A microscopic theory for antiphase boundary motion and its application to antiphase domain

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Appendix XVI B. Microbiological Examination of Non-sterile Products in BP2012

British Pharmacopoeia Volume VAppendicesAppendix XVI B. Microbiological Examination of Non-sterile Products1. Tests for Specified Micro-organisms1(Ph. Eur. method 2.6.13)1 INTRODUCTIONThe tests described hereafter will allow determination of the absence or limited occurrence of specified micro-organisms that may be detected under the conditions described.The tests are designed primarily to determine whether a substance or preparation complies with an established specification for microbiological quality. When used for such purposes, follow the instructions given below, including the number of samples to be taken, and interpret the results as stated below.Alternative microbiological procedures, including automated methods, may be used, provided that their equivalence to the Pharmacopoeia method has been demonstrated.2 GENERAL PROCEDURESThe preparation of samples is carried out as described in general chapter 2.6.12.If the product to be examined has antimicrobial activity, this is insofar as possible removed or neutralised as described in general chapter 2.6.12.If surface-active substances are used for sample preparation, their absence of toxicity for micro-organisms and their compatibility with inactivators used must be demonstrated as described in general chapter 2.6.12.3 GROWTH-PROMOTING AND INHIBITORY PROPERTIES OF THE MEDIA, SUITABILITY OF THE TEST AND NEGATIVE CONTROLSThe ability of the test to detect micro-organisms in the presence of the product to be tested must be established. Suitability must be confirmed if a change in testing performance, or the product, which may affect the outcome of the test is introduced.3-1 Preparation of test strainsUse standardised stable suspensions of test strains or prepare them as stated below. Seed lot culture maintenance techniques (seed-lot systems) are used so that the viable micro-organisms used for inoculation are not more than 5 passages removed from the original master seed-lot.3-1-1 Aerobic micro-organisms Grow each of the bacterial test strains separately in casein soya bean digest broth or on casein soya bean digest agar at 30-35 °C for 18-24 h. Grow the test strain for Candida albicans separately on Sabouraud-dextrose agar or in Sabouraud-dextrose broth at 20-25 °C for 2-3 days.— Staphylococcus aureus such as ATCC 6538, NCIMB 9518, CIP 4.83 or NBRC 13276;— Pseudomonas aeruginosa such as ATCC 9027, NCIMB 8626, CIP 82.118 or NBRC 13275;— Escherichia coli such as ATCC 8739, NCIMB 8545, CIP 53.126 or NBRC 3972;— Salmonella enterica subsp. enterica serovar Typhimurium, such as ATCC 14028 or, as an alternative,Salmonella enterica subsp. enterica serovar Abony such as NBRC 100797, NCTC 6017 or CIP 80.39;— Candida albicans such as ATCC 10231, NCPF 3179, IP 48.72 or NBRC 1594.Use buffered sodium chloride-peptone solution pH 7.0 or phosphate buffer solution pH 7.2 to make test suspensions. Use the suspensions within 2 h or within 24 h if stored at 2-8 °C.3-1-2 Clostridia Use Clostridium sporogenes such as ATCC 11437 (NBRC 14293, NCIMB 12343, CIP 100651) or ATCC 19404 (NCTC 532 or CIP 79.03) or NBRC 14293. Grow the clostridial test strain under anaerobic conditions in reinforced medium for clostridia at 30-35 °C for 24-48 h. As an alternative to preparing and then diluting down a fresh suspension of vegetative cells of Cl. sporogenes, a stable spore suspension is used for test inoculation. The stable spore suspension may be maintained at 2-8 °C for a validated period.3-2 Negative controlTo verify testing conditions, a negative control is performed using the chosen diluent in place of the test preparation. There must be no growth of micro-organisms. A negative control is also performed when testing the products as described in section 4. A failed negative control requires an investigation.3-3 Growth promotion and inhibitory properties of the mediaTest each batch of ready-prepared medium and each batch of medium prepared either from dehydrated medium or from ingredients.Verify suitable properties of relevant media as described in Table 2.6.13.-1.Test for growth promoting properties, liquid mediaInoculate a portion of the appropriate medium with a small number (not more than 100 CFU) of the appropriate micro-organism. Incubate at the specified temperature for not more than the shortest period of time specified in the test. Clearly visible growth of the micro-organism comparable to that previously obtained with a previously tested and approved batch of medium occurs.Test for growth promoting properties, solid mediaPerform the surface-spread method, inoculating each plate with a small number (not more than 100 CFU) of the appropriate micro-organism. Incubate at the specified temperature for not more than the shortest period of time specified in the test. Growth of the micro-organism comparable to that previously obtained with a previously tested and approved batch of medium occurs.Test for inhibitory properties, liquid or solid mediaInoculate the appropriate medium with at least 100 CFU of the appropriate micro-organism. Incubate at the specified temperature for not less than the longest period of time specified in the test. No growth of the test micro-organism occurs.Test for indicative propertiesPerform the surface-spread method, inoculating each plate with a small number (not more than 100 CFU) of the appropriate micro-organism. Incubate at the specified temperature for a period of time within the range specified in the test. Colonies are comparable in appearance and indication reactions to those previously obtained with a previously tested and approved batch of medium.3-4 Suitability of the test methodFor each product to be tested, perform the sample preparation as described in the relevant paragraph in section 4. Add each test strain at the time of mixing, in the prescribed growth medium. Inoculate the test strains individually. Use a number of micro-organisms equivalent to not more than 100 CFU in the inoculated test preparation.Perform the test as described in the relevant paragraph in section 4 using the shortest incubation period prescribed.The specified micro-organisms must be detected with the indication reactions as described in section 4.Any antimicrobial activity of the product necessitates a modification of the test procedure (see 4-5-3 of general chapter 2.6.12).If for a given product the antimicrobial activity with respect to a micro-organism for which testing is prescribed cannot be neutralised, then it is to be assumed that the inhibited micro-organism will not be present in the product.4 TESTING OF PRODUCTS4-1 Bile-tolerant gram-negative bacteria4-1-1 Sample preparation and pre-incubation Prepare a sample using a 1 in 10 dilution of not less than 1 g of the product to be examined as described in general chapter 2.6.12, but using casein soya bean digest broth as the chosen diluent, mix and incubate at 20-25 °C for a time sufficient to resuscitate the bacteria but not sufficient to encourage multiplication of the organisms (usually 2 h but not more than 5 h).4-1-2 Test for absence Unless otherwise prescribed, use the volume corresponding to 1 g of the product, as prepared in 4-1-1, to inoculate enterobacteria enrichment broth-Mossel. Incubate at 30-35 °C for 24-48 h. Subculture on plates of violet red bile glucose agar. Incubate at 30-35 °C for 18-24 h.The product complies with the test if there is no growth of colonies.4-1-3 Quantitative test4-1-3-1 Selection and subculture Inoculate suitable quantities of enterobacteria enrichment broth-Mossel with the preparation as described under 4-1-1 and/or dilutions of it containing respectively 0.1 g, 0.01 g and 0.001 g (or 0.1 mL, 0.01 mL and 0.001 mL) of the product to be examined. Incubate at 30-35 °C for 24-48 h. Subculture each of the cultures on a plate of violet red bile glucose agar. Incubate at 30-35 °C for 18-24 h.4-1-3-2 Interpretation Growth of colonies constitutes a positive result. Note the smallest quantity of the product that gives a positive result and the largest quantity that gives a negative result. Determine from Table 2.6.13.-2 the probable number of bacteria.4-2 Escherichia coli4-2-1 Sample preparation and pre-incubation Prepare a sample using a 1 in 10 dilution of not less than 1 g of the product to be examined as described in general chapter 2.6.12, and use 10 mL or the quantity corresponding to 1 g or 1 mL to inoculate a suitable amount (determined as described under 3-4) of casein soya bean digest broth, mix and incubate at 30-35 °C for 18-24 h.4-2-2 Selection and subculture Shake the container, transfer 1 mL of casein soya bean digest broth to 100 mL of MacConkey broth and incubate at 42-44 °C for 24-48 h. Subculture on a plate of MacConkey agar at 30-35 °C for 18-72 h.4-2-3 Interpretation Growth of colonies indicates the possible presence of E. coli. This is confirmed by identification tests.The product complies with the test if no colonies are present or if the identification tests are negative.4-3 Salmonella4-3-1 Sample preparation and pre-incubation Prepare the product to be examined as described in general chapter 2.6.12, and use the quantity corresponding to not less than 10 g or 10 mL to inoculate a suitable amount (determined as described under 3-4) of casein soya bean digest broth, mix and incubate at 30-35 °C for 18-24 h.4-3-2 Selection and subculture Transfer 0.1 mL of casein soya bean digest broth to 10 mL of Rappaport Vassiliadis Salmonella enrichment broth and incubate at 30-35 °C for 18-24 h. Subculture on plates of xylose, lysine, deoxycholate agar. Incubate at 30-35 °C for 18-48 h.4-3-3 Interpretation The possible presence of Salmonella is indicated by the growth of well-developed, red colonies, with or without black centres. This is confirmed by identification tests.The product complies with the test if colonies of the types described are not present or if the confirmatory identification tests are negative.4-4 Pseudomonas aeruginosa4-4-1 Sample preparation and pre-incubation Prepare a sample using a 1 in 10 dilution of not less than 1 g of the product to be examined as described in general chapter 2.6.12, and use 10 mL or the quantity corresponding to 1 g or 1 mL to inoculate a suitable amount (determined as described under 3-4) of casein soya bean digest broth and mix. When testing transdermal patches, filter the volume of sample corresponding to 1 patch of the preparation described under 4-5-1 in general chapter 2.6.12 through a sterile filter membrane and place in 100 mL of casein soya bean digest broth. Incubate at 30-35 °C for 18-24 h.4-4-2 Selection and subculture Subculture on a plate of cetrimide agar and incubate at 30-35 °C for 18-72 h.4-4-3 Interpretation Growth of colonies indicates the possible presence of P. aeruginosa. This is confirmed by identification tests.The product complies with the test if colonies are not present or if the confirmatory identification tests are negative.4-5 Staphylococcus aureus4-5-1 Sample preparation and pre-incubation Prepare a sample using a 1 in 10 dilution of not less than 1 g of the product to be examined as described in general chapter 2.6.12, and use 10 mL or the quantity corresponding to 1 g or 1 mL to inoculate a suitable amount (determined as described under 3-4) of casein soya bean digest broth and mix. When testing transdermal patches, filter the volume of sample corresponding to 1 patch of the preparation described under 4-5-1 in general chapter 2.6.12 through a sterile filter membrane and place in 100 mL of casein soya bean digest broth. Incubate at 30-35 °C for 18-24 h.4-5-2 Selection and subculture Subculture on a plate of mannitol salt agar and incubate at 30-35 °C for 18-72 h.4-5-3 Interpretation The possible presence of S. aureus is indicated by the growth of yellow/white colonies surrounded by a yellow zone. This is confirmed by identification tests.The product complies with the test if colonies of the types described are not present or if the confirmatory identification tests are negative.4-6 Clostridia4-6-1 Sample preparation and heat treatment Prepare a sample using a 1 in 10 dilution (with a minimum total volume of 20 mL) of not less than 2 g or 2 mL of the product to be examined as described in general chapter 2.6.12.Divide the sample into 2 portions of at least 10 mL. Heat 1 portion at 80 °C for 10 min and cool rapidly. Do not heat the other portion.4-6-2 Selection and subculture Use 10 mL or the quantity corresponding to 1 g or 1 mL of the product to be examined of both portions to inoculate suitable amounts (determined as described under 3-4) of reinforced medium for clostridia. Incubate under anaerobic conditions at 30-35 °C for 48 h. After incubation, make subcultures from each container on Columbia agar and incubate under anaerobic conditions at 30-35 °C for 48-72 h.4-6-3 Interpretation The occurrence of anaerobic growth of rods (with or without endospores) giving a negative catalase reaction indicates the presence of clostridia. This is confirmed by identification tests.The product complies with the test if colonies of the types described are not present or if the confirmatory identification tests are negative.4-7 Candida albicans4-7-1 Sample preparation and pre-incubation Prepare the product to be examined as described in general chapter 2.6.12, and use 10 mL or the quantity corresponding to not less than 1 g or 1 mL to inoculate 100 mL of Sabouraud-dextrose broth and mix. Incubate at 30-35 °C for 3-5 days.4-7-2 Selection and subculture Subculture