon from the probe pulse induces emission of another photon from the excited molecule,which returns to the ground state.The photon produced by stimulated emission is emitted in the exact same direction as the probe photon,and hence both will be detected.Note that the intensity of the probe pulse is so weak that the excited-state population is not affected appreciably by this process.Stimulated emission results in an increase of light intensity on the detector,corresponding to a negativeD A signal,as schematically indicated in Fig.1(dottedline).In many chromophores including bacteriochlo-rophyll(BChl),the Stokes shift may be so small that the stimulated emission band spectrally overlaps with ground-state bleach and merges into one band. (3)The third contribution is provided by excited-stateabsorption.Upon excitation with the pump beam, optically allowed transitions from the excited(pop-ulated)states of a chromophore to higher excited states may exist in certain wavelength regions,and absorption of the probe pulse at these wavelengths will occur.Consequently,a positive signal in the D A spectrum is observed in the wavelength region of excited-state absorption(Fig.1,solid line).Again,the intensity of the probe pulse is so weak that the excited-state population is not affected appreciably by the excited-state absorption process.(4)A fourth possible contribution to the D A spectrum isgiven by product absorption.After excitation of the photosynthetic,or more generally photobiological or photochemical system,reactions may occur that result in a transient or a long-lived molecular state,such as triplet states,charge-separated states,and isomerized states.The absorption of such a(transient)product will appear as a positive signal in the D A spectrum.A ground-state bleach will be observed at the wave-lengths where the chromophore on which the product state resides has a ground-state absorption.A well-known example of such a transient product state is the accessory bacteriochlorophyll(BChl)anion in the bacterial reaction center(RC),which acts as a transient intermediate in the electron transfer process from the primary donor P to the bacteriopheophytin(BPheo).The rise and decay of this species can be monitored through its speciﬁc product absorption at 1,020nm(Arlt et al.1993;Kennis et al.1997a). Pulse duration,time resolution,and spectral selectivity Laser pulses as short as5fs are now available for transient absorption spectroscopy(see,e.g.,Cerullo et al.(2002); and Nishimura et al.(2004)).A short pulse duration D t implies a large spectral bandwidth D v according to relation D t D v=0.44for Gaussian-shaped pulses.This relation is known as the time–bandwidth product.For instance,a10-fs pulse with a center wavelength of800nm has a spectral bandwidth of4.491013Hz at full-width at half maximum (FWHM),which corresponds to about100nm in this wavelength region.Thus,one has to make a trade-off between time resolution and spectral selectivity.Consider the example of the bacterial RC,which has the primary donor absorbing at860nm,the accessory BChls at 800nm,and the BPheos at760nm.With a10-fs pulse at 800nm,one would simultaneously excite all the cofactors. In order to selectively excite one of the cofactor pairs to study its excited-state dynamics,spectral narrowing to *30nm is required,which implies a longer excitation pulse of*30fs(Streltsov et al.1998;Vos et al.1997).For the photosystem II(PSII)RC,where the energy gaps between the pigments are signiﬁcantly smaller,the exci-tation bandwidth has to be narrowed even more to\10nm for selective excitation,with corresponding pulse durations of*100fs(Durrant et al.1992;Groot et al.1997).On very fast timescales,transient absorption signals have contributions from processes additional to those described in the previous section.These non-resonant contributions are often lumped together under the terms‘‘coherent arti-fact’’and‘‘cross-phase modulation.’’As transient absorp-tion signals result from light–matter interaction through the third-order non-linear susceptibility v(3)(Mukamel1995), non-sequential light interactions that do not represent pop-ulation dynamics of electronic states will contribute to the signals.Such undesired signals can be ignored by excluding the initial phases of the femtosecond dynamics from the data interpretation and analysis.On the other hand,they may be explicitly included in the analysis by considering their physical origin.In such a case,assumptions need to be made about the lineshapes and dephasing times of the chromo-phore in question(Novoderezhkin et al.2004).Cross-phase modulation effects are due to a change in the index of refraction of solvent and cuvette induced by the pump beam and give rise to oscillatory patterns around zero delay (Kovalenko et al.1999).These artifacts can in principle be subtracted from the data by recording an experiment in a cuvette with the solvent.Equipment:ampliﬁed Ti:sapphire laser systemsand optical parametric ampliﬁersGenerally speaking,two types of ultrafast transient absorption spectroscopy setups are widely used today for photosynthesis research,distinguished by the repetition rate and pulse energies at which they operate:theﬁrst type involves systems with a repetition rate of1–5kHz with a relatively high pulse energy.The second type involves systems with a repetition rate in the range40–250kHz with a relatively low pulse energy.In addition,the direct or cavity-dumped output from a Ti:sapphire oscillator has frequently been employed for transient absorption spec-troscopy,but will not be discussed here(Arnett et al.1999; Kennis et al.1997b;Nagarajan et al.1996;Streltsov et al. 1998;Vulto et al.1999).Theﬁrst type of spectroscopy typically provides the experimenter with excitation energies of5–100nJ,which when focused on150–200l m diameter(the regular focus-ing conditions in our laboratory)typically results in2–20% of the molecules being promoted to the excited state.This value is only approximate,since the accurate estimate of the excitation density depends on several factors,namely,the exact size of the focus,the concentration of the chromoph-ores,and their extinction coefﬁcient.The relatively high excitation densities achieved with these systems make them suitable to study complexes with a relatively small number of connected pigments such as pigments in solution(Billsten et al.2002;Cong et al.2008;De Weerd et al.2003;Nied-zwiedzki et al.2007;Polivka et al.1999),isolated reaction centers(De Weerd et al.2002;Holzwarth et al.2006a, 2006b;Wang et al.2007),isolated light-harvesting antenna complexes(Croce et al.2001;Gradinaru et al.2000,2001; Ilagan et al.2006;Krueger et al.2001;Papagiannakis et al. 2002,2003;Polı´vka et al.2002;Polivka and Sundstro¨m 2004;Zigmantas et al.2002),artiﬁcial antenna systems (Berera et al.2006,2007;Kodis et al.2004;Pan et al.2002), and photoreceptor proteins that bind only a single chromo-phore(Kennis and Groot2007;Wilson et al.2008).With appropriate detection schemes that involve multichannel detection on a shot-to-shot basis,signal detection sensitiv-ities of*10-5units of absorbance over a broad wavelength range can be achieved,implying that molecular species with a small extinction coefﬁcient or that accumulate in very low (transient)concentrations can be detected(Berera et al. 2006;Wilson et al.2008).A drawback of a1–5-kHz system is that with its relatively high excitation densities,multiple excited states may appear in a single multichromophoric complex,resulting in singlet–singlet annihilation processes among(B)Chls(Van Grondelle1985).With the laser systems that operate at40–250kHz,a lower pulse energy can be used for excitation with respect to the kHz systems owing to their higher repetition rate,which allows more laser shots to be averaged per unit time. Typically,pulse energies of0.5–10nJ are used,roughly corresponding to excited-state populations of\1–10%. Under the right circumstances,detection sensitivities of *10-6units of absorbance can be achieved.Accordingly, this kind of system has been used to study exciton migra-tion in large systems with many connected pigments such as chloroplasts and light-harvesting complex(LHC)II aggregates(Holt et al.2005;Ma et al.2003;Ruban et al. 2007).In addition,it has been used to examine exciton migration in isolated LH complexes under annihilation-free conditions(Monshouwer et al.1998;Novoderezhkin et al. 2004;Palacios et al.2006;Papagiannakis et al.2002). Drawbacks of this type of systems involve the shorter time between pulses(4–20l s),which may lead to the build-up of relatively long-lived species such as triplet or charge-separated states.In addition,multichannel detection on a shot-to-shot basis has been limited to14channels at such high repetition rates(Ruban et al.2007),although signiﬁ-cant strides are currently being made in our laboratory to resolve this limitation.Figure2shows a scheme of an ultrafast transient absorption setup,as it exists today in the Biophysics Lab-oratory of the Laser Center at the Vrije Universiteit(LCVU) in Amsterdam,The Netherlands.A broadband oscillator (Coherent Vitesse)generates pulses of*30fs duration with a wavelength of800nm,a bandwidth of*35nm at a repetition rate of80MHz.The pulses from the oscillator are too weak to perform any meaningful spectroscopy and therefore have to be ampliﬁed.Femtosecond pulse ampli-ﬁcation is not a trivial matter because at high energies,the peak power in a femtosecond pulse becomes so high that ampliﬁcation and pulse-switching media such as crystals and Pockels cells easily get damaged.A Pockels cell is an electro-optical device containing a crystal,such as potas-sium dihydrogenphosphate(KH2PO4),capable of switching the polarization of light when an electrical potential differ-ence is applied to it.In this way,the amount of stimulated emission from the laser cavity can be controlled.For this reason,femtosecond pulse ampliﬁcation is carried out through the chirped-pulse ampliﬁcation principle:the pulse from the oscillator(hereafter,referred to as‘‘seed pulse’’)is ﬁrst stretched to*200ps by a stretcher,which temporally delays the‘‘blue’’wavelengths within the pulse bandwidth of*35nm with respect to the‘‘red’’wavelengths by means of a grating pair.Then,the seed pulse is coupled into a regenerative ampliﬁer(Coherent Legend-UltraShort Pulse (USP)).There,the seed pulse travels through a Pockels cell which sets its polarization in such a way that it becomes trapped within the ampliﬁer’s cavity.On traveling back and forth in the cavity,it passes through a Ti:sapphire crystal that is pumped at1-kHz repetition rate by a diode-pumped Nd:YLF pump laser at527nm(Coherent Evolution,30W).At each passage through the crystal,the trapped seed pulse is ampliﬁed until saturation is reached.Then,the Pockels cell switches the polarization of the ampliﬁed pulse which results in its ejection from the ampliﬁer.The ampliﬁed pulse is compressed to*45fs by temporally synchronizing the ‘‘blue’’and‘‘red’’wavelengths within the pulse bandwidth, essentially the reverse of the‘‘stretching’’procedure.At this point,the output from the laser system is a40-fs pulse at an energy of2.5mJ,a center wavelength of800nm,a band-width of30nm,and a repetition rate of1kHz.In order to perform transient absorption spectroscopy with a Ti:sapphire laser alone,one is restricted to a wavelength region for the excitation pulse around800nm, allowing only the study of some BChl a-containing systems (Arnett et al.1999;Kennis et al.1997b;Nagarajan et al. 1996;Novoderezhkin et al.1999;Streltsov et al.1998; Vulto et al.1999).In order to shift the wavelength to other parts of the visible and near-IR spectra,optical parametric ampliﬁers(OPAs)or optical parametric generators(OPGs) are typically used.In an OPA,non-linear birefringent crystals such as beta barium borate(BBO)are pumped by the direct output of the ampliﬁed laser system at800nm or frequency-doubled pulses at400nm.The pump is tempo-rally and spatially overlapped with a white-light continuum in the crystal,and depending on the angle between the laser beam and the symmetry axis of the crystal,two particular wavelengths of the white-light continuum called‘‘signal’’and‘‘idler’’are ampliﬁed through the second-order non-linear polarizability of the crystal,of which the signal has the shortest wavelength and is routinely selected for further use.Since pump,signal,and idler beams have different polarizations,the group velocity of pump,signal,and idler beams can be made equal by varying the angle between the laser beam and the symmetry axis of the birefringent crystal.This allows energy from the pump beam to be converted to the signal and idler beams over a large propagation length up to millimeters.This is the so-called phase-matching condition.Conservation of energy requires that the sum of the frequencies of signal and idler add up to the frequency of the pump beam.Thus,800-nm-pumped OPAs operate in the near-InfraRed(IR)(1,100–1,600nm for the signal)while400-nm-pumped OPAs operate in the visible(475–750nm for the signal)ing the output of an OPA as a basis,essentially all wavelengths from the UltraViolet(UV)to mid-IR can be generated at relatively high pulse energies by using non-linear mixing processes such as frequency-doubling,sum-frequency generation,and difference-frequency generation in suitable non-linear crystals.Obviously,visible and near-IR light are the most useful wavelengths for the study of photosynthetic systems.In addition,mid-IR wavelengths are very useful for probing molecular vibrations of chlorophylls and carotenoids(Groot et al.2005,2007).The pulse duration out of the OPA roughly corresponds to that of the ampliﬁed Ti:sapphire laser system.The pulse energy from our regenerative laser ampliﬁer of2.5mJ allows simultaneous pumping of several OPAs.The latter option is important for experiments that require multiple pump pulses,such as pump–dump or pump–repump experiments(Kennis et al. 2004;Larsen et al.2003;Papagiannakis et al.2004).The transient absorption setupIn order to vary the time delay between the excitation and probe pulses,the excitation pulse generated by the OPA is sent through an optical delay line,which consists of a retroreﬂector mounted on a high-precision motorized computer-controlled translation stage.The translation stage employed in our experiments has an accuracy and repro-ducibility of0.1l m,which corresponds to a timing accu-racy of0.5fs.The delay line can be moved over80cm, implying that time delays up to5ns can be generated between excitation and probe beams.The excitation beam is focused in the sample to a diameter of130–200l m and blocked after the sample.In most cases,the polarization of the pump beam is set at the magic angle(54.7°)with respect to that of the probe to eliminate polarization and photoselection effects(Lakowicz2006).For the detection of the pump-induced absorbance changes,a part of the ampliﬁed800-nm light is focused on a sapphire or calciumﬂuoride plate(though other materials such as quartz,MgF2,water,and ethylene glycol can also be used)to generate a white-light continuum.In the absence of special precautions,the white-light continuum may range from*400to*1,100nm(depending on the material)and be used as a broadband probe;its intensity is so weak that it does not transfer an appreciable population from the ground to the excited state(or vice versa).It is focused on the sample to a diameter slightly smaller than the pump,spatially overlapped with the pump,collimated, and sent into a spectrograph.There,it is spectrally dis-persed and projected on a silicon diode array that consists of tens to hundreds of elements.The diode array is read out by a computer on a shot-to-shot basis,in effect measuring an absorption spectrum with each shot.Under some experimental conditions,detection with a diode array is not possible or appropriate.For instance,for many experiments in the near-IR and the UV,other detector types need to be employed that,in combination with the white-light continuum intensities at those wave-lengths,lack the sensitivity required for array detection.In these cases,single wavelength detection is often employed. In the mid-IR(*3–10l m),mercury cadmium telluride (MCT)arrays that consist of32or64elements are avail-able(Groot et al.2007).Another detection method in the visible spectrum employs a charge-coupled device(CCD) detector.Frequently,a reference beam is used to account for shot-to-shot intensityﬂuctuations in the white-light continuum.In such a case,the white-light continuum beam is split in two beams,the probe and the reference.The probe is overlapped with the pump beam in the sample, while the reference beam is led past the sample(or through the sample past the excited volume).The probe and ref-erence beams are then projected on separate diode arrays.During data collection,the probe beam is divided by the reference beam,which may lead to improved signal to noise because the intensityﬂuctuations of the white-light continuum are eliminated.By the nature of the white-light generation process,the white light is‘‘chirped’’on generation,i.e.,the‘‘blue’’wavelengths are generated later in time than the‘‘red’’wavelengths.The exact temporal properties depend on the speciﬁc generation conditions.Hence,the white-light continuum has an‘‘intrinsic’’group-velocity dispersion. When traveling through optically dense materials such as lenses and cuvettes,the group velocity dispersion in the white light readily increases to picoseconds.This effect can be minimized by using parabolic mirrors for collimation and focusing of the white-light beam between its point of generation and the sample.The group velocity dispersion may be accounted for in the data analysis and described by a polynomial function.Alternatively,the white-light con-tinuum can be compressed by means of a grating pair or prism pair in such a way that the‘‘red’’and‘‘blue’’wavelengths in the probe beam coincide in time.The instrument response function of this particular tran-sient absorption apparatus,which can be measured by fre-quency mixing in a non-linear crystal placed at the sample spot or by the transient birefringence in CS2or water,can usually be modeled with a Gaussian with a FWHM of 120fs.If required,the white-light continuum can be com-pressed down to*10fs by means of a grating pair or prism pair;in such a case,the instrument response function is generally limited by the duration of the pump pulse.For measurements at room temperature,the sample is placed in a1–2-mm quartz cuvette which is either con-nected to aﬂow system or mounted on a shaker to prevent exposure of the same excited volume to multiple laser shots and to prevent sample degradation.Collection of transient absorption spectraA transient absorption experiment proceeds as follows:the time delay between excitation and probe beams isﬁxed. Before reaching the sample,the excitation beam(that delivers a pulse every1ms)passes through a mechanical chopper that is synchronized to the ampliﬁer in such a way that every other excitation pulse is blocked.Thus,alter-nately the sample is being excited and not excited.Con-sequently,the white-light continuum that is incident on the detector diode array alternately corresponds to a‘‘pumped’’and‘‘unpumped’’sample,and the detector alternately measures the intensity of the probe beam of a‘‘pumped’’and‘‘unpumped’’sample,I(k)pumped and I(k)unpumped. I(k)pumped and I(k)unpumped are stored in separate buffers (while keeping the time delay between pump and probe ﬁxed),and a number of shots that is sufﬁcient for anacceptable signal-to-noise ratio is measured,usually103–104.With the shot-to-shot detection capability of the multichannel detection system,particular spectra that deviate from the average(‘‘outliers’’)can in real time be rejected during data collection,signiﬁcantly improving signal-to-noise ratio.A second white-light beam(the ref-erence beam)not overlapping with the pump pulse can also be used to further increase the signal-to-noise ratio.From the averaged values of I(k)pumped and I(k)unpumped,an absorbance difference spectrum D A(k)is constructed according toD AðkÞ¼ÀlogðIðkÞpumped =IðkÞunpumpedÞ:Then,the delay line is moved to another time delay between pump and probe,and the above procedure is repeated.In total,absorbance difference spectra at approximately100–200time points between0fs and*5ns are collected, along with absorbance difference spectra before time zero to determine the baseline.In addition,many spectra are collected around the time that pump and probe pulse overlap in time(‘‘zero delay’’)to enable accurate recording of the instrument response function.This whole procedure is repeated several times to test reproducibility,sample stability,and long-termﬂuctuations of the laser system.In this way,an entire dataset D A(k,s)is collected. Anisotropy experiments in transient absorption spectroscopyIn photosynthetic antennae and reaction centers,the pig-ments are bound in a well-deﬁned way.Energy and elec-tron transfer processes and pathways can be speciﬁcally assessed through the use of polarized excitation and probe beams.The time-dependent anisotropy is deﬁned asrðtÞ¼ðD A kðtÞÀD A?ðtÞÞ=ðD A kðtÞþ2D A?ðtÞÞ:With D A k(t),the time-dependent absorbance difference signal with pump and probe beams is polarized parallel, and with D A\(t),the time-dependent absorbance difference signal with pump and probe beams is polarized perpen-dicular.In light-harvesting antennae,the decay of r(t) indicates the elementary timescales of exciton migration, be it through incoherent hopping or exciton relaxation (Kennis et al.1997b;Nagarajan et al.1996;Novoderezhkin et al.1998;Savikhin et al.1994,1998,1999;Vulto et al. 1999;Vulto et al.1997).Energy transfer or exciton relaxation processes often occur among(pools of)Chls that have their absorption maxima at similar wavelengths. Consequently,these processes are associated with small spectral shifts of the D A spectra and are there-fore difﬁcult to observe under magic angle detection con-ditions.Through time-resolved anisotropy experiments,the timescales of such fast exciton migration events can accurately be determined.Data analysisIn time-resolved spectroscopic experiments,the very large amounts of data collected can be analyzed by global and target analysis techniques(Van Stokkum et al.2004).A typical time-resolved experiment D A(k,s)in fact consists of a collection of thousands of data points,i.e.,tens to hun-dreds wavelengths times one to two hundred data points.In order to extract valuable information,one could simply take slices of the data;for instance,one could take one wavelength and look at its evolution in time(a so-called kinetic trace),or one could plot the signal at different wavelengths for a given time point(a D A spectrum).This is normally theﬁrst stage of the data analysis where the experimentalist has a glimpse of an expected(or unex-pected)process.The next step in the data analysis is to apply the so-called global analysis techniques,in an attempt to distill the overwhelming amount of data into a relatively small number of components and spectra.In the most basic model,the femtosecond transient absorption data are globally analyzed using a kinetic model consisting of sequentially interconverting evolution-associated dif-ference spectra(EADS),i.e.,1?2?3?ÁÁÁin which the arrows indicate successive monoexponential decays of increasing time constants,which can be regarded as the lifetime of each EADS.Theﬁrst EADS correspond to the time-zero difference spectrum.This procedure enables a clear visualization of the evolution of the(excited)states of the system.Based on the insight obtained from this model and from the raw data,one can then take a further step in the analysis and apply a so-called target kinetic scheme. The EADS that follow from the sequential analysis are generally made up from a mixture of various molecular species.In general,the EADS may well reﬂect mixtures of molecular states.In order to disentangle the contributions from these molecular species and obtain the spectrum signature of the‘‘pure’’excited-and product state inter-mediates(the so-called species-associated difference spectra,SADS),a speciﬁc kinetic model must be applied in a so-called target analysis procedure.In this way,the energy and electron transfer mechanisms can be assessed in terms of a number of discrete reaction intermediates.A comprehensive review of global and target analysis tech-niques has been published(Van Stokkum et al.2004).In the next section,we illustrate a few examples of time-resolved experiments and data analysis.We will start with the description of elementary energy transfer processes in artiﬁcial systems followed by more complex examples in natural light-harvesting compounds.。