on a plate of Sabouraud-dextrose agar and incubate at 30-35 °C for 24 -48 h.4-7-3 Interpretation Growth of white colonies may indicate the presence of C. albicans. This is confirmed by identification tests.The product complies with the test if such colonies are not present or if the confirmatory identification tests are negative.The following section is given for information.5 RECOMMENDED SOLUTIONS AND CULTURE MEDIAThe following solutions and culture media have been found to be satisfactory for the purposes for which they are prescribed in the test for microbial contamination in the Pharmacopoeia. Other media may be used provided that their suitability can be demonstrated.Stock buffer solution Place 34 g of potassium dihydrogen phosphate in a 1000 mL volumetric flask, dissolve in 500 mL of purified water, adjust to pH 7.2 ± 0.2 with sodium hydroxide, dilute to 1000.0 mL with purified water and mix. Dispense into containers and sterilise. Store at 2-8 °C.Phosphate buffer solution pH 7.2 Prepare a mixture of stock buffer solution and purified water (1:800 V/V) and sterilise.Buffered sodium chloride-peptone solution pH 7.0Potassium dihydrogen phosphate 3.6 gDisodium hydrogen phosphate dihydrate7.2 g, equivalent to 0.067 M phosphateSodium chloride 4.3 gPeptone (meat or casein) 1.0 gPurified water1000 mLSterilise in an autoclave using a validated cycle.Casein soya bean digest brothPancreatic digest of casein17.0 gPapaic digest of soya bean 3.0 gSodium chloride 5.0 gDipotassium hydrogen phosphate 2.5 gGlucose monohydrate 2.5 gPurified water1000 mLAdjust the pH so that after sterilisation it is 7.3 ± 0.2 at 25 °C. Sterilise in an autoclave using a validated cycle. Casein soya bean digest agarPancreatic digest of casein15.0 gPapaic digest of soya bean 5.0 gSodium chloride 5.0 gAgar15.0 gPurified water1000 mLAdjust the pH so that after sterilisation it is 7.3 ± 0.2 at 25 °C. Sterilise in an autoclave using a validated cycle. Sabouraud-dextrose agarDextrose40.0 g Mixture of peptic digest of animal tissue and pancreatic digest of casein (1:1)10.0 g Agar15.0 g Purified water1000 mL Adjust the pH so that after sterilisation it is 5.6 ± 0.2 at 25 °C. Sterilise in an autoclave using a validated cycle. Potato dextrose agarInfusion from potatoes200 gDextrose20.0 gAgar15.0 gPurified water1000 mLAdjust the pH so that after sterilisation it is 5.6 ± 0.2 at 25 °C. Sterilise in an autoclave using a validated cycle. Sabouraud-dextrose brothDextrose20.0 g Mixture of peptic digest of animal tissue and pancreatic digest of casein (1:1)10.0 g Purified water1000 mLAdjust the pH so that after sterilisation it is 5.6 ± 0.2 at 25 °C. Sterilise in an autoclave using a validated cycle. Enterobacteria enrichment broth-MosselPancreatic digest of gelatin10.0 gGlucose monohydrate 5.0 g Dehydrated ox bile20.0 g Potassium dihydrogen phosphate 2.0 gDisodium hydrogen phosphate dihydrate8.0 gBrilliant green15 mgPurified water1000 mLAdjust the pH so that after heating it is 7.2 ± 0.2 at 25 °C. Heat at 100 °C for 30 min and cool immediately. Violet red bile glucose agarYeast extract 3.0 gPancreatic digest of gelatin7.0 gBile salts 1.5 gSodium chloride 5.0 gGlucose monohydrate10.0 gAgar15.0 gNeutral red30 mgCrystal violet 2 mgPurified water1000 mLAdjust the pH so that after heating it is 7.4 ± 0.2 at 25 °C. Heat to boiling; do not heat in an autoclave. MacConkey brothPancreatic digest of gelatin20.0 gLactose monohydrate10.0 gDehydrated ox bile 5.0 gBromocresol purple10 mgPurified water1000 mLAdjust the pH so that after sterilisation it is 7.3 ± 0.2 at 25 °C. Sterilise in an autoclave using a validated cycle. MacConkey agarPancreatic digest of gelatin17.0 gPeptones (meat and casein) 3.0 gLactose monohydrate10.0 gSodium chloride 5.0 gBile salts 1.5 gAgar13.5 gNeutral red30.0 mgCrystal violet 1 mgPurified water1000 mLAdjust the pH so that after sterilisation it is 7.1 ± 0.2 at 25 °C. Boil for 1 min with constant shaking then sterilise in an autoclave using a validated cycle.Rappaport Vassiliadis Salmonella enrichment brothSoya peptone 4.5 gMagnesium chloride hexahydrate29.0 gSodium chloride8.0 gDipotassium phosphate0.4 gPotassium dihydrogen phosphate0.6 gMalachite green0.036 gPurified water1000 mLDissolve, warming gently. Sterilise in an autoclave using a validated cycle, at a temperature not exceeding 115 °C. The pH is to be 5.2 ± 0.2 at 25 °C after heating and autoclaving.Xylose, lysine, deoxycholate agarXylose 3.5 gL-Lysine 5.0 gLactose monohydrate7.5 gSucrose7.5 gSodium chloride 5.0 gYeast extract 3.0 gPhenol red80 mgAgar13.5 gSodium deoxycholate 2.5 gSodium thiosulfate 6.8 gFerric ammonium citrate0.8 gPurified water1000 mLAdjust the pH so that after heating it is 7.4 ± 0.2 at 25 °C. Heat to boiling, cool to 50 °C and pour into Petri dishes. Do not heat in an autoclave.Cetrimide agarPancreatic digest of gelatin20.0 gMagnesium chloride 1.4 gDipotassium sulfate10.0 gCetrimide0.3 gAgar13.6 gPurified water1000 mLGlycerol10.0 mLHeat to boiling for 1 min with shaking. Adjust the pH so that after sterilisation it is 7.2 ± 0.2 at 25 °C. Sterilise in an autoclave using a validated cycle.Mannitol salt agarPancreatic digest of casein 5.0 gPeptic digest of animal tissue 5.0 gBeef extract 1.0 gD-Mannitol10.0 gSodium chloride75.0 gAgar15.0 gPhenol red0.025 gPurified water1000 mLHeat to boiling for 1 min with shaking. Adjust the pH so that after sterilisation it is 7.4 ± 0.2 at 25 °C. Sterilise in an autoclave using a validated cycle.Reinforced medium for clostridiaBeef extract10.0 gPeptone10.0 gYeast extract 3.0 gSoluble starch 1.0 gGlucose monohydrate 5.0 gCysteine hydrochloride0.5 gSodium chloride 5.0 gSodium acetate 3.0 gAgar0.5 gPurified water1000 mLHydrate the agar, dissolve by heating to boiling with continuous stirring. If necessary, adjust the pH so that after sterilisation it is 6.8 ± 0.2 at 25 °C. Sterilise in an autoclave using a validated cycle.Columbia agarPancreatic digest of casein10.0 gMeat peptic digest 5.0 gHeart pancreatic digest 3.0 gYeast extract 5.0 gMaize starch 1.0 gSodium chloride 5.0 gAgar, according to gelling power10.0-15.0 gPurified water1000 mLHydrate the agar, dissolve by heating to boiling with continuous stirring. If necessary, adjust the pH so that after sterilisation it is 7.3 ± 0.2 at 25 °C. Sterilise in an autoclave using a validated cycle. Allow to cool to 45-50 °C; add, where necessary, gentamicin sulfate corresponding to 20 mg of gentamicin base and pour into Petri dishes.2. Microbial Enumeration Tests1(Ph. Eur. method 2.6.12)1 INTRODUCTIONThe tests described hereafter will allow quantitative enumeration of mesophilic bacteria and fungi that may grow under aerobic conditions.The tests are designed primarily to determine whether a substance or preparation complies with an established specification for microbiological quality. When used for such purposes follow the instructions given below, including the number of samples to be taken, and interpret the results as stated below.The methods are not applicable to products containing viable micro-organisms as active ingredients.Alternative microbiological procedures, including automated methods, may be used, provided that their equivalence to the Pharmacopoeia method has been demonstrated.2 GENERAL PROCEDURESCarry out the determination under conditions designed to avoid extrinsic microbial contamination of the product to be examined. The precautions taken to avoid contamination must be such that they do not affect any micro-organisms that are to be revealed in the test.If the product to be examined has antimicrobial activity, this is insofar as possible removed or neutralised. If inactivators are used for this purpose, their efficacy and their absence of toxicity for micro-organisms must be demonstrated.If surface-active substances are used for sample preparation, their absence of toxicity for micro-organisms and their compatibility with inactivators used must be demonstrated.3 ENUMERATION METHODSUse the membrane filtration method or the plate-count methods, as prescribed. The most-probable-number (MPN) method is generally the least accurate method for microbial counts, however, for certain product groups with a very low bioburden, it may be the most appropriate method.The choice of method is based on factors such as the nature of the product and the required limit of micro-organisms. The chosen method must allow testing of a sufficient sample size to judge compliance with the specification. The suitability of the method chosen must be established.4 GROWTH PROMOTION TEST, SUITABILITY OF THE COUNTING METHOD AND NEGATIVE CONTROLS4-1 General considerationsThe ability of the test to detect micro-organisms in the presence of product to be tested must be established.Suitability must be confirmed if a change in testing performance, or the product, which may affect the outcome of the test is introduced.4-2 Preparation of test strainsUse standardised stable suspensions of test strains or prepare them as stated below. Seed lot culture maintenance techniques (seed-lot systems) are used so that the viable micro-organisms used for inoculation are not more than 5 passages removed from the original master seed-lot. Grow each of the bacterial and fungal test strains separately as described in Table 2.6.12.-1.Use buffered sodium chloride-peptone solution pH 7.0 or phosphate buffer solution pH 7.2 to make test suspensions; to suspend A. brasiliensis spores, 0.05 per cent of polysorbate 80 may be added to the buffer. Use the suspensions within 2 h or within 24 h if stored at 2-8 °C. As an alternative to preparing and then diluting a fresh suspension of vegetative cells of A. brasiliensis or B. subtilis, a stable spore suspension is prepared and then an appropriate volume of the spore suspension is used for test inoculation. The stable spore suspension may be maintained at 2-8 °C for a validated period of time.4-3 Negative controlTo verify testing conditions, a negative control is performed using the chosen diluent in place of the test preparation. There must be no growth of micro-organisms. A negative control is also performed when testing the products as described in section 5. A failed negative control requires an investigation.4-4 Growth promotion of the mediaTest each batch of ready-prepared medium and each batch of medium, prepared either from dehydrated medium or from the ingredients described.Inoculate portions/plates of casein soya bean digest broth and casein soya bean digest agar with a small number (not more than 100 CFU) of the micro-organisms indicated in Table 2.6.12.-1, using a separate portion/plate of medium for each. Inoculate plates of Sabouraud-dextrose agar with a small number (not more than 100 CFU) of the micro-organisms indicated in Table 2.6.12.-1, using a separate plate of medium for each. Incubate in the conditions described in Table 2.6.12.-1.For solid media, growth obtained must not differ by a factor greater than 2 from the calculated value for a standardised inoculum. For a freshly prepared inoculum, growth of the micro-organisms comparable to that previously obtained with a previously tested and approved batch of medium occurs. Liquid media are suitable if clearly visible growth of the micro-organisms comparable to that previously obtained with a previously tested and approved batch of medium occurs.4-5 Suitability of the counting method in the presence of product4-5-1 Preparation of the sample The method for sample preparation depends upon the physical characteristics of the product to be tested. If none of the procedures described below can be demonstrated to be satisfactory, an alternative procedure must be developed.Water-soluble products Dissolve or dilute (usually a 1 in 10 dilution is prepared) the product to be examined in buffered sodium chloride-peptone solution pH 7.0, phosphate buffer solution pH 7.2 or casein soya bean digest broth. If necessary, adjust to pH 6-8. Further dilutions, where necessary, are prepared with the same diluent.Non-fatty products insoluble in water Suspend the product to be examined (usually a 1 in 10 dilution is prepared) in buffered sodium chloride-peptone solution pH 7.0, phosphate buffer solution pH 7.2 or casein soya bean digest broth. A surface-active agent such as 1 g/L of polysorbate 80 may be added to assist the suspension of poorly wettable substances. If necessary, adjust to pH 6-8. Further dilutions, where necessary, are prepared with the same diluent.Fatty products Dissolve in isopropyl myristate, sterilised by filtration or mix the product to be examined with the minimum necessary quantity of sterile polysorbate 80 or another non-inhibitory sterile surface-active agent, heated if necessary to not more than 40 °C, or in exceptional cases to not more than 45 °C. Mix carefully and if necessary maintain the temperature in a water-bath. Add sufficient of the pre-warmed chosen diluent to make a 1 in 10 dilution of the original product. Mix carefully whilst maintaining the temperature for the shortest time necessary for the formation of an emulsion. Further serial tenfold dilutions may be prepared using the chosen diluent containing a suitable concentration of sterile polysorbate 80 or another non-inhibitory sterile surface-active agent.Fluids or solids in aerosol form Aseptically transfer the product into a membrane filter apparatus or a sterile container for further sampling. Use either the total contents or a defined number of metered doses from each of the containers tested.Transdermal patches Remove the protective cover sheets ('release liners') of the transdermal patches and place them, adhesive side upwards, on sterile glass or plastic trays. Cover the adhesive surface with a sterile porous。

fundamentals of microelectronics英文原版

The fundamentals of microelectronics refer to the basic principles and concepts that form the foundation of the field. Microelectronics deals with the study and application of small-scale electronic components, such as integrated circuits and transistors. This field has played a crucial role in the development of various technologies, including computers, smartphones, and medical devices.One of the key concepts in microelectronics is the idea of miniaturization. Microelectronic components are designed to be small and compact, allowing for increased functionality in a limited space. This miniaturization is made possible by advancements in semiconductor technology, which enables the production of smaller and more efficient electronic devices.Another fundamental principle is the understanding of electronic circuits. Microelectronics relies on the design and analysis of circuits that control the flow of electric current. These circuits can be composed of different components, such as resistors, capacitors, and inductors, which work together to perform specific tasks.The behavior of microelectronic devices is guided by the laws of physics, particularly quantum mechanics. At the nanoscale level, where microelectronics operates, particles exhibit quantum effects that can significantly impact the performance of electronic devices. Understanding these effects is essential for designing and optimizing microelectronic components.In addition to the physical principles, microelectronics also encompasses the study of fabrication techniques. The process of manufacturing microelectronic devices involves multiple steps, including deposition, lithography, etching, and doping. Each of these steps contributes to the creation of complex integrated circuits and other microelectronic components.The field of microelectronics also includes the study of electronic materials. Different materials exhibit unique properties that can be leveraged in microelectronic devices. For example, semiconductors, such as silicon, are widely used in microelectronics due to their ability to control the flow of electric current.Overall, the fundamentals of microelectronics cover a wide range of topics, including circuit design, semiconductor physics, fabrication techniques, and electronic materials. Understanding these principles is crucial for the development of new and innovative microelectronic devices that drive technological advancements in various industries.。