AccuStart

AccuStart™ Taq DNA Polymerase HiFiCat No. 95085-250 Size: 250 units Store at -25°C to -15ºC 95085-01K 1000 units95085-05K5000 unitsDescriptionAccuStart Taq DNA Polymerase HiFi is an enzyme mixture of recombinant Taq DNA polymerase preparation, a thermal stable DNA polymerase with 3’ 5’ exonuclease activity, and monoclonal antibodies that bind to the polymerase and keep it inactive before PCR thermal cycling (1). Upon heat activation (1 minute at 94ºC), the antibodies denature irreversibly, releasing fully active DNA polymerase. Non-specific extension of primers at low temperatures is a common cause of artifacts and poor sensitivity in PCR. The AccuStart automatic hot-start enables specific and efficient primer extension in the PCR process with the added convenience of room temperature reaction assembly. This enzyme mixture and optimized HiFi PCR buffer improves the fidelity of DNA synthesis approximately 6-fold higher than Taq DNA polymerase alone and enables amplification of DNA fragments up to 20-kb long (2). AccuStart Taq DNA Polymerase HiFi is a robust alternative PCR enzyme for both routine PCR applications as well as amplification of problematic templates.ComponentsAccuStart Taq DNA polymerase HiFi 5 units/µL in 50% glycerol, 20 mM Tris-HCl, 40 mM NaCl, 0.1 mM EDTA, and stabilizers.HiFi PCR Buffer (10X) 600 mM Tris-SO4 (pH 8.9), 180 mM (NH4)2SO450 mM magnesium sulfate 50 mM MgSO4Storage and StabilityStore components in a constant temperature freezer at -25°C to -15°C upon receipt.For lot specific expiry date, refer to package label, Certificate of Analysis or Product Specification Form.General PCR protocolThe following procedure is presented as general guideline for using AccuStart Taq DNA Polymerase HiFi in any PCR procedure. Cycling conditions, concentration of, primers, MgSO4, and dNTPs, and the amount of AccuStart Taq DNA Polymerase HiFi may need to be optimized. Preparation of a master mix cocktail that contains all components except DNA template when performing multiple PCRs with the same primer set. Reaction volume may be scaled to suit individual needs.Since as little as one molecule of DNA template can initiate the PCR process, it is important to take appropriate precautions to avoid contamination of reagents with DNA template and cross-sample contamination. Assemble reactions (without template) in a DNA-free area using dedicated pipettors and aerosol-resistant barrier tips. Add DNA template to reactions as the final step. Change gloves frequently. Ideally, the PCR workflow should be segregated into separate areas for reaction assembly, processing/addition of DNA template(s), and analysis of PCR products.Reaction AssemblyAdd the following components to a thin-walled PCR tube:Component Volume for 50-µL rxn. Final ConcentrationNuclease-free water variableHiFi PCR Buffer (10X) 5 µL 1x50 mM magnesium sulfate 2 µL 2 mM10 mM dNTP Mix 1 µL 200 µM each dNTPForward primer variable 100 – 500 nMReverse primer variable 100 – 500 nMAccuStart Taq DNA Polymerase HiFi 0.2 µL 1 unitDNA Template 5 – 10 µL variableFinal Volume (µL) 50 µLTemperature Cycling ProtocolIncubate the completed reaction mix in thermal cycler as follows:Initial denaturation: 94ºC, 1 minPCR cycling (20 – 40 cycles:) 94ºC, 15 to 20 s55 – 65ºC, 30s68ºC, 1 min per kb of product lengthHold 4ºC until processed for analysisFull activation of AccuStart Taq DNA Polymerase HiFi occurs within 30 seconds at 94ºC; however, complete denaturation of double-stranded DNA template is required to initiate the PCR process. Consequently, the initial denaturation time may require optimization depending on the nature and properties of a given target sequence. A 1-minute initial denaturation is sufficient for amplification of most templates. Amplification supercoiled DNA templates may require a longer initial denaturation time to fully denature the template prior to PCR cycling. Initial denature times should be kept to a minimum when amplifying long fragments to avoid temperature induced DNA damage (deamination, depurination, and strand cleavage). Quality ControlKit components are free of contaminating DNase and RNase. AccuStart Taq DNA Polymerase HiFi is functionally tested for amplification of a 20-kb fragment from human genomic DNA. Inhibition of polymerase activity by the AccuStart anti-Taq monoclonal antibodies is tested in an activity assay that measures polymerase inhibition relative to an uninhibited control.Unit definitionOne unit is defined as the amount of enzyme that will incorporate 10 nmol of dNTP into acid-insoluble material in 30 minutes at 74°C. References1Sharkey, D.J., Scalice, E.R., Christy, K.G., Atwood, S.M., Daiss, J.L. (1994) BioTechnology, 122 Barnes, W.M. (1994) Proc. Natl. Acad. Sci. USA 91, 2216.Limited Label LicensesUse of this product signifies the agreement of any purchaser or user of the product to the following terms:1.The product may be used solely in accordance with the protocols provided with the product and this manual and for use with components contained in the kitonly. QIAGEN Beverly, Inc. grants no license under any of its intellectual property to use or incorporate the enclosed components of this kit with any components not included within this kit except as described in the protocols provided with the product, this manual, and additional protocols available at . Some of these additional protocols have been provided by Quantabio product users. These protocols have not been thoroughly tested or optimized by QIAGEN Beverly, Inc.. QIAGEN Beverly, Inc. neither guarantees them nor warrants that they do not infringe the rights of third-parties.2.Other than expressly stated licenses, QIAGEN Beverly, Inc. makes no warranty that this kit and/or its use(s) do not infringe the rights of third-parties.3.This kit and its components are licensed for one-time use and may not be reused, refurbished, or resold.4.QIAGEN Beverly, Inc. specifically disclaims any other licenses, expressed or implied other than those expressly stated.5.The purchaser and user of the kit agree not to take or permit anyone else to take any steps that could lead to or facilitate any acts prohibited above. QIAGEN Beverly,Inc. may enforce the prohibitions of this Limited License Agreement in any Court, and shall recover all its investigative and Court costs, including attorney fees, in any action to enforce this Limited License Agreement or any of its intellectual property rights relating to the kit and/or its components.©2018 QIAGEN Beverly Inc. 100 Cummings Center Suite 407J Beverly, MA 01915Quantabio brand products are manufactured by QIAGEN, Beverly Inc.Intended for molecular biology applications. This product is not intended for the diagnosis, prevention or treatment of a disease.AccuStart is a trademark of QIAGEN Beverly Inc..。

智能融合cSoC：多通道FFT共享处理器使用FPGA纤维说明书

Application Note AC381February 20121© 2012 Microsemi Corporation SmartFusion cSoC: Multi-Channel FFT Co-Processor Using FPGA FabricTable of ContentsIntroductionThe SmartFusion ® customizable system-on-chip (cSoC) device integrates FPGA technology with a hardened ARM ® Cortex™-M3 processor based microcontroller subsystem (MSS) and programmable high-performance analog blocks built on a low power flash semiconductor process. The MSS consists of hardened blocks such as a 100 MHz ARM Cortex-M3 processor, peripheral direct memory access (PDMA), embedded nonvolatile memory (eNVM), embedded SRAM (eSRAM), embedded FlashROM (eFROM), external memory controller (EMC), Watchdog Timer, the Philips Inter-Integrated Circuit (I 2C),serial peripheral interface (SPI), 10/100 Ethernet controller, real-time counter (RTC), GPIO block, fabric interface controller (FIC), in-application programming (IAP), and analog compute engine (ACE).The SmartFusion cSoC device is a good fit for applications that require interface with many analog sensors and analog channels. SmartFusion cSoC devices have a versatile analog front-end (AFE) that complements the ARM Cortex-M3 processor based MSS and general-purpose FPGA fabric. The SmartFusion AFE includes three 12-bit successive approximation register (SAR) ADCs, one first order sigma-delta DAC (SDD) per ADC, high performance signal conditioning blocks, and comparators. The SmartFusion cSoCs have a sophisticated controller for the AFE called the ACE. The ACE configures and sequences all the analog functions using the sample sequencing engine (SSE) and post-processes the results using the post processing engine (PPE) and handles without intervention of Cortex-M3 processor.Refer to the SmartFusion Programmable Analog User’s Guide for more details.This application note describes the capability of SmartFusion cSoC devices to compute the Fast Fourier Transform (FFT) in real time. The Multi Channel FFT example design can be used in medical applications, sensor network applications, multi channel audio Spectrum analyzers, Smart Metering, and sensing applications (such as vibration analysis).This example design uses the Cortex-M3 processor in the SmartFusion MSS as a master and the FFT processor in the FPGA fabric as a slave. All three of the SmartFusion cSoC A2F500’s ADCs are used for data acquisition. The example design uses Microsemi’s CoreFFT IP and the advanced peripheral bus interface (CoreAPB3). A custom-made APB3 interface has been developed to connect CoreFFT with the MSS via CoreAPB3. The Cortex-M3 processor uses the PDMA controller in the MSS for the data transfer and thus helps to free up the Cortex-M3 processor instruction bandwidth.A basic understanding of the SmartFusion design flow is assumed. Refer to Using UART with SmartFusion - Microsemi Libero ® SoC and SoftConsole Flow Tutorial to understand the SmartFusion design flow.Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1Design Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2Design Description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2Implementing Multi Channel FFT on EVAL KIT BOARD . . . . . . . . . . . . . . . . . . . . . . . . . 7Running the Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9Appendix A – Design Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10SmartFusion cSoC: Multi-Channel FFT Co-Processor Using FPGA Fabric2Design OverviewThis design example demonstrates the capability of the SmartFusion cSoC device to compute the FFT for multiple data channels. The FFT computation is a complex task that utilizes extensive logic resources and computation time. In general, for N number of channels, N number of FFT IP’s are needed to be instantiated, which in turn utilize more logic resources on the FPGA. A way to avoid this limitation is to use the same FFT logic for multiple input channels.This design illustrates the implementation of a Multichannel FFT to process multiple data channels through a single FFT and store FFT points in a buffer. The FFT computes the input data read from each channel and stores the N-point result in the respective channel’s allocated buffer. The channel multiplexing is done once each channel buffer has been loaded with the FFT length.Computing frequency components for a real time data of six channels is described in this application note. For sampling the input signals the AFE is used and the complex FFT computation is implemented in the fabric of the SmartFusion cSoC device. The Cortex-M3 processor in the MSS of the SmartFusion cSoC handles the buffer management and channel muxing.Figure 1 depicts the block diagram of six channel FFT co-processor in FPGA fabric.Design DescriptionThe design uses CoreFFT for computing the FFT results. You can download the core generator for CoreFFT at /soc/portal/default.aspx?r=4&p=m=624,ev=60.The design example uses a 512-point and 16-bit FFT. A custom-made APB3 interface has been developed to connect CoreFFT IP with the MSS’s FIC. The CoreFFT output data is stored in a 512x32FIFO within the fabric. The FIFO status signals are given in Table 1 on page 3. The status signals indicate that FFT is ready to receive data and data is available in the output of FIFO. These status signals are mapped to the GPIOs in the MSS. The Cortex-M3 processor can read the GPIOs to handle flow control in the data transfer process from the MSS to CoreFFT.Figure 1 • Multi Channel FFT Block DiagramDesign Description3Figure 2 shows the block diagram of logic in the fabric with custom-made APB3 bus.The data valid signal (ifiD_valid) is generated in custom logic whenever the master needs to write data into the input buffer of the FFT to process through the APB3 interface. The FFT_IP_RDY signal indicates the status of the input buffer of the FFT. If the input buffer is full, the FFT_IP_RDY goes low. The master can read the FFT_IP_RDY signal to get the FFT input buffer status. The FFT generates the processed data with a data valid signal (ifoY_valid). The processed data is stored in the FIFO. When FIFO is not ready to receive output data, it can stop the data fetching from the FFT by pulling down the ifiRead_y signal. The status signal FFT_OP_RDY is used to indicate to the master that processed data is available in the FIFO. FFT_OP_RDY goes High whenever processed data is available in the FFT output buffer.The master can use AEMPTY_OUT or EMPTY_OUT to determine whether the FIFO is empty and all the processed data has been read. Refer to the CoreFFT Handbook for more details on architecture and interface signal descriptions.Three ADCs are configured to have two channels, each channel with 100 ksps sampling rate. The external memory is used for input and output buffers. For each channel, one input buffer having length double to the length of FFT i.e. 1024 words and one output buffer having length equal to the length of FFT i.e. 512 words are used. After each channel's input buffer has 512 points required for the full length of the FFT, each channel, one after the other, streams its points from the FIFO through the FFT. During the FFT computational period, the sampled data values of each channel are stored in the second half of the input buffer. Once the FFT computations for the First half of input buffer completes then the points in the second half of the input buffer will be streamed to FFT. This operation utilizes a ping-pong method. The Cortex-M3 processor is used for data management, that is, buffering the sampled points and data routing or muxing of these values to the FFT computation block. Sampling of the real time data is done by the ACE. The PDMA handles the data transfer between the external SRAM (eSRAM) buffers and CoreFFT logic in FPGA fabric.Figure 2 • CoreFFT with APB Slave InterfaceTable 1 • FIFO Status Signals with DescriptionsSignalDescription FFT_IP_RDYFFT is ready to receive the Input from the master processor FFT_OP_RDYProcessed data is ready in output buffer of FFT AEMPTY_OUTOutput FIFO is almost empty EMPTY_OUT Output FIFO is emptySmartFusion cSoC: Multi-Channel FFT Co-Processor Using FPGA Fabric4Figure 3 shows the implementation of multi channel FFT on the SmartFusion cSoC device.Hardware ImplementationThe MSS is configured with an FIC, clock conditioning circuit (CCC), GPIOs, EMC and a UART. The CCC generates 80 MHz clock, which acts as the clock source. The FIC is configured to use a master interface with an AMBA APB3 interface. Four GPIOs in the MSS are configured as inputs that are used to handle flow control in data transfer from MSS to FFT coprocessor. The EMC is configured for Region 0as Asynchronous RAM and port size as half word. The UART_0 is configured for printing the FFT values to the PC though a serial terminal emulation program.ADC0, ADC1, and ADC2 are configured with 12-bit resolution, two channels and the sampling rate is set to approximately 100 KHz. Figure 4 on page 5 shows the ACE configuration window.Figure 3 • Implementation of Multi Channel FFT on the SmartFusion cSoCDesign Description5The APB wrapper logic is implemented on the top of CoreFFT and connected to CoreAPB3. A FIFO of size 512*32 is used to connect to CoreFFT output.CoreAPB3 acts as a bridge between the MSS and the FFT coprocessor block. It provides an advanced microcontroller bus architecture (AMBA3) advanced peripheral bus (APB3) fabric supporting up to 16APB slaves. This design example uses one slave slot (Slot 0) to interface with the FFT coprocessor block and is configured with direct addressing mode. Refer to the CoreAPB3 Handbook for more details on CoreAPB3 IP .For more details on how to connect FPGA logic MSS, refer to the Connecting User Logic to the SmartFusion Microcontroller Subsystem application note.The logic in the FPGA fabric consumes 18 RAM blocks out of 24. We cannot use eSRAM blocks for implementing CoreFFT as the transactions between these SRAM blocks and FFT logic are very high and are time critical.Figure 5 on page 6 illustrates the multi channel FFT example design in the SmartDesign.Figure 4 • Configure ACESmartFusion cSoC: Multi-Channel FFT Co-Processor Using FPGA Fabric6Table 2 summarizes the logic resource utilization of the design on the A2F500M3F device.Software ImplementationThe Cortex-M3 processor continuously reads the values from ACE and stores the values into the input buffers. If the first 512 points are filled then the processor initiates the FFT process. In the FFT process,the input buffers are streamed one after other to the CoreFFT with the help of PDMA. Using another channel of PDMA the output of FFT is moved to the corresponding channel output buffers.During the FFT process the Cortex-M3 processor stores the sampled values into the second half of the input buffers. Once the FFT process completes the first half of input buffer, then the second half of the input buffer are streamed to CoreFFT.Figure 5 • SmartDesign Implementation of Multi Channel FFTTable 2 • Logic Utilization of the Design on A2F500M3FCoreFFTOther Logic in Fabric Total Ram Blocks14418 (75%)Tiles 78424718313 (72.1%)Implementing Multi Channel FFT on EVAL KIT BOARD7The CALL_FFT(int *) application programmable interface (API) initiates the PDMA to transfer input buffer data to the FFT in the fabric. Before initiating PDMA it checks for FFT whether or not it is ready to read the data. The CALL_FFT(int *) API also checks if the output FIFO is empty so that all the FFT out values have been already read. When the input buffer has points equal to the full length of FFT, then it will be called.The Read_FFT() API initiates the PDMA for reading the FFT output values from FIFO in fabric to the corresponding output buffer. After reading all the values it calls the CALL_FFT() API with the next channel buffer to compute the FFT for next channel. This is done for all channels. After completion of FFT computation for all channels, if the continuous variable is not defined, it will print the FFT output values on the serial terminal. When FFT_OP_READY interrupt occurs then this API will be called.The GPIO1_IRQHandler() interrupt service routine occurs on the positive edge of FFT_OP_READY signal. It calls Read_FFT() API. This interrupt mechanism is used to read the sample values continuously while computing the FFT.If continuous variable is defined, then the FFT is computed without any loss of data samples. If #define continuous line is commented then after every completion of FFT computation of all channels the FFT output is printed on serial terminal. The printed values are in the form of complex numbers.The ping-pong mechanism is used for input data buffer to store the samples continuously. For each channel the input buffer length is double of the full FFT length. While computing the FFT for the first half of the buffer, the new sample values are stored in the second half of the input buffer and while computing the FFT for second half of buffer, the new sample values are stored in first half of the input buffer.Customizing the Number of ChannelsYou can change the design depending on your requirement. Configure the ADC (Figure 4 on page 5)with the required number of channels and required sampling rate. In SoftConsole project change the parameter value NUM_CHANNELS according to the ADC configuration. Edit the main code for reading ADCs data into buffers according to ACE configuration.Throughput CalculationsThe actual time to get 512 samples with 100 ksps is 5.12 ms. Each channel is configured to 100 ksps, so for every 5.12 ms we will have 512 samples in the input buffers.The actual time taken to compute the FFT for each channel is the sum of time taken to transfer 512points to CoreFFT, FFT computation time, and time to read FFT output to the output buffer.•Total time for computing FFT = (time taken to receive 512 data + computational latency for 512points + time taken to store 512 data) = 512*5 + 23292 + 512*5 =28412 clks •Time to compute FFT for 6 channels = 28412*6 = 170472 clksTime to compute FFT for six channels is 2.1309 ms (If CLK is 80 MHz). It is less than half the sample rate of 5.12 ms.If only one channel is configured with maximum sampling rate (600 ksps) then time to get 512 samples with 600 ksps is 0.853 ms. Time to compute FFT for these 512 samples is 0.355 ms. If you configure three ADCs with maximum sampling rate (1800 ksps) then time to compute the FFT for these three channels will be 1.065 ms which is higher than the sampling time. In this there is a loss of some samples.The design works fine up to 1440 ksps.Implementing Multi Channel FFT on EVAL KIT BOARDTo implement the design on the SmartFusion Evaluation Kit Board the FFT must be 256 point and 8 bit because the A2F200 device has less RAM blocks and logic cells. The ADC channels must be selected for only ADC0 and ADC1. Figure 6 on page 8 shows the implementation of multi channel FFT on the SmartFusion cSoC (A2F200M3F) device.SmartFusion cSoC: Multi-Channel FFT Co-Processor Using FPGA Fabric8Table 3 summarizes the logic resource utilization of the design with 256 points 8-bit FFT on A2F200M3F device.Running the DesignProgram the SmartFusion Evaluation Kit Board or the SmartFusion Development Kit Board with the generated or provided *.stp file (refer to "Appendix A – Design Files" on page 10) using FlashPro and then power cycle the board.For computing continuous FFT values for the all six signals sampled through the ADCs, uncomment the line #define continuous in the main program. The FFT output values are stored in the rdata buffer. This buffer is updated for every computation of FFT.For printing the FFT values on serial terminal (HyperTerminal or PuTTy), comment the line #define continuous in the main program.Figure 6 • Implementation of Multi Channel FFT on the SmartFusion Evaluation Kit BoardTable 3 • Logic Utilization of the Design on A2F200M3F DeviceCoreFFTOther Logic in Fabric Total Ram Blocks718 (100%)Tiles 3201853286 (66%)Conclusion9Connect the analog inputs to the SmartFusion Kit Board with the information provided in Table 4.Invoke the SoftConsole IDE, by clicking on Write Application code under Develop Firmware in Libero ®System-on-Chip (SoC) project (refer to "Appendix A – Design Files") and launch the debugger. Start HyperTerminal or PuTTY with a baud rate of 57600, 8 data bits, 1 stop bit, no parity, and no flow control.If your PC does not have the HyperTerminal program, use any free serial terminal emulation program such as PuTTY or Tera Term. Refer to the Configuring Serial Terminal Emulation Programs Tutorial for configuring the HyperTerminal, Tera Term, or PuTTY .ConclusionThis application note describes the capability of the SmartFusion cSoC devices to compute the multi channel FFT. The Cortex-M3 processor, AFE, and FPGA fabric together gives a single chip solution for real time multi channel FFT system. This design example also shows the 6-channel data acquisition system.Table 4 • SettingsChannelEvaluation Kit Development Kit Channel 173 of J21 (signal header)ADC0 of JP4Channel 274 of J21 (signal header)ADC1 of JP4Channel 377 of J21 (signal header)77 of J21 (signal header)Channel 478 of J21 (signal header)78 of J21 (signal header)Channel 585 of J21 (signal header)Channel 686 of J21 (signal header)Figure 7 • FFT Output Data for 1 kHz Sinusoidal Signal on PUTTYSmartFusion cSoC: Multi-Channel FFT Co-Processor Using FPGA Fabric10Appendix A – Design FilesThe Design files are available for download on the Microsemi SoC Product Groups website:/soc/download/rsc/?f=A2F_AC381_DF.The design zip file consists of Libero SoC projects and programming file (*.stp) for A2F200 and A2F500.Refer to the Readme.txt file included in the design file for directory structure and description.51900249-0/02.12© 2012 Microsemi Corporation. All rights reserved. Microsemi and the Microsemi logo are trademarks of Microsemi Corporation. All other trademarks and service marks are the property of their respective owners.Microsemi Corporation (NASDAQ: MSCC) offers a comprehensive portfolio of semiconductor solutions for: aerospace, defense and security; enterprise and communications; and industrial and alternative energy markets. Products include high-performance, high-reliability analog and RF devices, mixed signal and RF integrated circuits, customizable SoCs, FPGAs, and complete subsystems. Microsemi is headquartered in Aliso Viejo, Calif. Learn more at .Microsemi Corporate HeadquartersOne Enterprise, Aliso Viejo CA 92656 USAWithin the USA: +1 (949) 380-6100Sales: +1 (949) 380-6136Fax: +1 (949) 215-4996。