阿尔布雷希特科塞尔

他的重要著作和论文，多数发表在《生理化学》杂志上。其中最重要的有《关于细胞核物质及其分解产物的研究》（1881年）、《生物化学的问题》（1908年）、《化学和生理学的关联》（1913年）、《生物化学入门》（1917年）等。他因为研究有关蛋白质、细胞化学及核酸方面的成就，获得了1910年的诺贝尔生理学及医学奖金。
阿尔布雷希特 ·科塞尔
德国生物化学家
目录
01 个人简介
02 部分作品
阿尔布雷希特·科塞尔（Ludwig Karl Martin Leonhard Albrecht Kossel ，1853年9月16日-1927年7月5 日），德国生化学家。他因细胞化学的研究（特别是蛋白质和核酸）获得1910年的诺贝尔生理学或医学奖。
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个人简介
阿尔布雷希特·科塞尔 1853年9月16日出生于德国的罗斯托克城。1871年进入斯特拉斯堡大学学习医学和生理化学。1878年获得医学博士学位。毕业后即在斯特拉斯堡生理学研究所工作，并在斯特拉斯堡大学任教。 1 8 8 3 年应杜波伊斯 ·雷孟德之聘，到柏林生理学研究所担任化学部门的领导人。 1 8 9 5 年任马尔堡大学教授和生理学研究所所长。1901年辞去马尔堡大学教职，不久任海德堡大学生理学教授、蛋白质研究所所长，1908年任海德堡大学副校长。此外，他还兼任过国际生理学会的会长，任过长达30年《生理学杂志》的编辑工作。由于他在医学和生化学方面的贡献，获得了英国剑桥大学等六所国内外著名大学授予的名誉博士学位，成为好几个国家的科学协会会员。1927年7月5日逝世于德国的海德堡，终年73岁。科塞尔毕生主要从事生理化学、特别是组织及细胞化学的研究。他是生化学这门独立学科的创始人之一。他发现了核酸构成成分，作为嘌呤体的腺嘌呤和鸟嘌呤，以及作为嘧啶体的硫胺酸(维生素B1)。在消化生物化学方面，他发现了白蛋白能分解成蛋白栋，而且在胰蛋白酶的作用下，蛋白栋产生多肽，这些物质都是由氨基酸构成的。是他介绍了氨基酸的蛋白质的基本结构的概念。