3GPP TS 36.331 V13.2.0 (2016-06)

3GPP TS 36.331 V13.2.0 (2016-06)Technical Specification3rd Generation Partnership Project;Technical Specification Group Radio Access Network;Evolved Universal Terrestrial Radio Access (E-UTRA);Radio Resource Control (RRC);Protocol specification(Release 13)The present document has been developed within the 3rd Generation Partnership Project (3GPP TM) and may be further elaborated for the purposes of 3GPP. The present document has not been subject to any approval process by the 3GPP Organizational Partners and shall not be implemented.This Specification is provided for future development work within 3GPP only. The Organizational Partners accept no liability for any use of this Specification. Specifications and reports for implementation of the 3GPP TM system should be obtained via the 3GPP Organizational Partners' Publications Offices.KeywordsUMTS, radio3GPPPostal address3GPP support office address650 Route des Lucioles - Sophia AntipolisValbonne - FRANCETel.: +33 4 92 94 42 00 Fax: +33 4 93 65 47 16InternetCopyright NotificationNo part may be reproduced except as authorized by written permission.The copyright and the foregoing restriction extend to reproduction in all media.© 2016, 3GPP Organizational Partners (ARIB, ATIS, CCSA, ETSI, TSDSI, TTA, TTC).All rights reserved.UMTS™ is a Trade Mark of ETSI registered for the benefit of its members3GPP™ is a Trade Mark of ETSI registered for the benefit of its Members and of the 3GPP Organizational PartnersLTE™ is a Trade Mark of ETSI currently being registered for the benefit of its Members and of the 3GPP Organizational Partners GSM® and the GSM logo are registered and owned by the GSM AssociationBluetooth® is a Trade Mark of the Bluetooth SIG registered for the benefit of its membersContentsForeword (18)1Scope (19)2References (19)3Definitions, symbols and abbreviations (22)3.1Definitions (22)3.2Abbreviations (24)4General (27)4.1Introduction (27)4.2Architecture (28)4.2.1UE states and state transitions including inter RAT (28)4.2.2Signalling radio bearers (29)4.3Services (30)4.3.1Services provided to upper layers (30)4.3.2Services expected from lower layers (30)4.4Functions (30)5Procedures (32)5.1General (32)5.1.1Introduction (32)5.1.2General requirements (32)5.2System information (33)5.2.1Introduction (33)5.2.1.1General (33)5.2.1.2Scheduling (34)5.2.1.2a Scheduling for NB-IoT (34)5.2.1.3System information validity and notification of changes (35)5.2.1.4Indication of ETWS notification (36)5.2.1.5Indication of CMAS notification (37)5.2.1.6Notification of EAB parameters change (37)5.2.1.7Access Barring parameters change in NB-IoT (37)5.2.2System information acquisition (38)5.2.2.1General (38)5.2.2.2Initiation (38)5.2.2.3System information required by the UE (38)5.2.2.4System information acquisition by the UE (39)5.2.2.5Essential system information missing (42)5.2.2.6Actions upon reception of the MasterInformationBlock message (42)5.2.2.7Actions upon reception of the SystemInformationBlockType1 message (42)5.2.2.8Actions upon reception of SystemInformation messages (44)5.2.2.9Actions upon reception of SystemInformationBlockType2 (44)5.2.2.10Actions upon reception of SystemInformationBlockType3 (45)5.2.2.11Actions upon reception of SystemInformationBlockType4 (45)5.2.2.12Actions upon reception of SystemInformationBlockType5 (45)5.2.2.13Actions upon reception of SystemInformationBlockType6 (45)5.2.2.14Actions upon reception of SystemInformationBlockType7 (45)5.2.2.15Actions upon reception of SystemInformationBlockType8 (45)5.2.2.16Actions upon reception of SystemInformationBlockType9 (46)5.2.2.17Actions upon reception of SystemInformationBlockType10 (46)5.2.2.18Actions upon reception of SystemInformationBlockType11 (46)5.2.2.19Actions upon reception of SystemInformationBlockType12 (47)5.2.2.20Actions upon reception of SystemInformationBlockType13 (48)5.2.2.21Actions upon reception of SystemInformationBlockType14 (48)5.2.2.22Actions upon reception of SystemInformationBlockType15 (48)5.2.2.23Actions upon reception of SystemInformationBlockType16 (48)5.2.2.24Actions upon reception of SystemInformationBlockType17 (48)5.2.2.25Actions upon reception of SystemInformationBlockType18 (48)5.2.2.26Actions upon reception of SystemInformationBlockType19 (49)5.2.3Acquisition of an SI message (49)5.2.3a Acquisition of an SI message by BL UE or UE in CE or a NB-IoT UE (50)5.3Connection control (50)5.3.1Introduction (50)5.3.1.1RRC connection control (50)5.3.1.2Security (52)5.3.1.2a RN security (53)5.3.1.3Connected mode mobility (53)5.3.1.4Connection control in NB-IoT (54)5.3.2Paging (55)5.3.2.1General (55)5.3.2.2Initiation (55)5.3.2.3Reception of the Paging message by the UE (55)5.3.3RRC connection establishment (56)5.3.3.1General (56)5.3.3.1a Conditions for establishing RRC Connection for sidelink communication/ discovery (58)5.3.3.2Initiation (59)5.3.3.3Actions related to transmission of RRCConnectionRequest message (63)5.3.3.3a Actions related to transmission of RRCConnectionResumeRequest message (64)5.3.3.4Reception of the RRCConnectionSetup by the UE (64)5.3.3.4a Reception of the RRCConnectionResume by the UE (66)5.3.3.5Cell re-selection while T300, T302, T303, T305, T306, or T308 is running (68)5.3.3.6T300 expiry (68)5.3.3.7T302, T303, T305, T306, or T308 expiry or stop (69)5.3.3.8Reception of the RRCConnectionReject by the UE (70)5.3.3.9Abortion of RRC connection establishment (71)5.3.3.10Handling of SSAC related parameters (71)5.3.3.11Access barring check (72)5.3.3.12EAB check (73)5.3.3.13Access barring check for ACDC (73)5.3.3.14Access Barring check for NB-IoT (74)5.3.4Initial security activation (75)5.3.4.1General (75)5.3.4.2Initiation (76)5.3.4.3Reception of the SecurityModeCommand by the UE (76)5.3.5RRC connection reconfiguration (77)5.3.5.1General (77)5.3.5.2Initiation (77)5.3.5.3Reception of an RRCConnectionReconfiguration not including the mobilityControlInfo by theUE (77)5.3.5.4Reception of an RRCConnectionReconfiguration including the mobilityControlInfo by the UE(handover) (79)5.3.5.5Reconfiguration failure (83)5.3.5.6T304 expiry (handover failure) (83)5.3.5.7Void (84)5.3.5.7a T307 expiry (SCG change failure) (84)5.3.5.8Radio Configuration involving full configuration option (84)5.3.6Counter check (86)5.3.6.1General (86)5.3.6.2Initiation (86)5.3.6.3Reception of the CounterCheck message by the UE (86)5.3.7RRC connection re-establishment (87)5.3.7.1General (87)5.3.7.2Initiation (87)5.3.7.3Actions following cell selection while T311 is running (88)5.3.7.4Actions related to transmission of RRCConnectionReestablishmentRequest message (89)5.3.7.5Reception of the RRCConnectionReestablishment by the UE (89)5.3.7.6T311 expiry (91)5.3.7.7T301 expiry or selected cell no longer suitable (91)5.3.7.8Reception of RRCConnectionReestablishmentReject by the UE (91)5.3.8RRC connection release (92)5.3.8.1General (92)5.3.8.2Initiation (92)5.3.8.3Reception of the RRCConnectionRelease by the UE (92)5.3.8.4T320 expiry (93)5.3.9RRC connection release requested by upper layers (93)5.3.9.1General (93)5.3.9.2Initiation (93)5.3.10Radio resource configuration (93)5.3.10.0General (93)5.3.10.1SRB addition/ modification (94)5.3.10.2DRB release (95)5.3.10.3DRB addition/ modification (95)5.3.10.3a1DC specific DRB addition or reconfiguration (96)5.3.10.3a2LWA specific DRB addition or reconfiguration (98)5.3.10.3a3LWIP specific DRB addition or reconfiguration (98)5.3.10.3a SCell release (99)5.3.10.3b SCell addition/ modification (99)5.3.10.3c PSCell addition or modification (99)5.3.10.4MAC main reconfiguration (99)5.3.10.5Semi-persistent scheduling reconfiguration (100)5.3.10.6Physical channel reconfiguration (100)5.3.10.7Radio Link Failure Timers and Constants reconfiguration (101)5.3.10.8Time domain measurement resource restriction for serving cell (101)5.3.10.9Other configuration (102)5.3.10.10SCG reconfiguration (103)5.3.10.11SCG dedicated resource configuration (104)5.3.10.12Reconfiguration SCG or split DRB by drb-ToAddModList (105)5.3.10.13Neighbour cell information reconfiguration (105)5.3.10.14Void (105)5.3.10.15Sidelink dedicated configuration (105)5.3.10.16T370 expiry (106)5.3.11Radio link failure related actions (107)5.3.11.1Detection of physical layer problems in RRC_CONNECTED (107)5.3.11.2Recovery of physical layer problems (107)5.3.11.3Detection of radio link failure (107)5.3.12UE actions upon leaving RRC_CONNECTED (109)5.3.13UE actions upon PUCCH/ SRS release request (110)5.3.14Proximity indication (110)5.3.14.1General (110)5.3.14.2Initiation (111)5.3.14.3Actions related to transmission of ProximityIndication message (111)5.3.15Void (111)5.4Inter-RAT mobility (111)5.4.1Introduction (111)5.4.2Handover to E-UTRA (112)5.4.2.1General (112)5.4.2.2Initiation (112)5.4.2.3Reception of the RRCConnectionReconfiguration by the UE (112)5.4.2.4Reconfiguration failure (114)5.4.2.5T304 expiry (handover to E-UTRA failure) (114)5.4.3Mobility from E-UTRA (114)5.4.3.1General (114)5.4.3.2Initiation (115)5.4.3.3Reception of the MobilityFromEUTRACommand by the UE (115)5.4.3.4Successful completion of the mobility from E-UTRA (116)5.4.3.5Mobility from E-UTRA failure (117)5.4.4Handover from E-UTRA preparation request (CDMA2000) (117)5.4.4.1General (117)5.4.4.2Initiation (118)5.4.4.3Reception of the HandoverFromEUTRAPreparationRequest by the UE (118)5.4.5UL handover preparation transfer (CDMA2000) (118)5.4.5.1General (118)5.4.5.2Initiation (118)5.4.5.3Actions related to transmission of the ULHandoverPreparationTransfer message (119)5.4.5.4Failure to deliver the ULHandoverPreparationTransfer message (119)5.4.6Inter-RAT cell change order to E-UTRAN (119)5.4.6.1General (119)5.4.6.2Initiation (119)5.4.6.3UE fails to complete an inter-RAT cell change order (119)5.5Measurements (120)5.5.1Introduction (120)5.5.2Measurement configuration (121)5.5.2.1General (121)5.5.2.2Measurement identity removal (122)5.5.2.2a Measurement identity autonomous removal (122)5.5.2.3Measurement identity addition/ modification (123)5.5.2.4Measurement object removal (124)5.5.2.5Measurement object addition/ modification (124)5.5.2.6Reporting configuration removal (126)5.5.2.7Reporting configuration addition/ modification (127)5.5.2.8Quantity configuration (127)5.5.2.9Measurement gap configuration (127)5.5.2.10Discovery signals measurement timing configuration (128)5.5.2.11RSSI measurement timing configuration (128)5.5.3Performing measurements (128)5.5.3.1General (128)5.5.3.2Layer 3 filtering (131)5.5.4Measurement report triggering (131)5.5.4.1General (131)5.5.4.2Event A1 (Serving becomes better than threshold) (135)5.5.4.3Event A2 (Serving becomes worse than threshold) (136)5.5.4.4Event A3 (Neighbour becomes offset better than PCell/ PSCell) (136)5.5.4.5Event A4 (Neighbour becomes better than threshold) (137)5.5.4.6Event A5 (PCell/ PSCell becomes worse than threshold1 and neighbour becomes better thanthreshold2) (138)5.5.4.6a Event A6 (Neighbour becomes offset better than SCell) (139)5.5.4.7Event B1 (Inter RAT neighbour becomes better than threshold) (139)5.5.4.8Event B2 (PCell becomes worse than threshold1 and inter RAT neighbour becomes better thanthreshold2) (140)5.5.4.9Event C1 (CSI-RS resource becomes better than threshold) (141)5.5.4.10Event C2 (CSI-RS resource becomes offset better than reference CSI-RS resource) (141)5.5.4.11Event W1 (WLAN becomes better than a threshold) (142)5.5.4.12Event W2 (All WLAN inside WLAN mobility set becomes worse than threshold1 and a WLANoutside WLAN mobility set becomes better than threshold2) (142)5.5.4.13Event W3 (All WLAN inside WLAN mobility set becomes worse than a threshold) (143)5.5.5Measurement reporting (144)5.5.6Measurement related actions (148)5.5.6.1Actions upon handover and re-establishment (148)5.5.6.2Speed dependant scaling of measurement related parameters (149)5.5.7Inter-frequency RSTD measurement indication (149)5.5.7.1General (149)5.5.7.2Initiation (150)5.5.7.3Actions related to transmission of InterFreqRSTDMeasurementIndication message (150)5.6Other (150)5.6.0General (150)5.6.1DL information transfer (151)5.6.1.1General (151)5.6.1.2Initiation (151)5.6.1.3Reception of the DLInformationTransfer by the UE (151)5.6.2UL information transfer (151)5.6.2.1General (151)5.6.2.2Initiation (151)5.6.2.3Actions related to transmission of ULInformationTransfer message (152)5.6.2.4Failure to deliver ULInformationTransfer message (152)5.6.3UE capability transfer (152)5.6.3.1General (152)5.6.3.2Initiation (153)5.6.3.3Reception of the UECapabilityEnquiry by the UE (153)5.6.4CSFB to 1x Parameter transfer (157)5.6.4.1General (157)5.6.4.2Initiation (157)5.6.4.3Actions related to transmission of CSFBParametersRequestCDMA2000 message (157)5.6.4.4Reception of the CSFBParametersResponseCDMA2000 message (157)5.6.5UE Information (158)5.6.5.1General (158)5.6.5.2Initiation (158)5.6.5.3Reception of the UEInformationRequest message (158)5.6.6 Logged Measurement Configuration (159)5.6.6.1General (159)5.6.6.2Initiation (160)5.6.6.3Reception of the LoggedMeasurementConfiguration by the UE (160)5.6.6.4T330 expiry (160)5.6.7 Release of Logged Measurement Configuration (160)5.6.7.1General (160)5.6.7.2Initiation (160)5.6.8 Measurements logging (161)5.6.8.1General (161)5.6.8.2Initiation (161)5.6.9In-device coexistence indication (163)5.6.9.1General (163)5.6.9.2Initiation (164)5.6.9.3Actions related to transmission of InDeviceCoexIndication message (164)5.6.10UE Assistance Information (165)5.6.10.1General (165)5.6.10.2Initiation (166)5.6.10.3Actions related to transmission of UEAssistanceInformation message (166)5.6.11 Mobility history information (166)5.6.11.1General (166)5.6.11.2Initiation (166)5.6.12RAN-assisted WLAN interworking (167)5.6.12.1General (167)5.6.12.2Dedicated WLAN offload configuration (167)5.6.12.3WLAN offload RAN evaluation (167)5.6.12.4T350 expiry or stop (167)5.6.12.5Cell selection/ re-selection while T350 is running (168)5.6.13SCG failure information (168)5.6.13.1General (168)5.6.13.2Initiation (168)5.6.13.3Actions related to transmission of SCGFailureInformation message (168)5.6.14LTE-WLAN Aggregation (169)5.6.14.1Introduction (169)5.6.14.2Reception of LWA configuration (169)5.6.14.3Release of LWA configuration (170)5.6.15WLAN connection management (170)5.6.15.1Introduction (170)5.6.15.2WLAN connection status reporting (170)5.6.15.2.1General (170)5.6.15.2.2Initiation (171)5.6.15.2.3Actions related to transmission of WLANConnectionStatusReport message (171)5.6.15.3T351 Expiry (WLAN connection attempt timeout) (171)5.6.15.4WLAN status monitoring (171)5.6.16RAN controlled LTE-WLAN interworking (172)5.6.16.1General (172)5.6.16.2WLAN traffic steering command (172)5.6.17LTE-WLAN aggregation with IPsec tunnel (173)5.6.17.1General (173)5.7Generic error handling (174)5.7.1General (174)5.7.2ASN.1 violation or encoding error (174)5.7.3Field set to a not comprehended value (174)5.7.4Mandatory field missing (174)5.7.5Not comprehended field (176)5.8MBMS (176)5.8.1Introduction (176)5.8.1.1General (176)5.8.1.2Scheduling (176)5.8.1.3MCCH information validity and notification of changes (176)5.8.2MCCH information acquisition (178)5.8.2.1General (178)5.8.2.2Initiation (178)5.8.2.3MCCH information acquisition by the UE (178)5.8.2.4Actions upon reception of the MBSFNAreaConfiguration message (178)5.8.2.5Actions upon reception of the MBMSCountingRequest message (179)5.8.3MBMS PTM radio bearer configuration (179)5.8.3.1General (179)5.8.3.2Initiation (179)5.8.3.3MRB establishment (179)5.8.3.4MRB release (179)5.8.4MBMS Counting Procedure (179)5.8.4.1General (179)5.8.4.2Initiation (180)5.8.4.3Reception of the MBMSCountingRequest message by the UE (180)5.8.5MBMS interest indication (181)5.8.5.1General (181)5.8.5.2Initiation (181)5.8.5.3Determine MBMS frequencies of interest (182)5.8.5.4Actions related to transmission of MBMSInterestIndication message (183)5.8a SC-PTM (183)5.8a.1Introduction (183)5.8a.1.1General (183)5.8a.1.2SC-MCCH scheduling (183)5.8a.1.3SC-MCCH information validity and notification of changes (183)5.8a.1.4Procedures (184)5.8a.2SC-MCCH information acquisition (184)5.8a.2.1General (184)5.8a.2.2Initiation (184)5.8a.2.3SC-MCCH information acquisition by the UE (184)5.8a.2.4Actions upon reception of the SCPTMConfiguration message (185)5.8a.3SC-PTM radio bearer configuration (185)5.8a.3.1General (185)5.8a.3.2Initiation (185)5.8a.3.3SC-MRB establishment (185)5.8a.3.4SC-MRB release (185)5.9RN procedures (186)5.9.1RN reconfiguration (186)5.9.1.1General (186)5.9.1.2Initiation (186)5.9.1.3Reception of the RNReconfiguration by the RN (186)5.10Sidelink (186)5.10.1Introduction (186)5.10.1a Conditions for sidelink communication operation (187)5.10.2Sidelink UE information (188)5.10.2.1General (188)5.10.2.2Initiation (189)5.10.2.3Actions related to transmission of SidelinkUEInformation message (193)5.10.3Sidelink communication monitoring (195)5.10.6Sidelink discovery announcement (198)5.10.6a Sidelink discovery announcement pool selection (201)5.10.6b Sidelink discovery announcement reference carrier selection (201)5.10.7Sidelink synchronisation information transmission (202)5.10.7.1General (202)5.10.7.2Initiation (203)5.10.7.3Transmission of SLSS (204)5.10.7.4Transmission of MasterInformationBlock-SL message (205)5.10.7.5Void (206)5.10.8Sidelink synchronisation reference (206)5.10.8.1General (206)5.10.8.2Selection and reselection of synchronisation reference UE (SyncRef UE) (206)5.10.9Sidelink common control information (207)5.10.9.1General (207)5.10.9.2Actions related to reception of MasterInformationBlock-SL message (207)5.10.10Sidelink relay UE operation (207)5.10.10.1General (207)5.10.10.2AS-conditions for relay related sidelink communication transmission by sidelink relay UE (207)5.10.10.3AS-conditions for relay PS related sidelink discovery transmission by sidelink relay UE (208)5.10.10.4Sidelink relay UE threshold conditions (208)5.10.11Sidelink remote UE operation (208)5.10.11.1General (208)5.10.11.2AS-conditions for relay related sidelink communication transmission by sidelink remote UE (208)5.10.11.3AS-conditions for relay PS related sidelink discovery transmission by sidelink remote UE (209)5.10.11.4Selection and reselection of sidelink relay UE (209)5.10.11.5Sidelink remote UE threshold conditions (210)6Protocol data units, formats and parameters (tabular & ASN.1) (210)6.1General (210)6.2RRC messages (212)6.2.1General message structure (212)–EUTRA-RRC-Definitions (212)–BCCH-BCH-Message (212)–BCCH-DL-SCH-Message (212)–BCCH-DL-SCH-Message-BR (213)–MCCH-Message (213)–PCCH-Message (213)–DL-CCCH-Message (214)–DL-DCCH-Message (214)–UL-CCCH-Message (214)–UL-DCCH-Message (215)–SC-MCCH-Message (215)6.2.2Message definitions (216)–CounterCheck (216)–CounterCheckResponse (217)–CSFBParametersRequestCDMA2000 (217)–CSFBParametersResponseCDMA2000 (218)–DLInformationTransfer (218)–HandoverFromEUTRAPreparationRequest (CDMA2000) (219)–InDeviceCoexIndication (220)–InterFreqRSTDMeasurementIndication (222)–LoggedMeasurementConfiguration (223)–MasterInformationBlock (225)–MBMSCountingRequest (226)–MBMSCountingResponse (226)–MBMSInterestIndication (227)–MBSFNAreaConfiguration (228)–MeasurementReport (228)–MobilityFromEUTRACommand (229)–Paging (232)–ProximityIndication (233)–RNReconfiguration (234)–RNReconfigurationComplete (234)–RRCConnectionReconfiguration (235)–RRCConnectionReconfigurationComplete (240)–RRCConnectionReestablishment (241)–RRCConnectionReestablishmentComplete (241)–RRCConnectionReestablishmentReject (242)–RRCConnectionReestablishmentRequest (243)–RRCConnectionReject (243)–RRCConnectionRelease (244)–RRCConnectionResume (248)–RRCConnectionResumeComplete (249)–RRCConnectionResumeRequest (250)–RRCConnectionRequest (250)–RRCConnectionSetup (251)–RRCConnectionSetupComplete (252)–SCGFailureInformation (253)–SCPTMConfiguration (254)–SecurityModeCommand (255)–SecurityModeComplete (255)–SecurityModeFailure (256)–SidelinkUEInformation (256)–SystemInformation (258)–SystemInformationBlockType1 (259)–UEAssistanceInformation (264)–UECapabilityEnquiry (265)–UECapabilityInformation (266)–UEInformationRequest (267)–UEInformationResponse (267)–ULHandoverPreparationTransfer (CDMA2000) (273)–ULInformationTransfer (274)–WLANConnectionStatusReport (274)6.3RRC information elements (275)6.3.1System information blocks (275)–SystemInformationBlockType2 (275)–SystemInformationBlockType3 (279)–SystemInformationBlockType4 (282)–SystemInformationBlockType5 (283)–SystemInformationBlockType6 (287)–SystemInformationBlockType7 (289)–SystemInformationBlockType8 (290)–SystemInformationBlockType9 (295)–SystemInformationBlockType10 (295)–SystemInformationBlockType11 (296)–SystemInformationBlockType12 (297)–SystemInformationBlockType13 (297)–SystemInformationBlockType14 (298)–SystemInformationBlockType15 (298)–SystemInformationBlockType16 (299)–SystemInformationBlockType17 (300)–SystemInformationBlockType18 (301)–SystemInformationBlockType19 (301)–SystemInformationBlockType20 (304)6.3.2Radio resource control information elements (304)–AntennaInfo (304)–AntennaInfoUL (306)–CQI-ReportConfig (307)–CQI-ReportPeriodicProcExtId (314)–CrossCarrierSchedulingConfig (314)–CSI-IM-Config (315)–CSI-IM-ConfigId (315)–CSI-RS-Config (317)–CSI-RS-ConfigEMIMO (318)–CSI-RS-ConfigNZP (319)–CSI-RS-ConfigNZPId (320)–CSI-RS-ConfigZP (321)–CSI-RS-ConfigZPId (321)–DMRS-Config (321)–DRB-Identity (322)–EPDCCH-Config (322)–EIMTA-MainConfig (324)–LogicalChannelConfig (325)–LWA-Configuration (326)–LWIP-Configuration (326)–RCLWI-Configuration (327)–MAC-MainConfig (327)–P-C-AndCBSR (332)–PDCCH-ConfigSCell (333)–PDCP-Config (334)–PDSCH-Config (337)–PDSCH-RE-MappingQCL-ConfigId (339)–PHICH-Config (339)–PhysicalConfigDedicated (339)–P-Max (344)–PRACH-Config (344)–PresenceAntennaPort1 (346)–PUCCH-Config (347)–PUSCH-Config (351)–RACH-ConfigCommon (355)–RACH-ConfigDedicated (357)–RadioResourceConfigCommon (358)–RadioResourceConfigDedicated (362)–RLC-Config (367)–RLF-TimersAndConstants (369)–RN-SubframeConfig (370)–SchedulingRequestConfig (371)–SoundingRS-UL-Config (372)–SPS-Config (375)–TDD-Config (376)–TimeAlignmentTimer (377)–TPC-PDCCH-Config (377)–TunnelConfigLWIP (378)–UplinkPowerControl (379)–WLAN-Id-List (382)–WLAN-MobilityConfig (382)6.3.3Security control information elements (382)–NextHopChainingCount (382)–SecurityAlgorithmConfig (383)–ShortMAC-I (383)6.3.4Mobility control information elements (383)–AdditionalSpectrumEmission (383)–ARFCN-ValueCDMA2000 (383)–ARFCN-ValueEUTRA (384)–ARFCN-ValueGERAN (384)–ARFCN-ValueUTRA (384)–BandclassCDMA2000 (384)–BandIndicatorGERAN (385)–CarrierFreqCDMA2000 (385)–CarrierFreqGERAN (385)–CellIndexList (387)–CellReselectionPriority (387)–CellSelectionInfoCE (387)–CellReselectionSubPriority (388)–CSFB-RegistrationParam1XRTT (388)–CellGlobalIdEUTRA (389)–CellGlobalIdUTRA (389)–CellGlobalIdGERAN (390)–CellGlobalIdCDMA2000 (390)–CellSelectionInfoNFreq (391)–CSG-Identity (391)–FreqBandIndicator (391)–MobilityControlInfo (391)–MobilityParametersCDMA2000 (1xRTT) (393)–MobilityStateParameters (394)–MultiBandInfoList (394)–NS-PmaxList (394)–PhysCellId (395)–PhysCellIdRange (395)–PhysCellIdRangeUTRA-FDDList (395)–PhysCellIdCDMA2000 (396)–PhysCellIdGERAN (396)–PhysCellIdUTRA-FDD (396)–PhysCellIdUTRA-TDD (396)–PLMN-Identity (397)–PLMN-IdentityList3 (397)–PreRegistrationInfoHRPD (397)–Q-QualMin (398)–Q-RxLevMin (398)–Q-OffsetRange (398)–Q-OffsetRangeInterRAT (399)–ReselectionThreshold (399)–ReselectionThresholdQ (399)–SCellIndex (399)–ServCellIndex (400)–SpeedStateScaleFactors (400)–SystemInfoListGERAN (400)–SystemTimeInfoCDMA2000 (401)–TrackingAreaCode (401)–T-Reselection (402)–T-ReselectionEUTRA-CE (402)6.3.5Measurement information elements (402)–AllowedMeasBandwidth (402)–CSI-RSRP-Range (402)–Hysteresis (402)–LocationInfo (403)–MBSFN-RSRQ-Range (403)–MeasConfig (404)–MeasDS-Config (405)–MeasGapConfig (406)–MeasId (407)–MeasIdToAddModList (407)–MeasObjectCDMA2000 (408)–MeasObjectEUTRA (408)–MeasObjectGERAN (412)–MeasObjectId (412)–MeasObjectToAddModList (412)–MeasObjectUTRA (413)–ReportConfigEUTRA (422)–ReportConfigId (425)–ReportConfigInterRAT (425)–ReportConfigToAddModList (428)–ReportInterval (429)–RSRP-Range (429)–RSRQ-Range (430)–RSRQ-Type (430)–RS-SINR-Range (430)–RSSI-Range-r13 (431)–TimeToTrigger (431)–UL-DelayConfig (431)–WLAN-CarrierInfo (431)–WLAN-RSSI-Range (432)–WLAN-Status (432)6.3.6Other information elements (433)–AbsoluteTimeInfo (433)–AreaConfiguration (433)–C-RNTI (433)–DedicatedInfoCDMA2000 (434)–DedicatedInfoNAS (434)–FilterCoefficient (434)–LoggingDuration (434)–LoggingInterval (435)–MeasSubframePattern (435)–MMEC (435)–NeighCellConfig (435)–OtherConfig (436)–RAND-CDMA2000 (1xRTT) (437)–RAT-Type (437)–ResumeIdentity (437)–RRC-TransactionIdentifier (438)–S-TMSI (438)–TraceReference (438)–UE-CapabilityRAT-ContainerList (438)–UE-EUTRA-Capability (439)–UE-RadioPagingInfo (469)–UE-TimersAndConstants (469)–VisitedCellInfoList (470)–WLAN-OffloadConfig (470)6.3.7MBMS information elements (472)–MBMS-NotificationConfig (472)–MBMS-ServiceList (473)–MBSFN-AreaId (473)–MBSFN-AreaInfoList (473)–MBSFN-SubframeConfig (474)–PMCH-InfoList (475)6.3.7a SC-PTM information elements (476)–SC-MTCH-InfoList (476)–SCPTM-NeighbourCellList (478)6.3.8Sidelink information elements (478)–SL-CommConfig (478)–SL-CommResourcePool (479)–SL-CP-Len (480)–SL-DiscConfig (481)–SL-DiscResourcePool (483)–SL-DiscTxPowerInfo (485)–SL-GapConfig (485)。

10 分钟教会你 UltraFAST

UltraFast是Xilinx在2013年底推出的一套设计方法学指导，旨在指引用户最大限度地利用现有资源，提升系统性能，降低风险，实现更快速且可预期的设计。

面向Vivado的UltraFast方法学的主体是UG949文档，配合相应的Checklist，随Vivado版本同时更新，用户可以在Xilinx的主页上免费下载。

目前，针对Vivado设计套件的UltraFast中文版也已经上市，另外一套全新的针对嵌入式可编程设计的 UltraFast嵌入式设计方法指南UG1046也已经在Xilinx官网上开放下载。

尽管UltraFast这个字眼经常在网上看到，不论官方还是其他媒体上说起Vivado 设计套件时也常常提到，但很多用户仍然对这个概念十分模糊，有不少人下载文档后看到300页的PDF顿时也失去了深入学习和了解的兴趣。

适逢《Vivado使用误区与进阶》系列连载半年多，大部分预先列好的主题也都已经按照计划完成，我们准备把这些短文集结为一本电子书，方便更多读者随手翻阅或是必要时用作设计参考。

借此机会，套用在Xilinx内部被誉为“Vivado之父”的产品营销总监 Greg Daughtry在去年第一届Club Vivado中所提出的“时序收敛十大准则” 的概念，试着用十分钟的篇幅来概括一下什么是UltraFast，以及怎样利用UItraFast真正帮助我们的FPGA设计。

时序设计的十大准则，基本上也涵盖了UltraFast设计方法指南的基本要点。

UG949中将FPGA设计分为设计创建、设计实现和设计收敛几大部分来讨论，除了介绍所有可用的设计方法和资源，更多的是一些高级方法学技巧，这些技巧基本上都跟时序收敛有关或是以时序收敛为目标，有些通用的方法和技巧甚至脱离了具体选用的FPGA器件的限制，适用于更广泛意义上的时序收敛。

最宝贵的是，所有这些UltraFast设计方法学技巧都来自一线技术支持人员的经验以及客户的反馈，是业界第一本真正意义上完全面向用户的指南，这一点只要你试着读过一两节UG949就会有明显感觉，所有其中提到的技巧和方法都具有很高的可操作性，可以带来立竿见影的效果。

飞秒激光微加工的研究进展

飞秒激光微加工的研究进展顾理;孙会来;于楷;赵方方【摘要】The article reviews the progress of micro-fabrication by femtosecond laser at home and abroad in recent years. Femtosecond laser pulses have undergone through the laboratory process to become a useful tool for material mi-cro-nano-processing in industrial field. In this paper, we introduce the process of femto-second laser precise micro-nanofabrication. Two different fabrication mechanisms are described which are laser ablation and two photo polymerization. Finally,the existing problems and future development of micro-manufacture by femtosecond laser are discussed.%综述了近年来国内外利用飞秒激光微加工的研究进展.飞秒激光脉冲作为材料微纳加工的一项工具,已经从实验室进入到工业化阶段.介绍了飞秒激光在微纳加工领域的一些研究情况,分别就飞秒激光烧蚀微加工以及双光子聚合加工进行了阐述.最后分析了飞秒激光微加工目前存在的问题及未来发展的主要方向.【期刊名称】《激光与红外》【年(卷),期】2013(043)001【总页数】5页(P14-18)【关键词】飞秒激光;微加工;烧蚀;双光子聚合【作者】顾理;孙会来;于楷;赵方方【作者单位】天津市现代机电装备重点实验室天津工业大学机械工程学院,天津300387;四川省制造与自动化重点实验室西华大学,四川成都610039;天津市现代机电装备重点实验室天津工业大学机械工程学院,天津300387;四川省制造与自动化重点实验室西华大学,四川成都610039;辽宁省铁岭港华燃气有限公司技术设备部,辽宁铁岭112000;天津市现代机电装备重点实验室天津工业大学机械工程学院,天津300387;四川省制造与自动化重点实验室西华大学,四川成都610039【正文语种】中文【中图分类】TN2491 引言激光作为20世纪最伟大的发明之一，自1960年Maiman利用红宝石实现的第一台激光器，已经经历了五十余年。

1 超连续脉冲MXFROG测量

1
State Key Laboratory of Precision Spectroscopy, East China Normal University, Shanghai 200062, China
2
jwu@
*hpzeng@
Abstract: We demonstrate that complex supercontinuum and few-cycle ultrashort laser pulses can be fully characterized by using a cross-correlation frequency-resolved optical gating with molecular alignment induced birefringence functioned as a gate. The temporal envelope and phase of the broadband supercontinuum pulse are retrieved by the principal component generalized projection algorithm. This technique shows advantages without phase-matching constraint that may limit the measurable spectral bandwidth, experimental robustness in operating through the whole transparent spectral region of the molecular gases, and intensity sensitivity to measure weak pulses which is inherited from the intrinsic linear process in recording the molecular birefringence induced polarization spectroscopy. Experimental measurements of a few-cycle pulse in the visible region of 525-725 nm confirm that the molecular alignment gating supports a full field characterization of the ultrashort pulse around 10 fs in duration.