Micro-encapsulation of Bifidobacterium lactis for incorporation into soft foods

Micro-encapsulation of Biﬁdobacterium lactis for incorporation into soft foodsL.D.McMaster*,S.A.Kokott1and P.Slatter21Department of Food and Agricultural Sciences,Cape Technikon(1),Flow Process Centre,Cape Technikon,P.O. Box652,8001Cape Town,South Africa2Department of Civil Engineering,Cape Technikon,P.O.Box652,8001Cape Town,South Africa*Author for correspondence:Tel.:+27-21-4603507,Fax:+27-21-4603424,E-mail:lynnb@ctech.ac.za Keywords:Beverages,Biﬁdobacterium lactis,microencapsulation,probiotics,rheologySummaryMicro-encapsulation of the probiotic micro-organism Biﬁdobacterium lactis isolated from a bio-yoghurt starter culture,was carried out using a mixture of hydrated gellan and xanthan gums.Rheological studies showed that the gum mix was suitable for encapsulation of ctis,for incorporation into soft foods/beverages.The gel behaved as a non-Newtonian material,and theﬂow curveﬁtted well to the Herschel–Bulkley model.The average yield stress of the gum was1.515Pa,indicating gum stability,and the yield stress range was1Pa over a temperature range of 35–50°C.Almost constant minimum gum viscosity occurred between46and61°C.Oval/round capsules were synthesized manually using a monoaxial gumﬂow through a27.5G bevelled needle,together with a superposed air stream(air knife technique).The diameter of the capsules,measured using laser diﬀractometry,varied from20to 2200l m,with50%of the capsules having a diameter of£637l m.Numbers of viable ctis in the capsules were estimated using high power ultrasound(20kHz).By using a concentrated inoculum of ctis,microcapsules containing log1011–12c.f.u.g)1were synthesized.Apart from the anaerobic culturing of ctis,all other work was done in the presence of atmospheric oxygen.The organism exhibited a high degree of oxygen tolerance.A 21-day survival study of immobilized cells in1M sodium phosphate buﬀer(pH7)stored at either4or22°C indicated that ctis survived in excess of log1011c.f.u.g)1microcapsule.This technique represents a suitable means of supplying viable probiotics to the food and/or pharmaceutical industries.Mathematical symbolsHerschel–Bulkley model:s0¼s yÀjÀn ys0yield stresss y shear stressj yﬂuid consistency indexnﬂow behaviour indexIntroductionThe consumption of foods containing probiotic organ-isms is becoming increasingly popular.Probiotic micro-organisms,generally bacteria,are deﬁned as micro-organisms which when consumed in certain numbers,exert health beneﬁts beyond nutrition(Lactic Acid Bacteria Industrial Platform,LABIP,cited by Guarner&Schaafsma1998).One group of organisms gaining credibility as probiotics is the genus Biﬁdobac-terium.The signiﬁcance and development of probiotic foods, and their use as alternatives to antibiotics,is predicted to increase in the future(Doyle2003;Kroger2003).In the European Union,consumption of probiotic foods increased40%during theﬁrst quarter of2004(Ross 2004).However,the provision and supply of foods containing viable Biﬁdobacterium in the numbers required for a probiotic organism to be beneﬁcial (RDA log108c.f.u.),has long been recognized to be problematic(Hughes&Hoover1991;Klaver et al.1993; Temmerman et al.2003).Two technologies used to supply Biﬁdobacterium in foods are freeze-and spray-drying.Although improvements in these methods have been reported(Siuta-Cruce&Goulet2001),the use of spray-and freeze-drying of biﬁdobacterium for incor-poration into foods for human consumption has shown that both the Biﬁdobacterium spp.and the carrier medium used in process inﬂuence survival of the organism(Lian et al.2002).Biﬁdobacterium cells under-going these treatments lose viability(Klaver et al.1993; Lian et al.2002).In addition,microbial injury duringWorld Journal of Microbiology&Biotechnology(2005)21:723–728ÓSpringer2005 DOI10.1007/s11274-004-4798-0both of these processes is common,and injured cells require time for repair in a suitable recovery medium (Ray2001).Bacteria supplied in foods as both freeze-and spray-dried cells require time to rehydrate and reconstitute in the human host.The human gastro-intestinal tract(GIT)is not an ideal environment for resuscitation of injured bacteria;hence it is likely that Biﬁdobacterium present as spray-or freeze-dried cells in probiotic foods do not all respond optimally under conditions associated with the gastro-intestinal tract. An alternate means of delivering living biﬁdobacteria directly to the GIT is through the use of micro-capsules. Unlike the freeze-and spray-drying processes where the dormant micro-organism is delivered directly into the environment,the capsule matrix surrounding the bacte-ria protects the viable organisms from the gastric juices of the upper gastro-intestinal tract,and once in the colon,capsules break down to release the living organ-isms over a period of time(Krasaekoopt et al.2003). Micro-encapsulation of probiotic biﬁdobacteria is re-ported in some cases to improve survival of the organism in various foods.Edible gums such as alginate, locust bean gum,j-carrageenan,xanthan and gellan are often used to immobilize bacterial cells(Sun&Griﬃths 2000;Siuta-Crus&Goulet2001;Kailasapathy2002; Truelstrup-Hansen et al.2002;Krasaekoopt et al. 2003).These gums form a soft microcapsule,suitable for inclusion into foods.The aim of this investigation was to provide micro-capsules of suitable dimensions synthesized from edible gums,in which a high concentration of living Biﬁdo-bacterium was present,for incorporation into soft foods and beverages.To achieve this,we undertook a preliminary rheological study of the gum/s of choice before proceeding with micro-encapsulation.We also wished to establish the best method for synthesising micro-capsules with a view to upgrading production to an industrial scale.For technological reasons,it was important to establish oxygen tolerance of the selected Biﬁdobacterium spp.A further objective was to establish the viability of immobilized cells when stored in a buﬀer, as a possible means of supplying the microcapsules to industry for incorporation into foods.Materials and methodsBacterial strains and culture conditionsBacterial cultures were propagated anaerobically at 37°C in a Bactron1.5anaerobic chamber(Shel Lab) at37°C in an environment consisting of hydrogen(4%), carbon dioxide(10%)and nitrogen(86%).Cells were grown in tryptone yeast glucose(TYG)media which contained the following,in g l)1:tryptone10(Biolab); yeast extract,5(Biolab);glucose,5(Saarchem);Tween 80,1.0(Sigma);NaCl,4.5;KCl,0.25;MgCl2Æ6H2O,0.15; KH2PO4,0.4;K2HPO4,0.2;NH4Cl,0.4(Saarchem); cystein HCl,0.5(Sigma);rezasurin,0.001%(Sigma).Agar(1.5%(w/v)(Biolab)was added for solid media. Biﬁdobacterium lactis was isolated from a yoghurt starter culture as described by Trindade et al.(2003).Rheology of the gum mixBased on work done elsewhere,gellan and xanthan were selected as the gums of choice,and were used as a mixture of0.75%w/v gellan and1%w/v xanthan(Sun &Grifﬁths2000).Immediately prior to the rheological studies,the gums were prepared and re-hydrated as described below.A Paar Physica MCR300rotational rheometer with a cone-plate measuring system was used for the gellan:xanthan gum mix to determine aﬂow curve in controlled shear rate mode,at shear rates of0.1–100s)1, and a temperature sweep at a constant shear rate of5s)1between30and60°C.In addition,a time dependency test,at a constant shear rate of5s)1at temperatures of30,40and45°C was done.Micro-encapsulation of ctisA variation of the method proposed by Sun&Grifﬁths (2000)was used.Gellan(0.1g),(Sigma)and xanthan (0.2g)(Sigma)were added to20ml distilled water.The solution was mixed using a magnetic stirrer(Ikamag) and heated to80°C for1h.The gel mix was then autoclaved at121°C ctis was grown in a Bactron1.5anaerobic chamber at37°C overnight in 250ml TYG broth to an OD6000.9–1.1.All subsequent procedures were done under aerobic conditions.Cells were harvested by centrifugation at8000·g for10min in a Beckman J21centrifuge at4°C.The pellet of cells was washed three times by resuspension in20ml aliquots of sterile distilled water,followed by centrifu-gation.Finally,the cells were suspended in sterile distilled water,to give a volume of2.5ml.One ml of this concentrate was mixed into20ml sterile gellan:xan-than gum at55°C.Microcapsules were generated by a bead entrapment method,using a superposed airﬂow together with mono-axial extrusion of the gum/bacteria mixture(Huebner&Buccholz1999;Park&Chang 2000).The gum/cell mix was manually extruded through a27.5G bevelled needle(Teruma)ﬁtted on to a sterile 20ml syringe(Promex).Gel and sterile airﬂow rates were10ml min)1and250ml s)1respectively.Micro-droplets formed by this method were hardened into spheres by free fall into a sterile0.1M CaCl2solution (100ml).After1h,beads were separated from the solution by asepticﬁltration through sterile Whatman ﬁlter paper No1.Microcapsules remaining on theﬁlter paper were washed thoroughly with sterile0.1M CaCl2. All procedures were carried out in a laminarﬂow hood. Estimation of microcapsule sizeThe diameter and size distribution of micro-capsules containing ctis were estimated by laser724L.D.McMaster et al.diﬀractometry using a Malvern Mastersizer S,version 2.19.The analysis model used was Polydisperse,and the presentation–30HD–‘‘standard wet’’for use with the Mie theory.Enumeration of ctisTo estimate numbers of ctis in microcapsules,0.1–0.2g of capsules were weighed into10ml of1M sodium phosphate buﬀer(NaH2PO446.3g l)1,NaH2-PO453.7g l)1),pH6.8.At predetermined time intervals, the mix was subjected to sonication to release ctis cells from the gel matrix.A Vibracell Ultrasonic Processor VCX750(20kHz)(Materials and Sonics) was used with a standard probe,with a tip diameter of 13mm generating a sound wave amplitude of105l m for15s.All samples were treated in20ml sterile cylindrical Polytop glass cylinders immersed in an ice water bath.After completion of the treatment,to estimate the number of released ctis,standard serial dilution and plate counts were done.Cells were diluted in2%buﬀered peptone water(Difco).Repre-sentative0.1ml volumes from dilutions were spread in triplicate/dilution onto TYG agar plates.These were incubated anaerobically at37°C for48h,and colonies were counted.Values obtained were expressed as either log10c.f.u.g)1or ml)1or as log surviving fraction.The log surviving fraction was calculated as N t/N0,where N0 and N t are the average number(N)of c.f.u.ml)1(free cells)or g)1(microcapsules)at the start of the experi-ment(0),and at time(t)respectively.Survival of immobilized ctis in1M sodium phos-phate buﬀer(pH6.8)Sodium phosphate buffer(1M)pH6.8was prepared as described.Known weights(0.2g)of entrapped ctis were added to10ml of sterile buﬀer.The solution was stored aerobically at either4or22°C for21days. Viable numbers of entrapped ctis were determined using the Vibracell Ultrasonic Processor VCX750 followed by serial plate dilution.Results and discussionBacterial strain isolatedUsing an AB yoghurt starter culture,Biﬁdobacterium lactis was isolated and used for all subsequent studies (Trindade et al.2003). ctis was chosen as the probiotic organism for encapsulation,as it is com-monly found in yoghurt starter cultures.The organism possesses attributes important in selecting a probiotic for commercial use.These include oxygen tolerance reported for up to24h exposure,fermentation of a wide variety of carbohydrates(Trindade et al.2003) and survival at pH2–3(Truelstrup-Hansen et al. 2002).Rheology of the gellan–xanthan gum mixWe elected to use gellan(0.75%)and xanthan(1%)for immobilizing materials as a combination of thesegums has been reported to have better technical prop-erties for micro-encapsulation than do alginate,j-carrageenan and locust bean gums(Sun&Grifﬁths2000).Although alginate is frequently used to micro-encapsulate probiotics,it has undesirable attributes,such as susceptibility to degradation by acids(Sun&Grifﬁths2000;Truelstrup-Hansen et al.2002;Kra-saekoopt et al.2003).Hence alginate micro-capsulesare likely to erode in the hydrochloric acid of thestomach and not reach the colon intact.Rheological studies used to determine the shear stressdata of the gellan:xanthan gum indicated that the gelbehaved as a non-Newtonian material,and theﬂowcurveﬁtted well to the Herschel–Bulkley models0¼s y)k y)n,which is used to describe theﬂow behav-iour of a yield pseudoplastic material(Figure1).Formicroencapsulation,Newtonian liquids such as water orhoney are unsuitable as encapsulating materials,asliquid droplets,not solid capsules,are formed duringextrusion.In order for successful encapsulation ofmicro-organisms to occur,the selected material shouldexhibit non-Newtonian properties i.e.be a relativelyviscous material with solid properties.The data reportedhere indicated that the gum mix of gellan and xanthansatisﬁed this requirement.The apparent yield stress calculated for the gellan–xanthan mix indicated that the gums would onlyﬂow ordeform plastically when external forces were greaterthan the internal structural forces inherent in the gum.The average yield stress of the gum mix was1.515Pa,avalue similar to that of raw meat batter(Steffe1996).This value indicated that the gum mix was stable andthat at lower stresses,the gel would behave as a solid.The yield–stress range was approximately1Pa over atemperature range of35–50°C(Figure2).Any materialselected for micro-encapsulation should be able totraverse the upper gastro-intestinal tract(GIT)intactto protect the bacteria,but on arrival in the colon,theyield stress of the gum should be such that theimmobilized bacteria are released into the humanhost.Micro-encapsulation of ctis725The immobilizing material should not be comprised of materials with a high yield stress,as probiotics encap-sulated in such a material would not be released.The value obtained in this study for the gellan–xanthan gum mix indicated that stresses associated with GIT move-ments/peristalsis,could release the bacteria in the colon. The average yield stress of the gum indicated that gellan:xanthan would behave as a solid under low mechanical stress conditions,but would easily be broken by shear stresses such as those associated with chewing. Therefore ctis enclosed in a gellan:xanthan micro-capsule would have to be supplied in soft foods not requiring mastication,to prevent breaking of the capsule in the mouth.The yield stress value is also important when considering the means of addition of the capsules to foods such that the capsules remain intact and are not disrupted during process.The shear viscosity of the gum mix maintained an almost constant minimum value at temperatures between 46and61°C(Figures2and3).These temperatures are within the range for survival of Biﬁdobacterium.It is recommended that encapsulation using gellan:xanthan as the support matrix should be done between46and 61°C.For industrial application of micro-encapsulation it is of importance that the shear viscosity is constant over a broad15°C range,as opposed to a limited range such as1–3°C.The latter values would require critical temperature control to maintain a constant shear vis-cosity immediately prior to the extrusion process during which microdroplets are formed.Maintenance of a constant minimum shear viscosity would enhance con-trol over microcapsule size and shape,as well as permit optimization/standardization of mixing procedures nec-essary for even dispersal of ctis in the viscous gum held at these temperatures,prior to extrusion.Micro-encapsulationUsing the methods described above,micro-encapsula-tion of ctis in the gellan–xanthan matrix was successful.Estimation of viable c.f.u.in the capsules over periods of time indicated that oxygen present in the superposed airﬂow during microencapsulation was not lethal to ctis(data not shown).Visual examination of microcapsules using a light microscope indicated that capsules were generally oval/ round in shape,with a dense core comprised of ctis cells(Figure4).When the capsules were broken open and stained by the Gram method,bacterial cells isolated from the core stained gram positive and demonstrated a typical biﬁdus morphology.The shape of the capsules was ing the manual air knife technique described,we were unable to generate a constant capsule shape.Although the irreg-ularity of microcapsule shape often reported for probi-otics immobilized in edible gums is related to process, this problem requires further elucidation,as microcap-sule shape could inﬂuence both viability and numbers of entrapped bacteria.Shape will also determineﬂow properties of the capsules,important for industrial process.The use of a constant temperature andgumFigure4.Phase contrast view of a single capsule(·500),with adensely packed core of ctis surrounded by the capsule supportmaterial.726L.D.McMaster et al.ﬂow rate during extrusion,not possible in our encap-sulation technique,should improve uniformity of shape. Despite having a constant rate of airﬂow,a wide variation in the size distribution of the capsules was noted.This ranged from a maximum diameter of 2200l m to a minimum of20l m,with50%of the capsules having a diameter of<637l m,measured using laser diﬀractometry(Table1).Variables,including temperature,gum-and airﬂow rates,needle gauge etc, will inﬂuence capsule diameter.Although we com-menced with a constant temperature of55°C(i.e. minimum gum viscosity),we were unable to maintain a constant temperature immediately prior to or during extrusion.Hence gumﬂow rate,as inﬂuenced by viscosity,varied.For industrial production of capsules, a uniform bead size is important.Data obtained from the rheology of the gum mix,will assist in attaining this.Sun &Griﬃths(2000),using B.infantis,reported an average gellan:xanthan bead diameter of3mm produced by a simple dropping of the mixture through a syringe with a 21G syringe needle.Krasaekoopt et al.(2003),reported an average diameter of2–5mm for capsules synthesized by extrusion.By using a smaller gauge needle with a bevelled tip,and a superposed airﬂow,we were able to reduce the average size of gellan:xanthan beads by80%. Measurement of particle size using laser diﬀractometry identiﬁed capsules with a diameter as small as20l m. Adikhari et al.(2003),using co-acervation/emulsion to micro-encapsulate B.longum B6in j-carrageenan, reported capsule diameters of22–350l m,measured using laser diﬀractometry.It is generally recognized that capsules generated by co-acervation are smaller than those manufactured by extrusion.However,there are disadvantages associated with co-acervation.These include the undesirable trapping of the emulsifying oil in the microcapsule,which can alter foodﬂavour and reduce bacterial viability.In addition,co-acervation is costly,and bacterial numbers in these capsules is low and random(Krasaekoopt et al.2003).Diameter of micro-capsules is inﬂuenced by many variables,and obtaining a uniform size of capsules within a batch when operating under non-standard conditions is diﬃcult.To manufac-ture capsules of both equal and optimum diameter will be complex,but necessary for successful incorporation into foods to minimize problems associated with cell viability and food texture.Little has been reported with regard to optimum capsule size for addition to foods.It is known that particles with a diameter below3l m are undetected by the tongue(Singer&Dunn1990).Therefore,above this value,capsules can impart a gritty texture to foods not normally associated with this sensation.Adikhari et al. (2003)reported that when microcapsules were added to yoghurt at10%(w/v),there was an unfavourable consumer response due to the resultant grainy texture. The10%content of encapsulated B.longum was neces-sary to provide the RDA of the organism.Ideally,a uniform capsule diameter is such that sensory qualities of the food are not altered,whilst simultaneously delivering therapeutic doses of probiotic.The diameter must not adversely aﬀect viability of the organism.Truelstrup-Hansen et al.(2002)reported that alginate capsules should have a diameter of at least100l m to prevent a reduction in Biﬁdobacterium viability in simulated acidic gastric juices.With milk as the surrounding medium,this was not noted.Hence determination of optimum uniform capsule size is complex.The optimum/ideal size of gellan:xanthan microcapsules is unknown,but we have demonstrated that it is possible to generate capsules of diﬀerent diameters,and to based on results obtained,we are currently developing a novel temperature-controlled procedure,without superposed airﬂow,to produce mi-crocapsules of100–200l m diameter.We intend to extend rheology studies to these capsules to gain insight into storage conditions which inﬂuence gum integrity, and therefore shear strength of the capsules. Enumeration of ctis in the capsulesThe use of the Vibracell VCX750to release viable cells from the immobilizing matrix was successful.The method is reproducible,rapid and there was no detect-able reduction in c.f.u.often reported for methods requiring lengthy extraction procedures for entrapped cells(Sun&Grifﬁths2000;Truelstrup-Hansen et al. 2002;Adikhari et al.2003).Using concentrated washed ctis cells in the stationary phase of growth,with the microencapsulation technique described,we consistently achieved a high loading of log1010–ctis g)1 microcapsules(results not shown).Hence to consume the RDA of ctis required0.1g microcapsules as a single serving.This amount would exert minimum eﬀect on the texture of a soft food or beverage.Table1.A single analysis of microcapsule diameter and size distribution as determined for0.2g capsules(wet weight)using a Malvern Mastersizer S.Diam (l m)Volume(%)Diam(l m)Volume(%)Diam(l m)Volume(%)Diam(l m)Volume(%)4.880.0026.200.05140.58 1.42754.238.375.690.0030.530.08163.77 1.71878.678.816.630.0035.560.12190.80 2.061023.668.387.720.0041.430.18222.28 2.491192.567.479.000.0048.270.25258.95 3.041389.33 6.1710.480.0056.230.35301.68 3.711618.57 4.7012.210.0165.510.47351.46 4.521885.64 3.2214.220.0176.320.62409.45 5.422196.77 1.7516.570.0188.910.78477.01 6.332559.230.2719.310.02103.580.97555.717.162981.510.0022.490.03120.67 1.18647.417.853473.450.00D(v,0.1)186.1;D(v,0.5)637.00;D(v,0.9)1387.15.D[3,2]367.71;D[4,3]658.76.D(v,0.1)10%of the microcapsules were below186.1l m indiameter.D(v,0.5)50%of the microcapsules were below637.00l m indiameter.D(v,0.9)90%of the microcapsules were below1387.15l m indiameter.D[3,2]surface area mean diameter of the sphere having the samesurface area as the real particle.Micro-encapsulation of ctis727Survival of ctis in1M sodium phosphate buﬀer (pH6.8)Storage of immobilized ctis for21days in1M sodium phosphate buﬀer under aerobic conditions indicated that loss of viability of the cells stored at4 or22°C was less than0.5log cycle at both temperatures (Figure5).Survival was improved by storage at4°C. At the commencement of the storage time,the number of ctis in the capsules was log1011.6c.f.u.g)1. Therefore after21days storage,the level of viable ctis in0.1g microcapsule s remained in excess of the RDA.Storage of the microcapsules at4°C in a sodium phosphate buﬀer(pH6.8)represents a possible means of supply of the capsules to the food industry.In conclusion,we report successful incorporation of high numbers of viable ctis in gellan xanthan micro-capsules.Rheology of the gum mix provided valuable data for application to standardization of procedures for both small-and large-scale industrial production of microcapsules.Shear stress data showed that the type of foods/beverages suitable as delivery vehicles cannot undergo mastication prior to consumption.Such foods would include beverages as well as soft ctis was not inactivated in the presence of oxygen,even after 21days.As maintenance of anaerobic conditions, required for growth of the organism,is unlikely during both microencapsulation and subsequent industrial processes,this attribute is technologically essential. AcknowledgementsThanks are due to Ms H.Divey,Dept Chemical Engineering,University of Cape Town for assistance with Malvern diffractometry analyses.We are grateful to Ms L.Francis,Dept of Civil Engineering,Cape Technikon,for rheological analyses.Financial assistance from the National Research Foundation and Cape Technikon Seed Research Fund is gratefully acknowledged.ReferencesAdikhari,K.,Mustapha,A.&Grun,I.U.2003Survival and metabolic activity of microencapsulated Biﬁdobacterium in stirred yoghurt.Journal of Food Science68,275–280.Doyle,M.P.2003Food research trends–2003and beyond.Food Technology56,44–45.Guarner,F.,&Schaafsma,G.J.1998Probiotics.International Journal of Food Microbiology39,237–238.Huebner,H.&Buccholz,R.1999Microencapsulation.In The Encyclopedia of Bioprocessing Technology;Fermentation,Biocatal-ysis and Bioseparation,vol4.eds.Flickinger,M.C.&Drew,S.pp.1785–1798.New York:John Wiley and Sons.Inc.ISBN0-471-13822-3.Hughes,D.B.&Hoover,D.G.1991Biﬁdobacteria:their potential for use in American dairy products.Food Technology45,74–83. 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Truelstrup-Hansen,L.,Allan-Wotjas,P.M.,Jin,Y.-L.&Paulson,A.T.2002Survival of Ca-alginate microencapsulated Biﬁdobacte-rium spp.in milk and simulated gastrointestinal conditions.Food Microbiology19,35–45.728L.D.McMaster et al.。