real-time PCR经典文献

Arya, Shergill, Williamson, Gommersall, Arya & Patel210Expert Rev. Mol. Diagn. 5(2), (2005)activity of Taq DNA polymerase (when the enzyme extended from an upstream primer into the region of the probe)degraded the probe into smaller fragments that could be differ-entiated from undegraded probe. This dependence on poly-merization ensured that cleavage of the probe occurred only if the target sequence was being amplified. After PCR, cleavage of the probe was measured by using thin-layer chromatography to separate cleavage fragments from intact probe.The introduction of dual-labeled oligonucleotide fluorogenic probes allowed the elimination of post-PCR processing for the analysis of probe degradation [21]. The probe has a reporter fluorescent dye at the 5´ end and a quencher dye attached to the 3´ end. Whilst the probe is intact, the close proximity of the quencher significantly decreases the fluorescence emitted by the reporter dye. A fluorescence signal is only emitted upon cleav-age of the probe, based on the fluorescence resonance energy transfer (FRET) principle [22].In the real-time quantitative TaqMan ® assay a fluorogenic nonextendable ‘TaqMan’ probe is used (FIGURE 1) [23]. The probe has a fluorescent reporter dye attached to its 5´ end and a quencher dye at its 3´ terminus. If the target sequence is present, the fluorogenic probe anneals downstream from one of the primer sites and is cleaved by the 5´ nuclease activity of the Taq polymerase enzyme during the extension phase of the PCR. Whilst the probe is intact, FRET occurs and the fluores-cence emission of the reporter dye is absorbed by the quenching dye. Cleavage of the probe by Taq polymerase during PCR sep-arates the reporter and quencher dyes, thereby increasing the fluorescence from the former. Additionally, cleavage removesthe probe from the target strand, allowing primer extension to continue to the end of template strand, thereby not interfering with the exponential accumulation of PCR product. Addi-tional reporter dye molecules are cleaved from their respective probes with each cycle, leading to an increase in fluorescence intensity proportional to the amount of amplicon produced.The various available chemistries for real-time PCR are described later in this review.Using any of the developed chemistries, the increase in fluor-escence emission during the PCR reaction can be detected in real time by a modified thermocycler. The computer software constructs amplification plots using the fluorescence emission data that are collected during the PCR amplification (FIGURE 2).FIGURE 2 demonstrates a representative amplification plot and defines the important terms associated with it.•Baseline: the baseline is defined as the PCR cycles in which a reporter fluorescent signal is accumulating but is beneath the limits of detection of the instrument. By default, the compu-ter software sets the baseline from cycles three to 15; however,this often needs to be changed manually.•∆Rn: a computer software program calculates a ∆Rn using the equation Rn = Rnf – Rnb, where Rnf is the fluorescence emission of the product at each time point and Rnb is the fluorescence emission of the baseline [23,24]. The ∆Rn values are plotted versus the cycle number. During the early cycles of PCR amplification, ∆Rn values do not exceed the baseline.•Threshold: an arbitrary threshold is chosen by the comput-ers, based on the variability of the baseline. It is calculated asten-times the standard deviation of the average signal of the baseline fluorescentsignal between cycles three to 15. A fluorescent signal that is detected above the threshold is considered a real signal that can be used to define the thresholdcycle (Ct) for a sample. If required, the threshold can be manually changed for each experiment so that it is in theregion of exponential amplification across all amplification plots.•Ct: this is defined as the fractional PCR cycle number at which the reporter fluor-escence is greater than the minimal detec-tion level (i.e., the threshold). The Ct is a basic principle of real-time PCR and is an essential component in producing accu-rate and reproducible data [1]. The pres-ence of more template at the start of the reaction leads to a fewer number of cycles reaching the point at which the fluores-cent signal is recorded as statistically sig-nificant above background [24]. This Ct value will always occur during the expo-nential phase of target amplification,which occurs during the early cycles ofBasic principles of real-time quantitative PCR 211PCR. As reaction components become limiting, the rate of tar-get amplification decreases until the PCR reaction is no longer generating template at an exponential rate (plateau phase) and there is little or no increase in PCR product. This is the main reason why Ct is a more reliable measure of starting copy number than an endpoint measurement of the amount of accumulated PCR product. During the exponential phase,none of the reaction components is limiting and therefore Ct values are very reproducible for replicate reactions with the same starting copy number.Standard & absolute quantitationT wo methods are available to quantify real-time PCR results,standard-curve or absolute quantitation and relative quantitation.Standard-curve or absolute quantitationAs shown by Higuchi and coworkers, the plot of the log of ini-tial target copy number for a set of known standards (five- or tenfold serial dilution) versus Ct is a straight line (the standard curve) [1]. Quantitation of the amount of target in the ‘unknown’ samples of interest is accomplished by measuring Ct and using the standard curve to determine starting copy number. The most common source of a known sample is a plas-mid for the gene of interest and the standard curve is generated based on a serial dilution of a starting amount. Another option,and easier to generate if a plasmid is unavailable, is the use of a synthetic single-stranded sense oligonucleotide for the entire amplicon. The advantage of this approach is that it significantly simplifies the process of obtaining a standard curve for ampli-cons up to 100 bp, which encompasses most real-time PCR amplicons. Furthermore, it is also less susceptible to bias when quantified by a spectrophotometer due to the relative purity of the oligonucleotide. Together with the greater precision of meas-urement of the standard and the possibility of calculating the moles of oligonucleotide (hence, number of copies), it is possi-ble to approximate the number of copies of a template in an unknown sample, although not in terms of absolute copy number. One final option for a standard curve is to use a cell line with a known copy number or expression level of the gene of interest. The standard curve method is used in circumstances when absolute quantitation is critical for the investigator (e.g.,when measuring a small number of genes in either a few or many samples [25,26]) and in quantitation of viral load [27–29].Relative quantitationRelative quantitation is also known as the comparative thresh-old method (2-∆∆Ct method). This method eliminates the need for standard curves and mathematical equations are used to cal-culate the relative expression levels of a target relative to a refer-ence control or calibrator such as a nontreated sample or RNA from normal tissue or a sample at time zero in a time-course study. The amount of target gene in the sample, normalized to an endogenous housekeeping gene and relative to the normal-ized calibrator, is then given by 2-∆∆Ct , where ∆∆Ct =∆Ct(sample) - ∆Ct(calibrator), and ∆Ct is the Ct of the targetgene subtracted from the Ct of the housekeeping gene. For this calculation to be valid and in order to obtain reliable results, it is imperative that the amplification efficiencies of the house-keeping and target gene are approximately equal and at or above 90%. This can be established by looking at how ∆Ct (of both sample and calibrator) varies with template dilution. If the plot of complementary DNA (cDNA) dilution versus ∆Ct is close to zero, it implies that the efficiences of the target and housekeeping genes are very similar. If a housekeeping gene cannot be found whose amplification efficiency is similar to the target, the standard curve method is then preferable. Alter-natively, new primers can be designed and/or optimized to achieve a similar efficiency for the target and housekeeping gene amplicons.Housekeeping genes & normalizationIn real-time quantitative PCR experiments specific errors will be introduced due to minor differences in the starting amount of RNA, quality of RNA or differences in efficiency of cDNA synthesis and PCR amplification. In order to minimize these errors and correct for sample-to-sample variation, a cellular RNA is simultaneously amplified with the target, which serves as an internal reference against which other RNA values can be normalized. The most common genes used for normalization,termed housekeeping genes, are β-actin, a cytoskeletal protein,glceraldehyde-3-phosphate dehydrogenase (GAPDH), a glyco-lytic enzyme [30], and ribosomal RNA (rRNA). These genes should theoretically be expressed at a constant level among dif-ferent tissues of an organism, at all stages of development, and their expression levels should also remain relatively constant in different experimental conditions. However, none of theseArya, Shergill, Williamson, Gommersall, Arya & Patel212Expert Rev. Mol. Diagn. 5(2), (2005)housekeeping genes are ideal. It has been shown that GAPDH expression levels are altered by glucose, insulin, heat shock and cellular proliferation and β-actin levels may also be modified by experimental treatments [31–35]. rRNA production is less likely to vary under conditions affecting mRNA transcription [36,37]. However, it is not always a good representative of total mRNA population in a cell as rRNA is expressed at a much higher level than mRNA. Other alternative housekeeping genes have been proposed but none have been entirely satisfac-tory and no single unequivocal reference gene has been identi-fied as yet. Some authors have suggested the use of several housekeeping genes in a single experiment and that the mean expression of these multiple housekeeping genes can be used for normalization [38].Importantly, selection of the housekeeping gene for each spe-cific experiment should be made very carefully as the reliability of the results depends on the choice of the most relevant house-keeping gene according to the cells of interest and specific experimental treatments.Amplicon detectionT wo general chemistries are available. These include double-stranded (ds)DNA-intercalating agents (DNA-binding dyes)and fluorescent probes. The former includes SYBR ® Green 1 or ethidium bromide and is the simplest and most cost-effectivemethod as amplicon-specific labeled hybridization probes are not required. SYBR Green 1 only fluoresces when intercalated into dsDNA. The intensity of the fluorescence signal is there-fore dependent on the quantity of dsDNA present in the reac-tion. The main disadvantage of this method is that it is not spe-cific since the dye binds to all dsDNAs formed during the PCR reaction (i.e., nonspecific PCR products and primer-dimers).With fluorogenic probes, nonspecific amplification due to mispriming or primer-dimer artifact does not generate signal as specific hybridization between probe and template is necessary for fluorescence emission. Also, fluorogenic probes can be labeled with different and distinguishable reporter dyes, thus allowing the detection of amplicons that may have been pro-duced by one or several primer pairs in a single PCR reaction –termed multiplex real-time PCR. However, different probes must be developed to detect different sequences. The various chemistries arenow described in more detail.dsDNA-intercalating agents(DNA-binding dyes)SYBR Green 1 is a nonsequence-specific fluorogenic minor groove DNA-binding dye that intercalates into dsDNA (it does not bind to single-stranded DNA)(FIGURE 3). SYBR Green 1 exhibits little fluorescence when unbound in solution but emits a strong fluorescent signal upon binding to dsDNA [39]. An increase in the fluorescence signal occurs duringpolymerization and this decreases when DNA is denatured.Fluorescent measurements are performed at the end of the elongation step of each PCR cycle to monitor the increasing amount of amplified DNA. The advantage of this technique is that it is relatively cheap as it can be used with any pair of primers for any target. However, as the presence of any dsDNA generates fluorescence, specificity of this assay is greatly decreased due to amplification of nonspecific PCR products and primer-dimers [40]. Generating and comparing melting curves (plotting fluorescence as a function of temperature)using the LightCycler™ (Roche Molecular Diagnostics; or RotorGene, Smart Cycler, iCycler, Mx4000) is one method of increasing the specificity of the reaction [40]. A characteristic melting peak at the melting temperature (T m) of the amplicon will distinguish it from amplification artifacts that melt at lower temperatures at broader peaks. It is possible to set the software to acquire fluorescence above the primer-dimers’ melt-ing temperature but below that of the target. Another control-lable problem is that longer amplicons create a stronger ually, SYBR Green is used in singleplex reactions; however,when coupled with melting point analysis, it can be used for multiplex reactions. The SYBR Green 1 reaction has been used for many applications (e.g., viral load detection [41] and cytokine quantifaction [42–44]).Hydrolysis probes (e.g., TaqMan probes)This chemistry has already been outlined earlier in this review (FIGURE 1). A forward and reverse primer and a probe are used.The efficiency of the assay is mainly dependent on 5´ to 3´nuclease activity – the most commonly used enzyme is Taq polymerase [20] but any enzyme with 5´ nuclease activity can be used [45]. The oligonucleotide probe has a covalently bonded fluorescent reporter dye and quencher dye at the 5´ and 3´ends, respectively. Various fluorescent reporter dyes are in use including 6-carboxyfluorescein (FAM), tetrachloro-6-carboxy-fluorescein (TET), hexacholoro-6-carboxyfluorescein (HEX),or VIC. Quenchers include either 6-carboxytetramethylrhod-amine (TAMRA) or 4-(dimethylaminoazo)benzene-4-carboxy-lic acid (DABCYL). When the probe is intact the proximity of the reporter and quencher dyes permits FRET, and fluorescenceBasic principles of real-time quantitative PCR 213emission does not occur. During PCR amplification the probe anneals to the tar-get and Taq polymerase cleaves the probe,allowing an increase in fluorescence emis-sion. The increase in fluorescence inten-sity is directly proportional to the amount of amplicon produced. The TaqManchemistry is the most widely used real-time PCR assay and has been used for multiple purposes [32,46,47].TaqMan minor groove-binding probes have more recently been developed. In this chemistry, the standard TAMRAquencher at the 3´ end is replaced by a nonfluorescent quencher and a minor groove-binder molecule is also incorpo-rated at the 3´ terminus. The latter stabi-lizes the probe–target complex by fold-ing into the minor groove of the dsDNA. Additionally, the Tm of theprobes is increased, allowing the use of very short oligoprobes (14 nucleotides in length) and providing more accurate allelic discrimination. Thus, TaqManminor groove-binding probes are ideal for detecting single nucleotide polymorphisms [48,49] and for the quantitative analysis of methylated alleles [50].Dual hybridization probes This method has been convincingly validated in studies using the LightCycler instrument (FIGURE 4). T wo hybridization probes are used – one carries a donor fluorophore at its 3´ end and the other is labeled with an acceptor fluorophore at its 5´end. After the denaturation step, both probes hybridize to their target sequence in a head to tail arrangement during the anneal-ing step. This brings the two dyes in close proximity allowing FRET. The donor dye in one of the probes transfers energy,allowing the other one to dissipate fluorescence at a different wavelength. The measured fluorescence is directly proportional to the amount of DNA synthesized during the PCR reaction.The specificity of this reaction is therefore increased as a fluor-escent signal is only detected when two independent probes hybridize to their correct target sequence. This method has been widely used for detection of minimal residual disease after therapy [51,52] and viral load quantification [53,54].Molecular beacons Molecular beacons also contain covalently bound fluorescent and quenching dyes at either end of a single-stranded DNA molecule. However, they are also designed to adopt a hairpin or stem-and-loop structure whilst free in solution to bring the fluorescent dye and the quencher in close proximity for FRET to occur (FIGURE 5) [55]. The loop portion of the molecule is complementary to the target nucleic acid molecule and the stem is formed by the annealing of complementary arm sequences on the ends of the probe sequence. The close prox-imity of the fluorophore and the quencher in this hairpin con-figuration suppresses reporter fluorescence. When the probe sequence in the loop hybridizes to a complementary nucleicacid target sequence during the annealing step, a conforma-tional change occurs that forces the stem apart. This results in a linear structure and thus separation of the flurophore from the quencher dye (FRET does not occur) and an increase in fluo-rescence emission. A new hybridization takes place in the annealing step of each cycle, and the intensity of the resultant fluorescence indicates the amount of accumulated amplicon at the end of the previous cycle. Molecular beacons remain intact during PCR and they must rehybridize to the target sequence each cycle for fluorescence emission. Molecular beacons are particularly suitable for identifying point mutations [56–58].ScorpionsSimilar to molecular beacons, scorpions adopt a stem-and-loop configuration with a 5´ fluorophore and 3´ quencher (FIGURE 6).The specific probe sequence is held within the hairpin loop,which is attached to the 5´ terminus of a PCR primer sequenceby a nonamplifiable monomer (termed the PCR stopper). Thischemical modification prevents PCR from copying the stem-loop sequence of the scorpion primer. During PCR, scorpion primers are extended to form amplicon. In the annealing phase,the specific probe sequence in the scorpion tail curls back to hybridize to the complementary target sequence in the ampli-con, thus opening up the hairpin loop. This prevents the fluor-escence from being quenched and a signal is observed [59]. As the tail of the scorpion and the amplicon are now part of the same strand of DNA, the interaction is intramolecular. TheArya, Shergill, Williamson, Gommersall, Arya & Patel214Expert Rev. Mol. Diagn. 5(2), (2005)benefits of Scorpions derive from the fact that the probe ele-ment is physically coupled to the primer element, which means that the reaction leading to signal generation is a unimolecular event. This contrasts with the bimolecular collisions required by other technologies such as TaqMan or molecular beacons.The benefits of a unimolecular rearrangement are significant in that the reaction is effectively instantaneous and the fluor-escence signal much stronger. Also, better discrimination and specificity are achieved using scorpions. Scorpion probes have been used for viral load and mutation detection [60,61].Duplex scorpions are a modification of scorpions. However,in contrast to scorpions (or molecular beacons), the fluorophore and quencher dye are separated onto different and complemen-tary oligonucleotides. The advantage of duplex scorpions is the significantly greater separation between the quencher and reporter fluorophore, which decreases fluorophore quenching when the probe is bound to the target, resulting in improved signal intensity compared with conventional scorpions [62].Primer, probe & amplicon designGreat care should go into the design of the assay. Primers,probes and amplicons are designed to very exacting specifica-tions and the TaqMan system provides its own primer/probe design software from Applied Biosystems known as Primer Express, which is probably the most widely used oligonucleo-tide design program for developing real-time quantitative PCR assays. Primer3, a free program from Massachusetts Institute of Technology (MA, USA), can also be used to generate good real-time PCR assays, including designs incorporating an internal hybridization probe.The amplicon for the PCR product should be as small as reasonably possible,usually 50–150 bp in length for designs using hybridization probes (and <300 bp for SYBR Green assays). Shorter amplicons amplify more efficiently and are more tol-erant of reaction conditions. The optimal length for single-stranded primers is 15–20bases with a G/C content of 20–80%.Their T m should be in the range of 68–70°C for TaqMan primers. Molecular beacon and hybridization probe-associated primers can have a wider range of T m, but the T m of any one pair should be similar (i.e., not differ by >1–2°C). Nonspecific priming is minimized by selecting primers that have only one or two G/Cs within the last 5 nucleotides at the 3´ end. If using a SYBR Green I approach, the PCR primers must not form an appreciable amount of primer-dimer bands. A melting curve anal-ysis of each product is needed to ensure that the fluorescent signal observed is from the desired PCR product. In mRNA expression assays using a hybridizationprobe, the probe sequence should span an exon/exon boundary if possible. Having the probe T m 8–10°C higher than that of the primers ensures that the probe is fully hybridized during primer extension. TaqMan probes should not contain a G at their 5´ends due to the quenching effect of a G in this position on reporter fluorescence, even after probe cleavage.Multiplex real-time PCRThe term multiplex real-time PCR is used to describe the use of multiple fluorogenic probes for the discrimination of multiple amplicons in a single tube. The main advantages of multiplexing over single-target analysis are the ability to provide internal con-trols, lower reagent costs and preservation of precious samples.The main restrictions of this technique have been the limited number of available fluorophores, fluorescence emission from quenching dyes and the common use in real-time instruments of a monochromatic light source. The introduction of nonfluor-escent quenchers, which have no inherent fluorescence, has been a breakthrough that has allowed an increase in the number of spec-trally discernable fluorogenic probes used per reaction. Initial real-time instrumentation contained optimized filters to minimize overlap of the emission spectra from the fluorophores. Newer sys-tems have used either multiple light-emitting diodes, which span the whole visible spectrum, or a tungsten lamp, which emits light over a broad range of wavelengths. However, despite these advancements, only four-color multiplex reactions are usually pos-sible [63,64], of which one color may be used for an internal con-trol. One recent advancement of note, which is the introduction of combinatorial fluorescence energy transfer tags [65,66], will help to boost the development of multiplex real-time PCR.Basic principles of real-time quantitative PCR 215Available equipmentThere are a variety of instruments available on the market, each of which has its own individual characteristics (TABLE 1). Great care should be taken when choosing which instrument to buy and it is important to match the instrument’s capabilities with laboratory needs. Cost should not be the only factor when mak-ing a choice; the cheaper models cannot compensate for the var-iance in the optics and therefore are not capable of detecting smaller differences. The higher throughput instrument may be more than is required.The ABI Prism ® 7700 Sequence Detection System (SDS)from Applied Biosystems was the first commercially availa-ble thermocycler for real-time PCR, but has now been dis-continued. Continuous fluorescence wavelength laser light detection from 500–660 nm allowed multiplex PCR. The ABI Prism 7700 has more recently been replaced by the ABI Prism 7900HT, which has similar specifications to the 7700SDS but is completely automated and designed espe-cially for very high-throughput applications (384 samples per run). Another recent introduction is the less expensive ABI Prism 7000 SDS. It retains the Peltier-based 96-well block thermal cycling format of the ABI 7700, but replaces the laser with a tungsten–halogen lamp that simultaneously illuminates all sample wells. The software supplied with the instrument is much more user friendly and is Microsoft ®Windows-based, which allows easy export of data and ampli-fication plots. One of the major advantages of the ABIinstruments is the collection of data from a passive reference signal to nor-malize each reaction for variances in the optics of the system. In addition,Applied Biosystems have launched the Applied Biosystems 7300 and 7500 Real Time PCR systems, which represent less expensive alternatives.The low-priced LightCycler from Roche Molecular Biochemicals induces fluorescence excitation by a blue light-emitting diode that is read by three sili-con photodiodes with different wave-length filters, allowing detection of spectrally distinct fluorophores. There-fore, multiplex PCR can be performed.A complete PCR run of 30–40 cycles is performed in 20–30 min; however,only a limited number of samples (max-imum 32) can be analyzed simultane-ously. As the LightCycler analyzes the specificity of the results by performing melting curves, it makes the use of dsDNA-binding dyes such as SYBR Green I more reliable. However, as sam-ples must be placed in capillaries as opposed to tubes, it is less practical for the investigator.The iCycler iQ from BioRad Instruments has a tungs-ten–halogen lamp allowing excitation of a wide range of fluorophores (400–700 nm). It is able to multiplex four differ-ent fluorophores per sample tube. Also, it has an optical mod-ule allowing fluorescence emission to be viewed during the course of PCR amplification. Furthermore, the 96 samples are tracked simultaneously, thereby providing a fast assay. A recently launched module allows it to amplify 384 samples at any one time.The Mx4000® Multiplex (Stratagene) can detect multiple fluorescence PCR chemistries, including TaqMan, hybridiza-tion probes and molecular beacons. The light source for the Mx4000 system is a quartz tungsten–halogen lamp that gen-erates a broad excitation range of 350–750 nm and there are four photomultiplier tubes with a detection range of 350–830nm. The instrument is ideal for performing multi-plex PCR. Importantly, the system contains an integrated per-sonal computer that operates independently from the instru-ment’s embedded microprocessor, which gives some protection against data loss.The Smart Cycler (Cepheid) also detects multiple fluor-escence PCR chemistries. With up to 96 independently pro-grammable reaction sites, the Smart Cycler can simultaneously run multiple experiments with different protocols and at differ-ent times. This enables multiple users to use the Smart Cycler simultaneously. Up to four different fluorophores can be detected in each reaction.。

qScript Ultra Flex Kit - IFU -132.1 REV 0 2说明书

qScript ® Ultra Flex KitDescriptionThe qScript Ultra Flex Kit is a complete, next-generation system that delivers rapid and efficient first-strand cDNA synthesis while providing flexibility in the choice of priming method. The kit features a stabilized 5x-concentrated reaction mix that provides all components for first-strand synthesis except RNA template and primers. A key component is a novel, state-of-the-art, RNase H deficient reverse transcriptase (RT) that was engineered for improved thermostability, velocity, processivity, and resistance to many common reaction inhibitors.The superior performance of this novel RT is further supported by proprietary replication accessory proteins and a recombinant mammalian RNase inhibitor protein. These features allow for reactions to be carried out at higher temperatures than standard reverse transcriptases, improving sensitivity and minimizing potential interference from blocking secondary structures. In addition, the improved synthesis speed and enhanced processivity allow for reactions to be complete in 10 minutes, with high yields of full-length cDNA products as long as 20 kb.The kit is supplied with both an anchored oligo(dT) solution and a modified random primer mixture that enhances cDNA yield with low input quantities or compromised RNA samples. Primer solutions include a proprietary enhancer compound that improves cDNA priming efficiency and protects RNA integrity during the optional denaturation step to destabilize RNA secondary structures. This enhancer is provided as a separate solution for use with your gene-specific primers. The kit is compatible with total RNA, polyA+ RNA, or viral RNA. The resulting cDNA product is directly compatible with real-time RT-qPCR methods or end-point RT-PCR. The length of cDNA product is dependent on the priming strategy and the quality of the RNA template. Greater length cDNA products can be obtained from oligo(dT) priming, in which primers specifically anneal to the 3’ poly(A) tails of mRNA, or by using gene-specific priming, in which primers anneal to a defined sequence. While strategies that utilize random primers produce shorter first strand cDNA, they are suitable for obtaining larger yields of product from the 5’-ends of RNA molecules and from classes of RNA biotypes that do not contain a 3’ poly(A) tail. Complete, unbiased first strand cDNA synthesis can be obtained with a mixed primer strategy, in which oligo(dT) and random primers are combined.Components95215-025 95215-100 5x qScript Ultra Reaction Mix1 x 100 µL1 x 400 µLOptimized master mix containing buffer, magnesium, dNTPs and qScript Ultra RTOligo(dT) primers1 x 50 µL1 x 200 µL10x concentrated in Enhancer solutionRandom primers1 x 50 µL1 x 200 µL10x concentrated in Enhancer solutionGSP Enhancer (10x) 1 x 50 µL 1 x 200 µL Nuclease-free Water1 x 1.5 mL2 x 1.5 mLCat No. 95215-025 Size: 25 x 20 µL reactions (1x 0.1 mL) Store at -25ºC to -15°C95215-100 100 x 20 µL reactions (1x 0.4 mL)Storage and StabilityStore components in a constant temperature freezer at -25°C to -15°C.After thawing, mix thoroughly before use.For lot specific expiry date, refer to package label, Certificate of Analysis or Product Specification Form. Standard Reaction Protocol1.Thaw components, mix and centrifuge before use. Hold on ice before use.2.Add the following to a thin-walled PCR tube or reaction plate on ice:Component Volume for 20 μL rxn.Final Concentration Nuclease-free water variableTemplate RNA variable 2.5 µg to 1 pg total RNA10x Oligo(dT) or 10X Random Primers or 1-10 µM Gene-specific primer(s) 2 µL1x Oligo(dT) or 1x Randomprimers or 0.1-1 µM Gene-specific primers(Optional) 10x GSP Enhancer, if using Gene-specificprimer(s)2 µL5x qScript Ultra Reaction Mix 4 µLFinal volume 20 µLNOTE: For multiple reactions, a master mix can be prepared with all components except template RNA and dispensed into 96-well plates or PCR tubes.3.Mix by gentle vortexing, then briefly centrifuge to collect contents.4.Incubate for:o 5-10 minutes at 25°C (only required if using random primers)o 10 minutes at 55°Co 5 min at 85°Co Hold at 4°C5.After the completion of cDNA synthesis, reactions can be used directly for endpoint RT-PCR or RT-qPCR analysis. It isrecommended that PCR reactions contain no more than 1/5 volume of the first-strand cDNA reaction. If desired, reactions can be diluted with TE buffer (10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA). Reaction can be stored at -20°C for future use. Two-Phase Reaction ProtocolFor improved yield of some long cDNA products, or if template is known to contain regions of secondary structure1.Thaw components, mix and centrifuge before use. Hold on ice before use.2.Add the following to a thin-walled PCR tube or reaction plate on ice:Component Volume for 20-μL rxn.Final ConcentrationNuclease-free water variableTemplate RNA variable 2.5 µg to 1 pg total RNA10x Oligo(dT) or 10X Random Primers or 1-10 µM Gene-specific primer(s) 2 µL1X Oligo(dT) or 1X Randomprimers or 0.1-1 µM Gene-specific primers(Optional) 10x GSP Enhancer, if using Gene-specificprimer(s)2 µL Final volume 16 µL3.Mix by gentle vortexing, then briefly centrifuge to collect contents.4.Incubate for 5 min at 65°C then immediately transfer to 4°C.5.Add to each reaction mixture:Component Volume for 20-μL rxn.Final Concentration5x qScript Ultra Reaction Mix 4 µL 1XFinal Volume (μL) 20 µL6.Mix by gentle vortexing, then briefly centrifuge to collect contents.7.Incubate for:o 5-10 minutes at 25°C (only required if using random primers)o 10 minutes at 55°Co 5 min at 85°Co Hold at 4°C8.After the completion of cDNA synthesis, reactions can be used directly for endpoint RT-PCR or RT-qPCR analysis. It isrecommended that PCR reactions contain no more than 1/5 volume of the first-strand cDNA reaction. If desired, reactions can be diluted with TE buffer (10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA). Reaction can be stored at -20°C for future use.Quality ControlFunctional PCR Assay for Flex Kit Reaction Mix (5X): Detection of ß-actin mRNA from 100 ng to 100 fg of total RNA. Coefficient of determination (R2 ) ≥ 0.990 with a slope analysis between -3.20 and -3.70. Single bands visible at 9 kb and 15 kb from 30 cycles of PCR using 100 ng Oligo-dT cDNA.Limited Label LicensesUse of this product signifies the agreement of any purchaser or user of the product to the following terms:1.The product may be used solely in accordance with the protocols provided with the product and this manual and for use withcomponents contained in the kit only. Quantabio, LLC. grants no license under any of its intellectual property to use or incorporate the enclosed components of this kit with any components not included within this kit except as described in the protocols provided with the product, this manual, and additional protocols available at . Some of these additional protocols have been provided by Quantabio product users. These protocols have not been thoroughly tested or optimized by Quantabio, LLC. Quantabio, LLC. neither guarantees them nor warrants that they do not infringe the rights of third-parties.2.Other than expressly stated licenses, Quantabio, LLC. makes no warranty that this kit and/or its use(s) do not infringe the rightsof third-parties.3.This kit and its components are licensed for one-time use and may not be reused, refurbished, or resold.4.Quantabio, LLC. specifically disclaims any other licenses, expressed or implied other than those expressly stated.5.The purchaser and user of the kit agree not to take or permit anyone else to take any steps that could lead to or facilitate anyacts prohibited above. Quantabio, LLC. may enforce the prohibitions of this Limited License Agreement in any Court, and shall recover all its investigative and Court costs, including attorney fees, in any action to enforce this Limited License Agreement or any of its intellectual property rights relating to the kit and/or its components.©2021 Quantabio, LLC. 100 Cummings Center Suite 407J Beverly, MA 01915Telephone number: 1-888-959-5165.Quantabio products are manufactured in Beverly, Massachusetts, Frederick, Maryland and Hilden, GermanyIntended for molecular biology applications. This product is not intended for the diagnosis, prevention or treatment of a disease.TrademarksqScript and ToughMix are registered trademark of Quantabio, LLC.。