产细菌素屎肠球菌的筛选鉴定及其抑菌特性

产细菌素屎肠球菌的筛选鉴定及其抑菌特性张明，罗强，魏婕，刘巧，罗璠*（西南民族大学生命科学与技术学院，青藏高原动物遗传资源保护与利用教育部重点实验室，四川成都610041）摘要：从采自四川红原的传统发酵酸乳中分离得到308 株疑似乳酸菌，通过96 孔板法初筛和平板打孔法复筛，筛选出1 株具有明显抑菌作用的菌株SC-Y112。

通过形态学观察、16S rDNA序列同源性分析和ropA管家基因序列同源性分析结合生理生化实验，鉴定该菌株为屎肠球菌（Enterococcus faecium）。

通过排酸及排除H2O2影响后，该菌株的发酵上清液仍具有明显抑菌活性，经蛋白酶处理后，抑菌效果明显消失或下降，说明发酵上清液中的抑菌成分具有蛋白质性质。

进一步探究该菌株所产细菌素的生物学特性、遗传稳定性及抑菌特性表明，该细菌素在菌株接种后6 h产生，12 h达到最高；菌株在连续传代86 代内，产细菌素能力较为稳定；细菌素在 pH 3.0～7.0的条件下稳定，在100 ℃处理30 min后仍具有抑菌活性；SC-Y112所产细菌素具有广谱抗菌性，对单核细胞性李斯特菌有明显的抑菌活性。