欧洲金属检测技术有限公司产品说明书

18G A K R 01Eurofins GeneScan Technologies GmbHTel:+***********************************/kitsThe information included in this publication is based on our current knowledge and was thoroughly verified. Nevertheless there is no guarantee for it to be up-to-date, complete and correct. Eurofins is not to be held liable for damages or injury, which are connected to the use of this information. Especially the confirmation of legaldetails is up to the user. All offers are non-binding and without obligation.GMO Ident Accelerated Growth SalmonAquAdvantage ®, a genetically modified salmon from AquaBounty Technologies, is approved for food in the USA and Canada. The introduction of a growth hormone-regulating gene from Pacific Chinook salmon leads to an increased growth of this genetically modified fish. In August 2017, AquaBounty Technologies announced that this GMO has been sold in Canada for food in the open market.AquAdvantage ®salmon is thus the firstgenetically engineered animal to be marketed for human consumption.The GMO testing kit GMO Ident RT IPCAccelerated Growth Salmon from Eurofins GeneScan Technologies offers the specific detection of the genetic construct present inAquAdvantage ®salmon.Kit Components• MasterMix for 96 reactions • Positive control DNA (Plasmid) • Negative control• Evaluation Sheet (provided by email)Kit Specifications• Specific detection of the genetic modificationpresent in AquAdvantage ®salmon• Second Kit for verification of positive results available• No false-negative results: inhibition control (internal positive control; IPC) included in every reaction• Can be used flexibly with common DNA-extraction methods.• Validated for the following real-time PCR cyclers:- Applied Biosystems® 7500 (Fast) - Agilent Mx3000P/Mx3005P™ - Bio-Rad CFX96 Touch™Ordering Information。

Quasi-phase-matching

Quasi-phase-matching for third harmonic generation in noble gases employing ultrasoundU. K. Sapaev,1 I. Babushkin,2 and J. Herrmann 1,∗1 Max-Born-Institutefor Nonlinear optics and Fast Spectroscopy, Max-Born-Str. 2a, Berlin D-12489, Germany 2 Weierstrass Institute, Mohrenstrasse 39, D-10117 Berlin, Germany∗ jherrman@mbi-berlin.deAbstract: We study a novel method of quasi-phase-matching for third harmonic generation in a gas cell using the periodic modulation of the gas pressure and thus of the third order nonlinear coefﬁcient in the axial direction created by an ultrasound wave. Using a comprehensive numerical model we describe the quasi-phase matched third harmonic generation of UV (at 266 nm) and VUV pulses (at 133 nm) by using pump pulses at 800 nm and 400 nm, respectively, with pulse energy in the range from 3 mJ to 1 J. In addition, using chirped pump pulses, the generation of sub-20-fs VUV pulses without the necessity for an external chirp compensation is predicted.© 2012 Optical Society of AmericaOCIS codes: (190.0190) Nonlinear optics; (190.4380) Nonlinear optics, four-wave mixing; (230.1040) Acousto-optical devices.References and links1. J. W. Ward and G. H. C. New, “Ultrabroadband phase-matched optical parametric generation in the ultraviolet by use of guided waves,” Phys. Rev. 185, 57–72 (1969). 2. G. Bjorklund, “Effects of focusing on third-order nonlinear processes in isotropic media,“ IEEE J. Quantum Electron. 11, 287–296 (1975). 3. R. Eramo and M. Matera, “Third-harmonic generation in positively dispersive gases with a novel cell,“ Appl. Opt. 33, 1691–1696 (1994). 4. T. Tamaki, K. Midirika, and M. Obara, “Phase-matched third-harmonic generation by nonlinear phase shift in a hollow ﬁber,“ Appl. Phys. B 67, 59–63 (1998). 5. D. S. Bethune and C. T. Retter, “Optical harmonic generation in nonuniform gaseous media with application to frequency tripling in free-jet expansions,“ IEEE J. Quantum Electron. 23, 1348–1360 (1987). 6. C. W. Siders, N. C. Turner, M. C. Downer, A. Babine, A. Stepanov, and A. M. Sergeev, “Blue-shifted thirdharmonic generation and correlated self-guiding during ultrafast barrier suppression ionization of subatmospheric density noble gases,“ J. Opt. Soc. Am. B 13, 330–336 (1996). 7. S. Backus, J. Peatross, Z. Zeek, A. Rundquist, G. Taft, M. M. Murnane, and H. C. Kapteyn, “16-fs, 1-μ J ultraviolet pulses generated by third-harmonic conversion in air,“ Opt. Lett. 21, 665–667 (1996). 8. S. A. Trushin, K. Kosma, W. Fub, and W. E. Schmid, “Sub-10-fs supercontinuum radiation generated by ﬁlamentation of few-cycle 800 nm pulses in argon,“ Opt. Lett. 32, 2432–2434 (2007). 9. N. Akozbek, A. Iwasaki, A. Becker, M. Scalora, S. L. Chin, and C. M. Bowden, “Third-harmonic generation and self-channeling in air using high-power femtosecond laser pulses,“ Phys. Rev. Lett. 89, 143901 (2002). 10. N. Kortsalioudakis, M. Tatarakis, N. Vakakis, S. D. Moustaizis, M. Franco, B. Prade, A. Mysyrowicz, A. A. Papadogiannis, A. Couairon, and S. Tzortzakis, ”Enhanced harmonic conversion efﬁciency in the self-guided propagation of femtosecond ultraviolet laser pulses in argon,” Appl. Phys. B 80, 211–214 (2005). 11. X. Yang, J. Wu, Y. Peng, Y. Tong, S. Yuan, L. Ding, Z. Xu, and H. Zeng, ”Noncollinear interaction of femtosecond ﬁlaments with enhanced third harmonic generation in air,” Appl. Phys. Lett. 95, 111103 (2009). 12. S. Suntsov, D. Abdollahpour, D. G. Papazoglou, S. Tzortzakis, ”Efﬁcient third-harmonic generation through tailored IR femtosecond laser pulse ﬁlamentation in air,” Opt. Express 17, 3190–3195 (2009).#173228 - $15.00 USDReceived 24 Jul 2012; revised 6 Sep 2012; accepted 6 Sep 2012; published 19 Sep 2012(C) 2012 OSA24 September 2012 / Vol. 20, No. 20 / OPTICS EXPRESS 2275313. S. Suntsov, D. Abdollahpour, D. G. Papazoglou, S. Tzortzakis, ”Filamentation-induced third-harmonic generation in air via plasma-enhanced third-order susceptibility,” Phys. Rev. A 81, 033817 (2010). 14. Y. Liu, M. Durand, A. Houard, B. Forestier, A. Couairon, A. Mysyrowicz, ”Efﬁcient generation of third harmonic radiation in air ﬁlaments: A revisit,” Opt. Commun. 284, 4706–4713 (2011). 15. C. G. Durfee, S. B. Margaret, M. Murnane, and H. C. Kapteyn, “Ultrabroadband phase-matched optical parametric generation in the ultraviolet by use of guided waves,” Opt. Lett. 22, 1565–1567 (1997). 16. P. Tzankov, O. Steinkellner, J. Zheng, M. Mero, W. Freyer, A. Husakou, I. Babushkin, J. Herrmann, and F. Noack, “High-power ﬁfth-harmonic generation of femtosecond pulses in the vacuum ultraviolet using a Ti:sapphire laser,” Opt. Express 15, 6389–6395 (2007). 17. I. V. Babushkin and J. Herrmann, “High energy sub-10 fs pulse generation in vacuum ultraviolet using chirped four wave mixing in hollow waveguides,” Opt. Express 16, 17774–17779 (2008). 18. J. A. Armstrong, N. Bloembergen, J. Ducuing, and P. S. Persham, ”Interactions Between Light Waves in a Nonlinear Dielectric,” Phys. Rev. 127, 1918–1939 (1962). 19. M. M. Fejer, G. A. Magel, D. H. Jundt, and R. L. Byer, ”Quasi-phase-matched second harmonic generation: tuning and tolerances,” IEEE J. Quantum Electron. QE-28, 2631–2654 (1992). 20. A. Paul, R. A. Bartels, R. Tobey, H. Green, S. Weiman, I. P. Christov, M. M. Murnane, H. C. Kapteyn, and S. Backus, “Quasi-phase-matched generation of coherent extreme-ultraviolet light,” Nature (London) 421, 51–54 (2003). 21. S. L. Voronov, I. Kohl, J. B. Madsen, J. Simmons, N. Terry, J. Titensor, Q. Wang, and J. Peatross, “Control of laser high-harmonic generation with counterpropagating light,” Phys. Rev. Lett. 87, 1339021 (2001). 22. X. Zhang, A. L. Lytle, T. Popmintchev, X. Zhou, H. C. Kapteyn, M. M. Murnane, and O. Cohen “Quasi-phasematching and quantum-path control of high-harmonic generation using counterpropagating light,” Nature Phys. 3, 270–275 (2007). 23. J. Herrmann, ”Apparatus and method for ampliﬁcation and frequency transformation of laser radiation using quasi-phase matching of four-wave mixing,” Patent: DE102009028819A1 (2011). 24. T. G. Leighton, “What is ultrasound?” Prog. in Bioph. and Molec. Biol. 93, 3–83 (2007). 25. A. Holm and H. W. Persson, “Optical diffraction tomography applied to airborne ultrasound,” Ultrasonics 31, 259–265 (1993). 26. J. A. Gallego-Juarez and L. Gaete-Garreton, “Experimental study of nonlineairity in free progressive acoustic waves in air at 20 kHz,” J. De Phys. 40, C8-336–340 (1979). 27. H. Tijdeman, “On the propagation of sound wave in cylindrical tubes,” J. Sound Vibr. 39, 1–13 (1975). 28. E. Rodarte, G. Singh, N. R. Miller, and P. Hrnjak, “Sound attenuation in tubes due to visco-thermal effects,” J. Sound Vibr. 231, 1221–1241 (2000). 29. T. D. Rossing, Handbook of Acoustics (Springer, 2007). 30. M. Iskhakovich, General Acustics (Nauka, Moscow, 1973). 31. L. J. Bond, C. Chiang, and C. M. Fortunko, “Absorption of ultrasonic waves in air at high frequencies (10-20 MHz),” J. Acoust. Soc. Am. 92, 2006–2015 (1992). 32. Z. Song, Y. Qin, G. Zhang, S. Cao, D. Pang, L. Chai, Q. Wanga, Z. Wangb, and Z. Zhang, “Femtosecond pulse propagation in temperature controlled gas-ﬁlled hollow ﬁber,” Opt. Commun. 281, 4109–4113 (2008). 33. M. Mlenjnek, E. M. Wright, and J. V. Moloney, “Femtosecond pulse propagation in argon: A pressure dependence study,” Phys. Rev. Lett. 58, 4903–4910 (1998). 34. A. Couairon, M. Franco, G. Mechain, T. Olivier, B. Prade, and A. Mysyrowicz, “Femtosecond ﬁlamentation in air at low pressures: Part I: Theory and numerical simulations,” Opt. Commun. 58, 265–273 (2006). 35. G. P. Agrawal, Nonlinear Fiber Optics (Academic press, USA, 2001). 36. A. Couairon and A. Mysyrowiczb, “Femtosecond ﬁlamentation in transparent media,” Phys. Repor. 441, 47-189 (2007).1.IntroductionThird harmonic generation (THG) in gases is a method with one of the simplest setup allowing to generate picosecond and femtosecond pulses in the UV and VUV spectral ranges. Compared with alternative schemes employing solid-state crystals several disadvantages can be avoided by using gases such as low damage threshold, strong dispersion, bandwidth limitations and restrictions of the spectra to the range above 200 nm. For intensities below the ionization threshold THG cannot take place for pump beams focused into a gas cell because the emitted third harmonic (TH) before the focus cancels the one emitted after the focus [1, 2] due to destructive interference. This problem can be avoided by placing the gas cell before or after the focus [1,2], by using a differentially pumped gas cell, a narrow gas jet [3], or hollow waveguides [4, 5]. THG during ultrafast ionization with higher efﬁciencies has been generated by focusing the#173228 - $15.00 USD Received 24 Jul 2012; revised 6 Sep 2012; accepted 6 Sep 2012; published 19 Sep 2012(C) 2012 OSA24 September 2012 / Vol. 20, No. 20 / OPTICS EXPRESS 22754pump beam inside a chamber with a noble gas with intensities far in excess of that necessary for ionization [6–8]. TH has also been generated during ﬁlamentation of femtosecond pulses in air and argon [9–14]. Several papers reported that the efﬁciency of THG in a ﬁlament can be increased by using a second intercepting IR pulse [11–14], which can be explained by the quenching of interference effects [14]. One of the main difﬁculties, limiting the efﬁciency in frequency conversion in the above described methods is the problem to realize phase-matching. In UV and VUV pulse generation in hollow waveguides by four-wave mixing phase-matching can be realized using the anomalous dispersion of the ﬁber [15–17]. However, the small diameter of the capillary limits the pulse energy and leads to more complexity in practical realization. In the case of frequency conversion in solid nonlinear crystals an alternative approach, quasi-phase matching (QPM), is used. It exploits a periodic modulation of the nonlinear susceptibility to correct the linear phase mismatch [18, 19]. For the application in high-order harmonic generation in gas-ﬁlled hollow waveguides QPM was demonstrated by using modulated hollow-core waveguides [20] or using counter-propagating light [21, 22]. In this paper, we investigate a novel technic for quasi-phase matching by ultrasound in THG. As presented in Fig. 1 in this scheme a cell ﬁlled with a noble gas is excited by a pump pulse and an ultrasound wave which modulates the gas pressure and thus the third order nonlinearity of the gas. For an ultrasound wave-number approximately equal to the module of the linear phase mismatch between the fundamental and the generated TH QPM can be realized, greatly increasing the conversion efﬁciency. Since the beam diameter is not limited as in hollow waveguides, this method allows the use of very high pump energies with larger diameters for frequency transformation into the VUV. Here we study THG in an argon-ﬁlled cell with the pump wavelength at 800 nm for UV and at 400 nm for VUV pulses generation with moderate and high pump energies up to the joule level. Using a comprehensive nonlinear model including the inﬂuence of dispersion, diffraction, third-order nonlinearity, ionization as well as the sound loss, we show that the conversion efﬁciency can be increased as a result of QPM. In particular, we show the possibility that by using pre-chirped femtosecond pump pulses at 400 nm sub-20 fs VUV pulses can be generated. Intriguingly, due to the normal dispersion of the gas the generated VUV pulse at the output is nearly unchirped and thus does not need any additional chirp compensation stage.(a)1.2 1(b)0.3 0.25(c)Ωs [MHz]] β [cm s400 600 800 1000 12000.8 0.6 0.4 0.2 0−10.2 0.15 0.10.05 0λ [nm]0.2 0.4 0.6 0.8Ω [MHz]11.2Fig. 1. QPM for THG using an ultrasound wave. (a) - The basic scheme: the pump with frequency ω is sent into a tube where an ultrasound wave is exited to achieve the QPM. At the output, third harmonic 3ω is observed. (b) - Dependence of the ultrasound frequency Ωs on the pump wavelength λ for THG QPM in argon at 1 atm. (c) - The absorption rate of the ultrasound wave in argon at the normal conditions in dependence on its frequency Ωs .#173228 - $15.00 USDReceived 24 Jul 2012; revised 6 Sep 2012; accepted 6 Sep 2012; published 19 Sep 2012(C) 2012 OSA24 September 2012 / Vol. 20, No. 20 / OPTICS EXPRESS 227552.Quasi-phase matching using ultrasoundQPM is a technic to correct the phase mismatch between interacting waves without matching the phase velocities. QPM in periodically poled nonlinear crystals is realized by a nonlinear structure in which the sign of the nonlinear susceptibility is periodically reversed throughout the medium [18, 19]. However, QPM can also be realized by a periodic modiﬁcation of the nonlinear coefﬁcient without sign change. A method for QPM in isotropic gases was described in [23], in which an ultrasound transducer in a gas cell excites ultrasound waves which periodically change the pressure or, in other words, the particles number density and therefore the third-order nonlinear susceptibility. The ultrasound wavevector Ks required to achieve QPM of interacting waves in the cell is approximately equal to the phase-mismatch |Δk| due to dispersion of the corresponding waves. In this paper we study in detail THG as a simplest prototypical interaction (see Fig. 1). In this case the phase mismatch is given by Δk = k3ω − 3kω , where kω is the pump wavevector with the frequency ω and k3ω is the TH wavevector with the frequency 3ω . The dependence of the ultrasound frequency Ωs = 2π /Ks on the pump wavelength λ = 2π /ω is shown in Fig. 1(b). As seen for argon QPM for THG with a pump pulse at 800 nm requires an ultrasound frequency of Ωs ≈ 22 kHz and for a pump pulse at 400 nm we ﬁnd Ωs ≈ 300 kHz. For high intensities the Kerr effect introduces an additional phase shift. Ultrasound waves can be efﬁciently generated by the application of piezoelectric generators. In liquids, high power ultrasound waves up to tens of MHz are heavily used in many applications in technology, medicine, biology and chemistry [24]. In contrast, in gases typical highpower ultrasound devices rarely exceed ∼ 40 kHz limit. However, some ultrasonic applications such as nondestructive testing require airborn ultrasound from 100 kHz up to 1 MHz. In such applications, the pressure amplitudes p in a pulsed regime can achieve ∼ 10−2 atm inside the area of ∼ 1 cm2 [25]. Even using relatively common airborn high-power ultrasound source with frequency around 20 kHz, higher frequencies can be obtained as harmonics of the fundamental one. In particular, in [26], high power (200 W) cw ultrasound with high pressure level ≈ 160 dB (this corresponds to p ∼ 10−2 atm) was excited in air at atmospheric pressure for a transducer with resonant frequency Ωs = 20 kHz. Due to nonlinearities of generation and propagation, the ultrasound contained higher harmonics; in particular, the contribution of fourth-order harmonic (Ωs = 80 kHz) in this wave was estimated to be up to ≈ 130 dB ( p ≈ 5 × 10−4 atm). The propagation of sound wave in a tube is relatively well studied [27, 28]. If the viscous effects from the walls can be neglected (the case of “wide tube”), the fundamental sonic mode in a waveguide is homogeneous across the direction transverse to the propagation one. Such mode have also a free-space dispersion and negligible waveguide-induced losses. In the case when viscosity effects becomes important (the case of “narrow tube”), the situation changes. Viscosity leads to the occurring of a boundary layer, with pressure variations decreasing to zero at the walls, and also to additional losses. The role of viscosity is determined by the soΩs called shear wave number s = D 2 ν [27, 28] where D is the tube diameter, ν is the kinematic viscosity. Viscosity effects become important for s ≤ 1. For typical parameters considered in the present article (atmospheric pressure, Ωs = 0.3 MHz) and assuming reasonable tube diameter of ≈ 1 cm ( [25]) we obtain s ∼ 3 × 103 , that is the approximation of “wide tube” is very well applicable. For the above mentioned parameters, the losses induced by the tube itself are of order of 5 × 10−2 cm−1 , according the model introduced in [28]. Even in a free space the high frequency ultrasound has noticeable decay, which is in the case of noble gas can be described by the formula [29, 30]:βs =Ω2 s 8π 2 ρ0 c3 s4 κ η + (γ − 1) 3 Cp,(1)where ρ0 is the gas atomic density, cs is the sound speed, κ is the thermal conductivity, η is the#173228 - $15.00 USD Received 24 Jul 2012; revised 6 Sep 2012; accepted 6 Sep 2012; published 19 Sep 2012(C) 2012 OSA24 September 2012 / Vol. 20, No. 20 / OPTICS EXPRESS 22756shear viscosity, γ = Cp /Cv is the rate of the heat capacity at constant pressure (Cp ) and constant volume (Cv ). The dependence of the loss coefﬁcient βs on the ultrasound frequency Ωs for argon is shown in Fig. 1(c). Although the losses given by the classical formula Eq. (1) are underestimated for liquids and multi-atomic gases, Eq. (1) works still reasonably good for noble gases [29–31]. According to the facts mentioned above, the gas pressure can be described in a simple way as a decaying plane wave along the propagation axis z: P(x, y, z) = Po + p(x, y)e−βs z cos(KS z). Here Po is the background pressure and p(x, y) is the ultrasound amplitude, which is constant inside the tube ( x2 + y2 ≡ r < D/2) and zero otherwise (the “wide tube” approximation discussed above) that is, p(x, y) ≡ p = const. The propagation of the pump and TH in the gas-ﬁlled tube excited by the ultrasound wave can be described by a comprehensive model taking into account diffraction, dispersion, third-order nonlinearity, gas ionization effects and periodically changing pressure. Using the slowly varying envelope approximation the amplitudes of the pump Aω (x, y, z, t ) and the TH A3ω (x, y, z, t ) as well as the free electron density ρ (x, y, z, t ) in time t and space are given by:∂ Aω 2 iΔkz + Mω Aω = iγω (z) Aω |Aω |2 + 2 |A3ω |2 − Γω + A∗ , (2) ω A3ω e ∂z ∂ A3ω −iΔkz + M3ω A3ω = iγ3ω (z) A3ω |A3ω |2 + 2 |Aω |2 − Γ3ω + A3 /3 , (3) ωe ∂z ∂ρ ρ = (ρo (z) − ρ ) σKω |Aω |2Kω + σK3ω |A3ω | 2K3ω + σω (z) |Aω |2 + σ3ω (z) |A3ω |2 . ∂t Ui (4)Here γm (z) = 3π m2 χ (3) (z) is the third order nonlinear coefﬁcient (m = ω and 3ω for the fundao 2c2 km mental and TH, correspondingly); χ (3) (z) is the third-order nonlinear susceptibility, depending on the pressure P (and hence on z); Δk is the linear phase mismatch for the gas pressure P0 . Mω , M3ω and Γω , Γ3ω are deﬁned as:o ) + νm (z) Mm = −i (km (z) − kmigm (z) ∂ 2 i ∂ ∂2 ∂2 + − + 2 , 2 2 ∂t 2 ∂ t 2km (z) ∂ x ∂y β σm (z) K o (1 + iωm Γm = τc (z)) + m (ρo (z) − ρ ) |Am |2Km −2 , 2 2(5) (6)where νω = 0 and ν3ω (z) = 1/Vω (z) − 1/V3ω (z); Vm (z) and gm (z) are the group velocities and o are the wave numbers for the group velocity dispersions of the interacting pulses; km (z) and km (z-dependent) pressure P and for the background pressure P0 , respectively; σm (z) is the cross section of inverse Bremsstrahlung; τc (z) is the free-carrier collision time; σKm is the ionization cross section, where Km ≡< Ui /h ¯ ωm + 1 > here also Ui is the ionization potential of the gas; βKm is the multiphoton ionization coefﬁcient, which is deﬁned as βKm = Km h ¯ ωm σKm ; ρo (z) is the density of neutral atoms [32, 33]. The coefﬁcients km (z), γm (z), Vm (z), gm (z), σm (z), τc (z), are assumed here to be proportional to the pressure (and thus varying along the z-coordinate) [32–34]. Let us ﬁrst consider the simplest approximation assuming an un-depleted pump pulse and neglecting diffraction, dispersion, ionization (Γm = 0) and loss, but taking into account the nonlinear self-phase modulation of the pump. Then, the on-axis QPM condition for the ultrasound wave vector Ks is: 2 o 2 (7) KS = Δk + 3γω |Ao ω | − 2γ3ω |Aω |#173228 - $15.00 USDReceived 24 Jul 2012; revised 6 Sep 2012; accepted 6 Sep 2012; published 19 Sep 2012(C) 2012 OSA24 September 2012 / Vol. 20, No. 20 / OPTICS EXPRESS 22757where Ao ω is the ﬁeld amplitude of the pump at the input. If this condition is fulﬁlled and the weak non-phase matched contributions are neglected one obtains for the intensity of the third harmonic I3ω : cεo (γ3ω pz)2 o 6 |Aω | . (8) I3ω (z) ≈ 12 Remarkably, I3ω does not depend on the background pressure P0 but only on the ultrasound amplitude p. For a more exact treatment we solved Eqs.(2) − (4) numerically using the split-step method [35] with the fast Fourier transform in time and 2D transverse space dimensions to calculate the linear part of the equations and the ﬁfth-order Runge-Kutta method for the nonlinear one. In the solution of Eq. (4) the fourth-order Runge-Kutta method were used. The results of numerical simulations of Eqs. 2-4 for bandwidth-limited femtosecond and chirped picosecond pump pulses at 800 nm as well as at 400 nm are presented in the Chapter 3. 3. 3.1. Results and their discussion UV pulse generation by using 800 nm pump pulsesFirst, we studied the proposed method for a bandwidth-limited 800 nm Gaussian pump pulse with a duration (FWHM) τω = 700 fs, a radius rω = 0.05 cm and an energy 3 mJ (corresponding to the input intensity Io ≈ 1 TW/cm2 and power Pω ≈ 3.97 GW) with a sound amplitude of p = 0.01 atm. The required ultrasound frequency necessary to fulﬁll the QPM condition according Eq. (7) is Ωs = 22.24 kHz. For these parameters the self-focusing distance is z f ≈ 11.01 m, the critical power of self-focusing is Pcrit ≈ 3.94 GW and the walk-off length is Lν = τω /ν ≈ 345 m. Therefore one can expect a relatively long propagation distance without beam collapsing, temporal walk-off or formation of a ﬁlament [36]. The results for these parameters are presented in Fig. 2. Figure 2(a) shows the results of the analytical formula Eq. (8) (red dashed curve) and of the numerical simulations (red solid curve) for the efﬁciency of THG deﬁned as η3ω (z) = |A3ω (z, x, y, t )|2 dxdydt / |Aω (z = 0, x, y, t )|2 dxdydt . One can see from the Fig. 2(a) that QPM results in the efﬁcient conversion to the TH at the optimum ultrasound frequency, which is 27 times larger than without ultrasound (green curve). The self-focusing effect is relatively weak as seen from the evolution of pump intensity (blue curve in Fig. 2(b)) and beam radius (red curve in Fig. 2(d)), while that spatial proﬁle of the TH shows a good beam quality (Fig. 2(c)).0.02 0.015 (a) 0.01 0.008 (b) 2 1.8 1.6 1.4 1.2 100 200 z [cm] 1 3 x 10−3(c)0.05(d)I [TW/cm 2]η3ω[%]0.01 0.005 0 0r [cm]0.2 0 −0.2 y [mm]0.006 0.004 0.002I ω[TW/cm ][TW/cm 2]22 103ωI3ω0 0.2 0 x [mm] −0.2100 200 z [cm]3000 0−0.050100 200 z [cm]Fig. 2. THG for a 0.7 ps pump pulse at 800 nm with 3 mJ energy. In (a) the conversion efﬁciency η3ω calculated analytically by Eq. (8) (red-dashed) and numerically (red-solid) in dependence on the propagation distance z (the result in the absence of ultrasound is shown by the green curve); in (b) the evolution of the peak intensity of the pump (blue) and the TH (red); in (c) the spatial intensity proﬁle of the TH at the output and in (d) the change of the radii of the pump (red) and the TH (blue) are presented.Figure 3 shows the results for the case of a bandwidth-limited pump pulse at 800 nm with a high energy of 1 J, a duration of τω = 1 ps (transform-limited), a radius of rω = 0.5 cm and a#173228 - $15.00 USD Received 24 Jul 2012; revised 6 Sep 2012; accepted 6 Sep 2012; published 19 Sep 2012(C) 2012 OSA24 September 2012 / Vol. 20, No. 20 / OPTICS EXPRESS 227580.12 (a) 0.16 5(b)60 50 40 30 20 10 100 200 z [cm] 300 0 (c) 1.50.5(d)I 3ω[TW/cm ]η3ω[%]0.08 0.06 0.04 0.02 0 0 100 200 z [cm] 3004 3 2 1 0 0I ω[TW/cm ][TW/cm 2]22r [cm]0.5 0 −0.5 y [mm]1 0.5 0 0.5 0 −0.5 x [mm]0I3ω−0.50100 200 z [cm]Fig. 3. THG for a 1 ps pump pulse at 800 nm with 1 J energy. The description of (a)-(d) and the curves are analogous as Fig. 2.sound amplitude of p = 0.01 atm. For these parameters one can calculate: Io ≈ 2.4 TW/cm2 , z f ≈ 247 cm, Lν = τω /ν ≈ 487 cm. In this case the power of the pump pulse Pω ≈ 940 GW is much larger than the critical power of self-focusing Pcrit ≈ 3.94 GW, therefore as seen in Fig. 3(d) after a propagation distance of about 2.5 m the pump beam radius signiﬁcantly decreases and its intensity increases. The efﬁciency increases up to η3ω ≈ 0.12%, but due to the change of the pump intensity the QPM condition Eq. (7) is violated after this distance. Without ultrasound (green curve) the efﬁciency is at ∼ 2.5 m 18 times smaller, but after self-focusing distance it increases signiﬁcantly due to the increase of the pump intensity. 3.2. VUV pulse generation by using 400 nm pump pulsesUsing THG with pump pulses at 400 nm allows frequency transformation into the VUV spectral range at 133 nm. Nowadays, generation of such pump pulses with high energy by second harmonic generation in nonlinear crystals from near-infrared ones is a standard method. Here we study THG with a bandwidth-limited pump pulse at 400 nm with 0.3 mJ energy, duration of τω = 1.4 ps, beam radius of rω = 0.03 cm, pump intensity Io ≈ 1.4 TW/cm2 and sound amplitude of p = 0.01 atm. As one can see from the Fig. 4, the conversion efﬁciency increases up to the propagation length of about 50 cm. The saturation of THG is caused by the strong self-focusing effect with the formation of a ﬁlament. The strong increase of the pump intensity leads to the violation of the optimum QPM condition, which terminates the frequency conversion. Figure 4(b) and 4(d) illustrates the well known dynamics of the formation of a ﬁlament after approximately ∼ 50 cm propagation. As seen the combined action of the optical Kerr effect, multiphoton absorption and ionization leads to focusing and defocusing cycles with very small quasi-periodicity [36]. This highly dynamic process leads to aperiodic spikes in the free electron density (Fig. 4.(c)) and recurrent, aperiodic intensity variations (Fig. 4(b)) of the fundamental (blue curve) and the TH (red curve). The resolution in the numerical simulation of Fig. 4. is about 0.2 mm. Next, we investigate TGH with a higher pump energy of 0.1 J with the duration of τω = 1 ps, radius rω = 0.1 cm (Io ≈ 6 TW/cm2 ) and the sound wave amplitude p = 0.01 atm. As can be seen from the Fig. 5. the conversion efﬁciency of THG up to ≈ 0.02% can be obtained. However, in this case a multiﬁlamentation takes place. It appears because the input peak power (Pω ≈ 93 GW) is much larger than the critical power (Pcrit ≈ 0.9 GW) [36]. 3.3. Sub-20 fs VUV pulse generation by using chirped 400 nm pump pulsesThe method under consideration can be combined with a stretching of the pump pulses to much longer durations. This allows to reduce the peak intensity to a range where deleterious nonlinear effects do not play a role. Using negatively chirped pump pulses the generated chirp of the TH can be compensated by normal dispersive elements. In the simulation shown in Fig. 6 and Fig. 7 we assume a negatively chirped pump pulse with a duration of τω = 1 ps and 0.3 mJ energy#173228 - $15.00 USDReceived 24 Jul 2012; revised 6 Sep 2012; accepted 6 Sep 2012; published 19 Sep 2012(C) 2012 OSA24 September 2012 / Vol. 20, No. 20 / OPTICS EXPRESS 22759x 10 (a) 5 4η3 ω[%]−30.4 (b)100 2x 10 (c)17(d) 0.02[TW/cm 2 ]I ω[TW/cm ] −3 ρ [cm ]21.5 1r [cm]3 2 1 0 0 20 40 z [cm] 600.2500I3ω0.5 0 0 0 0 0 20 40 60−0.02 0 20 40 602040 z [cm]60z [cm]z [cm]Fig. 4. THG for a 1.4 ps pump pulse at 400 nm with 3 mJ energy. In (a) the conversion efﬁciency calculated analytically by Eq. (8) (dashed) and numerically (solid); in (b) the evolution of the peak intensity of the fundamental (blue) and the TH (red); in (c) the maximum electron density and in (d) the change of radii of the fundamental (red) and the TH (blue) are presented.Fig. 5. THG for a 1 ps pump pulse at 400 nm with 0.1 J energy. In (a) the conversion efﬁciency calculated analytically by Eq. (8) (dashed) and numerically (solid); in (b) the evolution of the peak intensity of the fundamental (blue) and the TH (red); in (c) the spatial intensity proﬁle of the TH at the output are presented.obtained after phase-modulation from a bandwidth-limited one of 20 fs duration. The beam radius is rω = 0.25 cm, the input intensity is Io ≈ 2.87 TW/cm2 and the sound amplitude is p = 0.01 atm. As one can see, the conversion efﬁciency is limited to the same level as in the previous example. However, the duration of both pump and TH pulses decrease signiﬁcantly because of chirp compensation due to propagation in the normal-dispersive argon gas (Fig. 7(a)). The duration of the generated VUV pulse at 133 nm is reduced down to 18 fs (Fig. 7(c)) and its pulse energy is ∼ 3 μ J. This self-compression is caused by a chirp compensation during propagation due to normal dispersion of the gas.x 10 (a)−34η3 ω[%]3 2 1 0 0 10z [cm] 20 300.16 (b) 0.14 0.12 0.1 0.08 0.06 0.04 0.02 0 010z [cm]203080 70 60 50 40 30 20 10 0(c) 0.12 0.1 0.08 0.06 0.04 0.02 0.05 0 x [mm]−0.05 0 −0.05 y [mm][TW/cm 2 ]0.02 0.01r [cm](d)[TW/cm 2 ]I ω[TW/cm 2 ]03ω3ω−0.01 −0.02 0.05 0 z [cm]20IFig. 6. THG for a negatively chirped pump with the energy of 3 mJ. (a) The THG efﬁciency η3ω in dependence on z; (b) Peak intensity of the fundamental (blue) and the TH (red) pulses versus z; (c) Spatial proﬁle of the TH at the output; (d) Change of radii of the fundamental (red) and TH (blue) with z.#173228 - $15.00 USDReceived 24 Jul 2012; revised 6 Sep 2012; accepted 6 Sep 2012; published 19 Sep 2012(C) 2012 OSA24 September 2012 / Vol. 20, No. 20 / OPTICS EXPRESS 22760I。