关键词：发酵酸乳；屎肠球菌；细菌素；抑菌Screening for and Identification for Bacteriocin-producing Enterococcus faecium and Its Antibacterial PropertiesZHANG Ming, LUO Qiang, WEI Jie, LIU Qiao, LUO Fan*(Key Laboratory of Qinghai-Tibet Plateau Animal Genetic Resource Reservation and Exploitation, Ministry of Education, College of Life Science and Technology, Southwest Minzu University, Chengdu610041, China)Abstract: A total of308 strains suspected of being Lactobacillus were isolated from traditional fermented yoghurt collected from Hongyuan, Sichuan province, and were screened by 96-well plate method and agar well diffusion method. Among these strains, SC-Y112 was selected for its obvious antibacterial effect. It was identified as Enterococcus faecium by morphological observation, 16S rDNA and ropA housekeeping gene sequencing analysis, and physiological and biochemical tests. After removing the influence of organic acid and hydrogen peroxide, the fermentation supernatant of the strain still had considerable antibacterial activity. The antibacterial effect decreased and even disappeared after being treated with protease, which indicated that the antibacterial components in the fermentation supernatant were proteins. Further, the biological characteristics, genetic stability and bacteriostatic characteristics of the bacteriocin produced by the strain were investigated, which showed that the bacteriocin was produced at 6 h and reached the maximum at 12 h after inoculation; the bacteriocin production ability was relatively stable within 86 consecutive generations; the bacteriocin was stable under the condition of pH 3.0-7.0 and heat-treatment at 100 ℃ for 30 min. The bacteriocin had a wide antibacterial spectrum, especially against Listeria monocytogenes.Keywords: fermented yogurt; Enterococcus faecium; bacteriocin; inhibitionDOI:10.7506/spkx1002-6630-20191213-147中图分类号：Q93.3 文献标志码：A 文章编号：1002-6630（2021）06-0171-07引文格式：张明, 罗强, 魏婕, 等. 产细菌素屎肠球菌的筛选鉴定及其抑菌特性[J]. 食品科学, 2021, 42(6): 171-177. DOI:10.7506/ spkx1002-6630-20191213-147. ZHANG Ming, LUO Qiang, WEI Jie, et al. Screening for and identification for bacteriocin-producing Enterococcus faecium and its antibacterial properties[J]. Food Science, 2021, 42(6): 171-177. (in Chinese with English abstract) DOI:10.7506/ spkx1002-6630-20191213-147. 收稿日期：2019-12-13基金项目：西南民族大学研究生“创新型科研项目”（CX2017SZ046）第一作者简介：张明（1993—）（ORCID: 0000-0003-4252-5342），男，硕士研究生，研究方向为饲料微生物。

microabscesses 名词解释

microabscesses1. IntroductionMicroabscesses are small, localized collections of pus that form within tissues as a result of infection or inflammation. These tiny abscesses typically measure less than 1 cm in diameter and are often found in organs such as the liver, lungs, or skin. While they may be small in size, microabscesses can have significant clinical implications and require prompt diagnosis and treatment.2. CausesMicroabscesses can be caused by various factors, including bacterial, fungal, or parasitic infections. Common pathogens that can lead to microabscess formation include Staphylococcus aureus, Streptococcus species, Escherichia coli, and Candida albicans. In some cases, microabscesses may also develop as a result of non-infectious conditions, such as autoimmune diseases or inflammatory processes.3. PathogenesisThe formation of microabscesses begins with the introduction of an infectious agent or the activation of the immune system. In response to the presence of pathogens or inflammatory stimuli, immune cells, such as neutrophils and macrophages, are recruited to the site of infection or inflammation. These immune cells release various chemical mediators, including cytokines and chemokines, which promote the recruitment and activation of additional immune cells.As the immune response progresses, neutrophils and macrophages engulfand destroy the invading pathogens. This process leads to the release of enzymes and toxic substances that can cause tissue damage. Additionally, the accumulation of dead immune cells and debris forms the core of the microabscess. Surrounding this core, there is a layer of inflammatory cells that attempt to contain the infection or inflammation.4. Clinical PresentationThe clinical presentation of microabscesses can vary depending on their location and underlying cause. In some cases, microabscesses may be asymptomatic and only discovered incidentally during imaging studies. However, when symptoms are present, they may include localized pain, swelling, redness, and warmth. Systemic symptoms, such as fever, chills, and malaise, may also be present, especially in cases of widespread microabscess formation.5. DiagnosisDiagnosing microabscesses can be challenging due to their small size and nonspecific clinical presentation. Imaging studies, such as ultrasound, computed tomography (CT), or magnetic resonance imaging (MRI), are often used to visualize the presence of microabscesses. These imaging modalities can help identify the location, size, and number of microabscesses, as well as any associated complications.In some cases, a tissue biopsy may be necessary to confirm the diagnosis. During a biopsy, a small sample of the affected tissue is obtained and examined under a microscope. The presence of inflammatory cells, necrosis, and microorganisms can provide valuable information regarding the underlying cause of the microabscesses.6. TreatmentThe treatment of microabscesses involves addressing the underlying cause and promoting healing of the affected tissues. In cases of bacterial infection, appropriate antibiotics are prescribed to target the specific pathogens involved. Antifungal or antiparasitic medications may be necessary in cases of fungal or parasitic microabscesses, respectively.Surgical drainage or aspiration of the microabscesses may be required in certain situations, particularly if there is a large collection of pusor if the microabscesses are causing significant symptoms or complications. Additionally, supportive measures, such as pain management and wound care, may be necessary to promote recovery and prevent further infection.7. ComplicationsIf left untreated, microabscesses can lead to various complications. For example, microabscesses in the liver can progress to liver abscesses, which may rupture or spread to other organs. Microabscesses in the lungs can cause pneumonia or lung abscesses. In some cases, microabscesses can also lead to the formation of septic emboli, which are small infected clots that can travel through the bloodstream and cause infections in distant sites.ConclusionMicroabscesses are small collections of pus that form within tissues asa result of infection or inflammation. They can have significantclinical implications and require prompt diagnosis and treatment. Understanding the causes, pathogenesis, clinical presentation, diagnosis, and treatment of microabscesses is crucial for healthcare professionalsin providing appropriate care to affected individuals and preventing complications.。

将科学知识用英语表达出来作文

将科学知识用英语表达出来作文Title: The Essence of Scientific Knowledge.In the vast expanse of human thought and inquiry, scientific knowledge stands as a beacon of rationality and progress. It is the foundation upon which we build our understanding of the world, explaining phenomena, predicting outcomes, and enabling us to make informed decisions. The journey of scientific discovery is an ongoing quest, one that involves observation, hypothesis, experimentation, and refinement.At its core, scientific knowledge is built on observation. Scientists are constantly on the lookout for patterns, trends, and anomalies in nature. These observations are the raw materials for further investigation. They might come from the tiniest particles in a laboratory experiment or the vastest reaches of the universe, but they all play a crucial role in shaping our understanding.Once observations are made, scientists form hypothesesto explain them. These hypotheses are testable explanations for why something happens the way it does. They are notmere guesses but are backed by existing evidence and theory. The scientific method demands that hypotheses befalsifiable, meaning there should be a way to prove them wrong. This ensures that scientific knowledge is always subject to scrutiny and refinement.Experimentation is the next step in the scientific process. Scientists design experiments to test their hypotheses under controlled conditions. These experiments might involve manipulating variables, collecting data, or making observations. The goal is to see if the hypothesis stands up to scrutiny and if it can predict or explain observed phenomena.If the experiments support the hypothesis, it begins to gain acceptance within the scientific community. However, even then, it remains tentative and subject to further testing and refinement. Science is an ongoing dialoguebetween observation, theory, and experiment, with knowledge constantly evolving and adapting as new evidence becomes available.Scientific knowledge is also cumulative. It builds upon the work of previous scientists, who have madecontributions to our understanding of the world. This cumulative nature of science means that knowledge accumulates over time, with each new discovery adding to our understanding of the universe.Moreover, scientific knowledge is objective. It is based on observable facts and reproducible experiments, rather than subjective opinions or individual beliefs. This objectivity is crucial for ensuring that scientificfindings can be trusted and relied upon. It allows us to make predictions, design technologies, and develop policies based on solid evidence.In conclusion, scientific knowledge is the foundation of our understanding of the world. It is built on observations, hypotheses, experiments, and cumulativelearning. Its objectivity and adaptability make it a powerful tool for explaining phenomena, predicting outcomes, and informing decision-making. As we continue to exploreand discover more about the universe, the importance of scientific knowledge will only grow.。

反对伪科技的作文英语

反对伪科技的作文英语I am strongly against pseudo-science. Pseudo-science refers to beliefs or practices that are presented as scientific, but lack supporting evidence or plausibility. It is important to differentiate between genuine scientific research and pseudo-science, as the latter can be misleading and potentially harmful.英文，I believe that pseudo-science is a serious issue that needs to be addressed. For example, there are many so-called "miracle cures" that are promoted as scientific breakthroughs, but in reality, they are not backed by credible scientific evidence. People may be drawn to these false promises and end up wasting their time and money, or even worse, putting their health at risk.中文，我认为伪科学是一个需要解决的严重问题。