Script One-Step qRT-PCR Kit 产品说明书

95057 / IFU-034.1 REV 021Script ® One-Step qRT-PCR KitCat. No 95057-050 Size: 50 x 50-µL reactions Store at -25°C to -15°C95057-200200 x 50-µL reactionsDescriptionThe qScript One-Step qRT-PCR Kit is a convenient and highly sensitive solution for reverse transcription quantitative PCR (RT-qPCR) of RNA templates using hybridization probe detection chemistries such as TaqMan ® 5’-hydrolysis probes or molecular beacons on real-time quantitative PCR systems that do not require an internal reference dye. cDNA synthesis and PCR amplification are carried out in the same tube without opening between procedures. The system has been optimized to deliver maximum RT-PCR efficiency, sensitivity, and specificity, enabling unbiased co-amplification of low copy transcripts in the presence of higher copy reference genes. The proprietary reaction buffer has been specifically formulated to maximize activities of both reverse transcriptase and Taq DNA polymerase while minimizing the potential for primer-dimer and other non-specific PCR artifacts. Highly specific amplification is crucial to successful qRT-PCR as non-specific product(s) can compete for amplification of the target sequence and impair PCR efficiency. A key component of this kit is AccuStart Taq DNA polymerase, which contains monoclonal antibodies that bind to the polymerase and keep it inactive prior to the initial PCR denaturation step. Upon heat activation at 95°C, the antibodies denature irreversibly, releasing fully active, unmodified Taq DNA polymerase. Instrument CompatibilityDifferent real-time PCR systems employ different strategies for the normalization of fluorescent signals and correction of well-to-well optical variations. It is critical to match the appropriate qPCR reagent to your specific instrument. The qScript One-Step qRT-PCR Kit does not contain an internal reference dye. Please consult the following table, or visit our web site at to find an optimized kit for your instrument platform(s).ReagentCat NosCompatible Real-Time PCR SystemsqScript One-Step qRT-PCR Kit, ROX 95058-050 95058-200 Applied Biosystems 7000, 7300, 7700, 7900, 7900HT, 7900HT Fast, StepOne™, StepOnePlus™ qScript One-Step qRT-PCR Kit, Low ROX 95059-050 95059-200 Applied Biosystems 7500, 7500 Fast, ViiA™ 7 Stratagene MX4000™, MX3005P™, MX3000P™qScript One-Step qRT-PCR Kit95057-050 95057-200Bio-Rad CFX96™, CFX384™,iCycler iQ ®, iQ™5, MyiQ™ Opticon™, MiniOpticon™, Chromo4™Cepheid Smart Cycler ®; Qiagen/Corbett Rotor-Gene ® Eppendorf Mastercycler ® ep realplex Roche Applied Science LightCycler ® 480Components ReagentDescriptionqScript One-Step Reverse Transcriptase Optimized 50X formulation of recombinant MMLV reverse transcriptase for one-step RT-PCR.One-Step Master Mix (2X) 2X reaction buffer containing dNTPs, magnesium chloride, AccuStart Taq DNA polymerase, and stabilizers Nuclease-free waterStorage and StabilityStore components in a constant temperature freezer at -25°C to -15°C upon receipt. Repeated freezing and thawing of the reaction mix is not recommended.For lot specific expiry date, refer to package label, Certificate of Analysis or Product Specification Form.95057 / IFU-034.1 REV 022Guidelines for One-Step qRT-PCR Thaw all components, except qScript One-Step RT, at room temperature. Mix vigorously, and then centrifuge to collect contents to thebottom of the tube before using. Place all components on ice after thawing. To maximize specificity, reactions should be assembled on ice. AccuStart Taq DNA polymerase is inactive prior to high temperatureactivation; however, qScript One-Step reverse transcriptase is active at lower temperatures. First-strand synthesis can be carried out between 42°C and 52°C. Optimal results are generally obtained with a 10-minute incubation at 48 – 50°C. We recommend a 5 minute incubation at 95°C to fully inactivate the RT prior to PCR cycling. Preparation of a reaction cocktail is recommended to reduce pipetting errors and maximize assay precision. Assemble the reactioncocktail with all required components except RNA template and dispense equal aliquots into each reaction tube. Add RNA to each reaction as the final step. Addition of sample as 5 to 10-µL volumes will improve assay precision. Suggested input quantities of template are: 1 pg to 1 µg total RNA; 10 fg to 100 ng poly A(+) RNA; 10 to 1x108 copies viral RNA. After sealing each reaction, vortex gently to mix contents. Centrifuge briefly to collect components at the bottom of the reaction tube.Reaction Assembly Component Volume for 50-µL rxn. Final Concentration One-Step Master Mix (2X) 25 µl 1X Forward primer Variable 400 – 900 nM Reverse primer Variable 400 – 900 nM Probe Variable 50-200 nM Nuclease-free water Variable RNA template 5 – 10 µL VariableqScript One-Step RT 1 µL1X Final Volume (µL) 50 µLNote : For smaller reaction volumes (i.e. 25-µL reactions), scale all components proportionally. Reaction ProtocolIncubate complete reaction mix in a real-time thermal detection system as follows: cDNA Synthesis48 – 50°C, 10 min Initial denaturation95°C, 5 min PCR cycling (30 - 45 cycles) 95°C, 10 to 15s55 – 60°C, 30 to 60s (data collection step)Quality ControlKit components are free of contaminating DNase and RNase. The qScript One-Step qRT-PCR Kit is functionally tested in RT-qPCR. Kinetic analysis must demonstrate linear resolution over six orders of dynamic range (r 2 > 0.995) and a PCR efficiency > 90%Limited Label LicensesUse of this product signifies the agreement of any purchaser or user of the product to the following terms:1. The product may be used solely in accordance with the protocols provided with the product and this manual and for use with components contained in the kitonly. QIAGEN Beverly, Inc. grants no license under any of its intellectual property to use or incorporate the enclosed components of this kit with any components not included within this kit except as described in the protocols provided with the product, this manual, and additional protocols available at . Some of these additional protocols have been provided by Quantabio product users. These protocols have not been thoroughly tested or optimized by QIAGEN Beverly, Inc.. QIAGEN Beverly, Inc. neither guarantees them nor warrants that they do not infringe the rights of third-parties.2. Other than expressly stated licenses, QIAGEN Beverly, Inc. makes no warranty that this kit and/or its use(s) do not infringe the rights of third-parties.3. This kit and its components are licensed for one-time use and may not be reused, refurbished, or resold.4. QIAGEN Beverly, Inc. specifically disclaims any other licenses, expressed or implied other than those expressly stated.5. The purchaser and user of the kit agree not to take or permit anyone else to take any steps that could lead to or facilitate any acts prohibited above. QIAGEN Beverly, Inc. may enforce the prohibitions of this Limited License Agreement in any Court, and shall recover all its investigative and Court costs, including attorney fees, in any action to enforce this Limited License Agreement or any of its intellectual property rights relating to the kit and/or its components.©2018 QIAGEN Beverly Inc. 100 Cummings Center Suite 407J Beverly, MA 01915 Quantabio brand products are manufactured by QIAGEN, Beverly Inc.Intended for molecular biology applications. This product is not intended for the diagnosis, prevention or treatment of a disease.qScriptis a registered trademark of QIAGEN Beverly, Inc.TaqMan is a registered trademark of Roche Molecular Systems, Inc. LightCycler is a registered Trademark of Roche. Applied Biosystems, StepOne, StepOnePlus, ViiA, and ROX are trademarks Life Technologies Corporation. Stratagene, MX3000P, MX3005P and MX4000 are trademarks of Agilent Technologies, Inc. Mastercycler is a trademark of Eppendorf. Rotor-Gene is a registered trademark of Qiagen GmbH. SmartCycler is a trademark of Cepheid. CFX96, CFX384, iCycler iQ, iQ5, MyiQ, Opticon, MiniOpticon and Chromo4 are trademarks of Bio-Rad Laboratories.。

Quanta Biosciences qScript XLT cDNA SuperMix 说明书

*Note: These conditions are provided as a general guideline and support maximum cDNA yield and sensitivity for global gene expression profiling. Specific applications may benefit from modified reaction conditions. qScript XLT can be used at temperatures up to 54°C for RNA with stable secondary structure(s). However, higher temperatures can compromise cDNA yield for other RNAs. Shorter incubation times (30 min) can be applied when using <500 ng of total RNA template without compromising cDNA yield. qScript ™ XLT cDNA SuperMixDescription qScript XLT cDNA SuperMix is a next-generation tool for first-strand cDNA synthesis, providing a highly sensitive and easy-to-use solution for two-step RT-PCR and RT-qPCR. qScript XLT is an engineered M-MLV reverse transcriptase with reduced RNase H activity and improved activity and stability at higher temperatures. Combined with a precise mixture of reaction components, this SuperMix enables superior results over a wide dynamic range of input RNA, with up to 8-fold higher sensitivity than cDNA synthesis kits utilizing an RNase H(+) reverse transcriptase (RT). This 5X concentrated master mix provides all necessary components (except RNA template) for first-strand synthesis including: buffer, dNTPs, MgCl 2, primers, RNase inhibitor protein, qScript XLT reverse transcriptase and stabilizers. The unique blend of oligo (dT) and random primers in the qScript XLT cDNA SuperMix captures unbiased representation of all RNA sequences into cDNA product (including the 5’-end, 3’-end, or central regions of long RNAs) and works exceptionally well with a wide variety of RNA templates. However, due to the relatively short average length of random-primed first-strand product, amplicon length for conventional RT-PCR applications should be limited to 1 kb or less.Components qScript XLT cDNA SuperMix 5X reaction buffer containing optimized concentrations of MgCl 2, dNTPs (dATP, dCTP, dGTP, dTTP), recombinant RNase inhibitor protein, qScript reverse transcriptase, random primers, oligo(dT) primer and stabilizers. Storage and Stability Store components in a constant temperature freezer at -25°C to -15°C upon receipt For lot specific expiry date, refer to package label, Certificate of Analysis or Product Specification Form.Reaction Assembly Place components on ice. Mix, and then briefly centrifuge to collect contents to the bottom of the tube before using.ComponentVolume for 20-μL rxn. Final Concentration qScript XLT cDNA SuperMix (5X)4 μL 1X RNA templatevariable (2 μg to 10 pg total RNA) RNase/DNase-free water variable Total Volume (μL)20 µL Note: for smaller reaction volumes (i.e. 10-μL reactions), scale components proportionally.Reaction Protocol•Combine reagents in 0.2-mL micro-tubes or 96-well plate sitting on ice. •After sealing each reaction, vortex gently to mix contents. Centrifuge briefly to collect components at the bottom of the reaction tube. • Incubate:5 minutes at 25ºC60 minutes at 42ºC*5 minutes at 85ºCHold at 4ºC • Use 1/10th (or less) of the first-strand product as template for PCR / qPCR amplification. The optimal amount of cDNA for PCR can vary depending on the amount of starting RNA template, choice of detection chemistry, and abundance of the specific target sequence. The high yield of cDNA resulting from reactions containing ≥ 500 ng of total RNA can overwhelm detection of specific product by SYBR® Green I qPCR. If required, dilute cDNA product with 10 mM Tris-HCl (pH 8.0), 0.1 mM EDTA. Store first-strand product at -20ºC.Cat No. 95161-025 Size: 25 x 20-µL reactions (1 x 100 µL) Store at -25ºC to - 15°C95161-100100 x 20-µL reactions (1 x 400 µL) 95161-500 500 x 20-µL reactions (2 x 1 mL)Guidelines for Reverse Transcription-qPCRMinus RT-controls: Accurate quantification of gene expression by RT-qPCR requires testing and reporting the extent of contamination of genomic DNA in each RNA sample for each gene of interest. The presence of trace amounts of gDNA does not usually interfere with quantification of high copy reference genes. However, it can have a significant contribution on signal for low copy genes. Even when using primers that are separated by intronic sequence or bridge exon junctions, the presence of genomic DNA can produce positive signals from amplification of pseudogene or off-target PCR product. Therefore, it is important to always include the appropriate “no RT” or “minus RT” control reactions in your experimental design.Since the reverse transcriptase is an integral component of qScript XLT cDNA SuperMix, it is not feasible to construct a formal cDNA synthesis control that includes all components except the RT. The most direct method to test for the presence of genomic DNA is to bypass the RT step and use an equivalent amount of the RNA preparation directly for PCR amplification. For example: if you start with1 µg of total RNA for cDNA synthesis and use 1/10th of the first-strand reaction as template for qPCR; then use 100 ng of total RNA as template for the minus RT-control qPCR. Any signal from the RNA only reaction is attributable to the presence of genomic DNA. DNase digestion of total RNA: Trace levels of genomic DNA can obscure accurate quantification, particularly when the specific gene(s) of interest are low copy. PerfeC T a® DNase I is a high purity, recombinant DNase I preparation that is free of any contaminating RNases. It provides a simple and rapid solution to eliminate residual genomic DNA that is directly compatible with qScript XLT cDNA SuperMix, or other first-strand synthesis kits. The supplied Reaction Buffer and proprietary Stop Buffer support a simple heat-kill step that permanently inactivates all trace levels of DNase activity before the cDNA synthesis step. Heat-kill procedures used by other DNase I reagents are ineffective and not compatible with qScript XLT cDNA SuperMix. Residual, or renatured, DNase will degrade cDNA product and alter apparent expression levels. If using other sources of RNase-free DNase I, it is essential to remove all traces of DNase activity before proceeding with first-strand synthesis. Suitable RNA purification methods include phenol:chloroform extraction followed by ethanol precipitation, or the use of chaotropic salts and a silica-based RNA purification cartridge or column. Please call technical support at (800) 364-2149 or visit our web site at if you require additional information or protocols.Quality ControlKit components are free of contaminating DNase and RNase. qScript XLT cDNA SuperMix is functionally tested in reverse transcription quantitative PCR (RT-qPCR). First-strand synthesis is performed in triplicate on each dilution of a log-fold serial dilution of HeLa cell total RNA from 1 pg to 1 µg. One-tenth of each first-strand reaction is used for qPCR amplification. Kinetic analysis must demonstrate linear resolution over five orders of dynamic range (r2 > 0.990) and a PCR efficiency > 90%.Limited Label LicensesUse of this product signifies the agreement of any purchaser or user of the product to the following terms:1.The product may be used solely in accordance with the protocols provided with the product and this manual and for use with components contained in the kitonly. Quantabio, LLC. grants no license under any of its intellectual property to use or incorporate the enclosed components of this kit with any components not included within this kit except as described in the protocols provided with the product, this manual, and additional protocols available at . Some of these additional protocols have been provided by Quantabio product users. These protocols have not been thoroughly tested or optimized by Quantabio, LLC. Quantabio, LLC. neither guarantees them nor warrants that they do not infringe the rights of third-parties.2.Other than expressly stated licenses, Quantabio, LLC. makes no warranty that this kit and/or its use(s) do not infringe the rights of third-parties.3.This kit and its components are licensed for one-time use and may not be reused, refurbished, or resold.4.Quantabio, LLC. specifically disclaims any other licenses, expressed or implied other than those expressly stated.5.The purchaser and user of the kit agree not to take or permit anyone else to take any steps that could lead to or facilitate any acts prohibited above. Quantabio,LLC. may enforce the prohibitions of this Limited License Agreement in any Court, and shall recover all its investigative and Court costs, including attorney fees, in any action to enforce this Limited License Agreement or any of its intellectual property rights relating to the kit and/or its components.This product is covered by US patent 7,470,515, US patent 7,638,612 and other patents pending in the United States and Europe. The purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer. The buyer is not authorized to sell or otherwise transfer this product, any of its components to a third party. The purchase of this product does not authorize the purchaser to use the product or any of its components for manufacture of commercial product. For information on obtaining a license to this product for purposes other than research, contact Licensing Department, Quantabio, LLC. 100 Cummings Center Suite 407J Beverly, MA 01915; Telephone number: 1-888-959-5165.©2021 Quantabio, LLC. 100 Cummings Center Suite 407J Beverly, MA 01915.Quantabio products are manufactured in Beverly, Massachusetts, Frederick, Maryland and Hilden, Germany.Intended for molecular biology applications. This product is not intended for the diagnosis, prevention or treatment of a disease.qScript is a trademark of Quantabio, LLC. PerfeC T a is a registered Trademark of Quantabio, LLC. SYBR is a registered trademark of Molecular Probes, Inc.。

Agilent Brilliant III QPCR StepOne Plus QRG 说明书

Stratagene Products 1Brilliant III Ultra-Fast QPCR Master MixQuick Reference Guide for the ABI StepOnePlusReal-Time PCR SystemAgilent Technologies This quick reference guide provides an optimized protocol for usingthe Stratagene Brilliant III Ultra-Fast QPCR Master Mix with theStepOnePlus Real-Tim e PCR System from Applied Biosystem s. Fordetailed instructions, refer to the full product m anual.Prepare theReactions 1Dilute the reference dye 1:50 using nuclease-free PCR-g rade water.2Prepare the experimental reactions by combining the components of thereag ent mixture in the order listed in the table below. Prepare a sing lereag ent mixture for replicate reactions (plus at least one reactionvolume excess) using multiples of each component.3Gently mix the reag ent mixture without creating bubbles, thendistribute the mixture to the experimental reaction tubes.4Add x μl of experimental DNA to each reaction to bring the finalreaction volume to 20 μl. The table below lists a sug g ested quantityrang e for different DNA templates.*Refers to RNA input amount during cDNA synthesis5Mix the reactions without creating bubbles, then centrifug e briefly.Reagent Mixture Nuclease-free PCR-grade water to bring final volume to 20 μl (including DNA)10 μl of 2× QPCR Master Mix x μl of experimental probe at optimized concentration (150–600 nM)x μl of upstream primer at optimized concentration (200–600 nM)x μl of downstream primer at optimized concentration (200–600 nM)0.3 μl of diluted reference dyeDNAQuantity per reaction Genomic DNA5 pg – 100 ng cDNA 0.1 pg – 100 ng*2Brilliant III Ultra-Fast QPCR Master Mix Set Up theQPCR Plate andThermal Profile 1From the Home screen of the StepOnePlus software, click Advanced Setup .2Complete the Setup screens for a new experiment as needed.On the Experiment Properties screen, select TaqMan Reagents and theFast ram p speed.On the Run Method screen, set the reaction volum e to 20 μl and adjustthe therm al profile according to the im age below .Run the PCRProgram 1Place the reactions in the StepOnePlus instrument.2On the Run screen, click START RUN .Analyze Data 1Analyze the results of the run as needed for your experiment.Notice to Purchaser Practice of the patented 5´ Nuclease Process requires a license from Applied Biosystems. The purchase of this product includes an immunity from suit under patents specified in the product insert to use only the amount purchased for the purchaser’s own internal research when used with the separate purchase of Licensed Probe. No other patent rights are conveyed expressly, by implication, or by estoppel. Further information on purchasing licenses may be obtained from the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.Manual Part Number 5990-3054, Revision A©Agilent Technologies, Inc. 2010Product InformationCatalog #600880, 400 reactionsCatalog #600881 4000 reactions Ordering Information By phone (US only*): 800-424-5444, x3On the web: Technical Services By phone (US only*): 800-894-1304, x2Byemail:*************************For other countries, please contact your local sales representative at /chem/contactus。

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Ultrafast Header Processing in All-OpticalPacket Switched-NetworkHoa Le Minh, Student Member,IEEE, Z. Ghassemlooy, Senior Member, IEEE,and Wai Pang Ng., Member, IEEEOptical Communications Research GroupSchool of Informatics, Engineering and TechnologyNorthumbria University, Newcastle upon Tyne, NE1 8ST, UKEmail: h.le-minh@, fary.ghassemlooy@, wai-pang.ng@Phone: +44 (0)191 227 4902, Fax: +44 (0)191 227 3684ABSTRACTHeader processing in all-optical domain in ultra-fast photonics network is challenging issue. In this paper, we present modelling and simulation of all-optical header processing based on pulse position modulation (PPM) format for 100Gbit/s packet-switched network composed up to 256 nodes. Both the header address codeword (8-bit) and the routing table are converted into PPM format, where the later offers reduced entries compared with the existing routing table. Simulation results show that matching of 256 8-bit header address with routing table entries can be performed effectively and as fast as possible using all optical AND gates.Keywords: Codeword processing, routing table, pulse-position1. INTRODUCTIONIn all-optical packed-switched network, packet header processing at node level is a challenging task especially when it is carried out in all-optical domain. This is due to the complexity and unavailability, at present, of powerful all-optical devices such as optical memory, optical logic gates array etc. A number header processing techniques have been proposed based on all-optical correlators [1, 2], optical serial-to-parallel conversion combined with CMOS processor [3], optical label switching [4-6], all-optical logic circuits [7]. Most of these schemes can help to resolve the data packet address matching problem when the network size is small (i.e. small size routing table). For a larger size network, with a few hundred nodes and each node having a large routing table with hundreds entries, the task of matching the data packet address with routing table with large entries become a challenging one. Header processing schemes employing all-optical logic gates are based on active nonlinear device such as semiconductor optical amplifier (SOA) [8, 9], which has long recovery time (~ 1 ns) after each comparison. Therefore to carry out a large number of comparisons in optical domain with a minimum processing time, one needs either to employ a considerable number of gates or a small number of all-optical logic gates to perform sequential comparison. However, it is un-feasible in the current real systems.In this paper, we propose an ultra-fast 8-bit header-codeword processor in an all-optical network with up to 256 nodes. The node has two output ports and its routing table has 256 entries. The packet header address and node routing table are converted into a pulse-position modulation (PPM) format, thus making address-matching task much simpler where a single comparison is required. The proposed PPM formatting scheme also makes to the network becoming more flexible.2. HEADER PROCESSINGFigure 1 show schematic block diagram a typical edge/core node (or router) with the proposed header processing units. In Fig. 1, optical packet is applied to the clock and header extraction units via a 1×3 splitter. Symmetrical Mach-Zehnder (SMZ) switch reported in [10] has been used to extract both the clock and header information. The former is used to for node synchronisation, whereas the header information (8-bit address return-to-zero codeword) is fist converted into the pulse position modulation format before being compared with the data in the routing table. In PPM format, a frame of fixed length with 256 slots is generated where a single short pulse is located in one of 256 slots (frame C). The position of the pulse corresponds to the decimal value of the address codeword. For example for codeword of “10111010” with decimal weight of 186, a pulse is located at slot 186. The routing table is also constructed based on the PPM format, in this case with only two entries corresponding to two output ports of the node. Each routing entries consists of a frame of one or up to 255 pulses (frames E1 and E2 for entry 1 and 2, respectively) corresponding to the number of address patterns required for switching packet to a particular output port. Thus using just two routing entries it is possible to carry out 256-addressmatching, which considerably less than the existing schemes where 256 routing entries. The matching process is simply carried out by using a pair of all-optical SMZ-based AND gates by comparing the frame C (i.e. the control input) with routing table entries (either frame E1 or E2 is input to SMZ). The AND gate functions as a switch when the control pulse (frame C) overlaps with one of slots in E1 or E2, thus switching one pulse, if it existed in E1 or E2, to the switched output. The output of the AND gate is used to control the main all-optical switch, where the delayed optical packet is forwarded to its intended destination. Packets could be deleted (i.e. not routed) when identical indexed slots in both routing table entries are filled by bit “0”. The node could be set to be in broadcast or multicast mode by ensuring that the matching bits appear in both identical indexed slots of E1 and E2 entries.DDFigure 1. All-optical packet header processor3. CODEWORD AND ROUTING TABLE IN PPM FORMATThe schematic diagram of PPM conversion unit for 8-bit address codeword is shown in Fig. 2. It consists of serial connection of eight 1×1 all-optical switching stages and a number of optical delay lines. The header address codeword C = [c7c6c5c4c3c2c1c0], where c0 is the least significant bit (LSB), is used as the control signal to enable switching of AND gates. A single optical pulse, generated by the clock recovery unit, is applied to the input of the first switch, emerging from one of the output ports depending on the state of address codeword, until it reaches the last stage. At the stage (i+1)th, if bit c i = 1, the optical pulse emerging from the upper output port of the switch will pass through an optical delay line with 2i×T s seconds delay, where T s is the slot duration. Otherwise, when c i = 0, the pulse is emerges from the lower output port before entering the next stage without any delay. Depending on the codeword combination, a single pulse with duration T s is finally located in an exact slot location within a PPM frame that corresponds to the codeword decimal weight.Figure 2. PPM conversion unit: 8-bit codeword to 256-slot frameIn general, slots in entries of the PPM based routing table (PPRT) could be populated by the pattern generators provided full synchronisation is established. If PPM based routing table has M entries, then it is necessary to have M pattern generators. For simplicity, the simulated PPM based routing table entries in this paper are composed by multiplexing different delayed pulses into two 256-slot frames.4. SIMULATION RESULTS AND DISCUSSIONSUsing Virtual-Photonic simulation package VPI, simulations were carried out for a number of optical data packets. Each data packet contained a single clock bit, 8-bit header address codeword and 8-bit payload. The payload length can be chosen longer, but for purpose of this simulation work, we only choose the payload length of 8 bits. A guard band of 1-bit duration of “0” is used to separate clock, header address codeword and payload. The data packet bit rate is 100 Gbits/s with its pulse power is 1 mW. The pulse full width half magnitude (FWHM) and the PPM frame slot width T s were set at 1ps and 5ps, respectively.PPM based frames E1 and E2 corresponding to entries 1 and 2, respectively can have at least one pulse or up to 255 pulses. In the simulation, a number of data packets with random address codeword having decimal weight of 186, 48, 93, 214, 0, 255, 21, 112, 85, 99, 200, 67, 3, 143, 32, 18, 164, 78, 69, 28, 166, 39, 100, 153, 19, 245, see in Fig. 3, were generated. For frame E1, the pulses appear at positions 0, 18, 19, 21, 31, 48, 67, 69, 78, 99, 153, 186, 200, 245. Therefore a data packet with its address codeword matching a bit in E1 will be switched to the output port 1 of the node. For frame E2, pulses will be located at positions 3, 28, 32, 85, 93, 100, 112, 143, 164, 166, 214, 255.Figure 3. Data packets sent to node. Boxed inset, 8-bit codeword has decimal weight 186The output pulse stream observed at the AND gates output as a result of matching the address with the routing table entries are shown in Fig. 4. All the test cases gave correct matching results at the outputs of appropriate AND gates. In which outputs of AND gate 1 and 2 correspond to the matching of C with E1 and E2, respectively. From Fig. 4, it is evident that the matching pulses have varying intensities. This is due to the gain profiles of switching stages in Fig. 2 were not kept strictly constant during 256×T s, thus resulting in gain variation seen by different codeword patterns. Here we consider the worst case scenario where the codeword is 0D to find out the intensity fluctuation when the pulse has just passed through switching stage via the lower port of SMZ switch with less gain than the gain induced during the transmitting mode of SMZ switch (output is the upper port). In the case of 0D, the AND gate output signal with 10 mW of power is capable of controlling the main all-optical switch to allow packets being routed to its target destination output. However, in the case of very low intensity control pulse, post-amplifier for gain compensation may be employed to ensure proper power of control signal of the main optical switch.Figure 4. Matching results of incoming data packet header codewords with PPRT entriesAn ideal switch should normally have no residual power at the undesired output ports (portion of the signal emerging from non-target output). However, real switches would always have certain amount of crosstalk at undesired outputs. Thus this residual power coming from each stage of PPM conversion unit in Fig. 2 will be amplified and accumulated through switching stages. Although at each stage this crosstalk is suppressed, but for the case with a large number of switching stages (8 stages) there is a small amount crosstalk that appears at the output of the last stage, see small pulses of Fig. 5 (b), on either sides of desired pulse positioned in an PPM frame. However, these small pulses can be neglected due to their low intensities (~ 100 µW) compared to the desired PPM pulse shown in Fig. 5(a)(a) (b)Figure 5. (a) Outputs at the last stage of PPM conversion unit, (b) Small crosstalk, zoomed from (a)5. CONCLUSIONSUltrafast all-optical header processing in optical packet-switched network will allow complex and large packet-switched networks operating entirely in optical domain using single wavelength source becoming a real possibility. 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