例如，有许多所谓的“奇迹疗法”被宣传为科学突破，但实际上它们并没有可靠的科学证据支持。

人们可能会被这些虚假承诺所吸引，最终浪费时间和金钱，甚至更糟的是，危害自己的健康。

法布里珀罗基模共振英文

法布里珀罗基模共振英文The Fabryperot ResonanceOptics, the study of light and its properties, has been a subject of fascination for scientists and researchers for centuries. One of the fundamental phenomena in optics is the Fabry-Perot resonance, named after the French physicists Charles Fabry and Alfred Perot, who first described it in the late 19th century. This resonance effect has numerous applications in various fields, ranging from telecommunications to quantum physics, and its understanding is crucial in the development of advanced optical technologies.The Fabry-Perot resonance occurs when light is reflected multiple times between two parallel, partially reflective surfaces, known as mirrors. This creates a standing wave pattern within the cavity formed by the mirrors, where the light waves interfere constructively and destructively to produce a series of sharp peaks and valleys in the transmitted and reflected light intensity. The specific wavelengths at which the constructive interference occurs are known as the resonant wavelengths of the Fabry-Perot cavity.The resonant wavelengths of a Fabry-Perot cavity are determined bythe distance between the mirrors, the refractive index of the material within the cavity, and the wavelength of the incident light. When the optical path length, which is the product of the refractive index and the physical distance between the mirrors, is an integer multiple of the wavelength of the incident light, the light waves interfere constructively, resulting in a high-intensity transmission through the cavity. Conversely, when the optical path length is not an integer multiple of the wavelength, the light waves interfere destructively, leading to a low-intensity transmission.The sharpness of the resonant peaks in a Fabry-Perot cavity is determined by the reflectivity of the mirrors. Highly reflective mirrors result in a higher finesse, which is a measure of the ratio of the spacing between the resonant peaks to their width. This high finesse allows for the creation of narrow-linewidth, high-resolution optical filters and laser cavities, which are essential components in various optical systems.One of the key applications of the Fabry-Perot resonance is in the field of optical telecommunications. Fiber-optic communication systems often utilize Fabry-Perot filters to select specific wavelength channels for data transmission, enabling the efficient use of the available bandwidth in fiber-optic networks. These filters can be tuned by adjusting the mirror separation or the refractive index of the cavity, allowing for dynamic wavelength selection andreconfiguration of the communication system.Another important application of the Fabry-Perot resonance is in the field of laser technology. Fabry-Perot cavities are commonly used as the optical resonator in various types of lasers, providing the necessary feedback to sustain the lasing process. The high finesse of the Fabry-Perot cavity allows for the generation of highly monochromatic and coherent light, which is crucial for applications such as spectroscopy, interferometry, and precision metrology.In the realm of quantum physics, the Fabry-Perot resonance plays a crucial role in the study of cavity quantum electrodynamics (cQED). In cQED, atoms or other quantum systems are placed inside a Fabry-Perot cavity, where the strong interaction between the atoms and the confined electromagnetic field can lead to the observation of fascinating quantum phenomena, such as the Purcell effect, vacuum Rabi oscillations, and the generation of nonclassical states of light.Furthermore, the Fabry-Perot resonance has found applications in the field of optical sensing, where it is used to detect small changes in physical parameters, such as displacement, pressure, or temperature. The high sensitivity and stability of Fabry-Perot interferometers make them valuable tools in various sensing and measurement applications, ranging from seismic monitoring to the detection of gravitational waves.The Fabry-Perot resonance is a fundamental concept in optics that has enabled the development of numerous advanced optical technologies. Its versatility and importance in various fields of science and engineering have made it a subject of continuous research and innovation. As the field of optics continues to advance, the Fabry-Perot resonance will undoubtedly play an increasingly crucial role in shaping the future of optical systems and applications.。

微流控芯片技术及质谱技术用于细菌耐药性检测及耐药机制研究

Journal of China Pharmaceutical University 2023，54（6）：695 - 705学报微流控芯片技术及质谱技术用于细菌耐药性检测及耐药机制研究张冬雪，乔亮*（复旦大学化学系，复旦大学生物医学研究院，上海 200433）摘要细菌耐药性严重影响全球公共卫生安全。

抗生素错用和滥用不仅没有达到治疗细菌感染性疾病的效果，反而会刺激细菌发生DNA损伤修复反应（SOS反应），加剧细菌耐药性的进化和耐药菌的传播。

本文聚焦于耐药菌，简明介绍细菌耐药性与SOS反应，系统概述了质谱技术、微流控技术及其联用技术在细菌检测及细菌耐药机制研究中的应用。

本文为细菌耐药性相关的药物靶点挖掘及新药开发提供理论参考，以期发展细菌耐药性快速检测新方法和抑菌新方法，推动临床细菌感染性疾病的诊断与治疗。

关键词细菌耐药；耐药机制；微流控技术；质谱检测；组学分析中图分类号O65；R318 文献标志码 A 文章编号1000 -5048（2023）06 -0695 -11doi：10.11665/j.issn.1000 -5048.2023060203引用本文张冬雪，乔亮.微流控芯片技术及质谱技术用于细菌耐药性检测及耐药机制研究［J］.中国药科大学学报，2023，54（6）：695–705.Cite this article as：ZHANG Dongxue，QIAO Liang. Microfluidic chip and mass spectrometry-based detection of bacterial antimicrobial resis⁃tance and study of antimicrobial resistance mechanism［J］.J China Pharm Univ，2023，54（6）：695–705.Microfluidic chip and mass spectrometry-based detection of bacterial antimi⁃crobial resistance and study of antimicrobial resistance mechanism ZHANG Dongxue, QIAO Liang*Department of Chemistry, and Institutes of Biomedical Sciences, Fudan University, Shanghai 200433, ChinaAbstract Bacterial antimicrobial resistance (AMR) is a globally serious problem that threatens public health security.Misuse and abuse of antibiotics cannot achieve the effect of treating bacterial infectious diseases, but will trigger the SOS response of bacteria, exacerbating the evolution of bacterial AMR and the spread of resistant bacteria.This article focuses on antibiotic-resistant bacteria, briefly introduces the pathogenesis of bacterial AMR and SOS response, and systematically summarizes the determination and mechanism study of bacterial AMR based on microfluidics and mass spectrometry.This article provides theoretical basis for AMR-related drug target mining and new drug development, aiming to develop new methods for rapid detection of bacterial AMR and new methods for bacteria inhibition, and promote the diagnosis and treatment of clinical bacteria infectious diseases. Key words bacterial antimicrobial resistance; mechanism of antimicrobial resistance; microfluidics; mass spec⁃trometry detection; omics analysisThis study was supported by China Postdoctoral Science Foundation (No.2022M720806)细菌是最常见的病原微生物之一，是引起大部分感染性疾病的重要原因。

Antibiotics (anti-microbials)

However, many bacteria are now resistant to antibiotics and some are resistant to all known agents.
ቤተ መጻሕፍቲ ባይዱ New drugs are continually being introduced to combat evolving patterns of resistance.
Disruption of the microbial cell membrane
Essentially, affect cell membrane transportation in and out
Increases permeability of membrane
External influences have greater effect Microbe death
Interrupt this leads to new microbes being susceptible to external influences
Cell ruptures Microbe death
Eg. Penicillinsm cephalosporins, vancomycin and bacitracin
Antibiotics (anti-microbials)
Dr Gareth Noble (and Dr Sue Jordan)
Introduction
Kock & Pasteur over a century
Discovered ‘cause and effect’ Once found, needed an agent to be created to handle them

microcassette词源

在古代文字的年代，微型录音带技术出现之前，人们使用各种方法来进行录音，比如用钢丝、磁带、磁片等等。

但这些方法存在诸多不便，比如录音设备太大、重，难以携带，并且录音的时候容易有噪音干扰。

于是，20世纪60年代，美国一家公司开发出了微型录音带技术，解决了以上问题，大大提高了录音的便携性和质量。

这种技术的诞生，使得人们可以方便地进行录音，并大大促进了现代信息时代的发展。

“microcassette”一词源自英语，由“micro”（微小的）和“cassette”（盒式录音磁带）两部分组成。

从词源来看，“microcassette”一词直接展现了这种技术产品的主要特点——微小。

微型录音带技术是当时录音技术发展的一个重要里程碑，也是当时科技创新的一项重要成果。

这个词的词源反映了人们对技术的不断创新和进步。

微型录音带技术的发展，不仅让人们的生活变得更加便利，也推动了很多行业的发展。

在新闻业、法律业、学术研究等领域，微型录音带技术的应用极大地提高了工作效率和信息记录的准确性。

微型录音带技术也深刻地改变了音乐产业，推动了录音技术的不断创新，使得音乐的传播和保存更加便捷和高效。

从个人来讲，我对微型录音带技术的词源有着特别的兴趣。

这个词源富有创新和技术感，让我想起了人类对科技不断探索和创新的精神。

微型录音带技术的诞生，让我对科技的发展和应用有了更深刻的认识和理解。

从这个词源出发，我对微型录音带技术的发展历程和影响有了更多的思考和研究，也更加关注当下科技的发展和应用。

微型录音带技术的词源“microcassette”反映了当时技术创新和应用的特点，展现了人类对科技不断探索和创新的精神。

这种技术的应用，不断推动了各行业的发展，也促进了人类社会的进步。

我对这个词源的个人理解，也让我对科技的发展和应用有了更深刻的认识和思考。

希望未来，科技能够更好地造福人类，让我们的生活变得更加便利和美好。

微型录音带技术的词源“microcassette”展现了当时技术创新和应用的特点，也反映了人类对科技不断探索和创新的精神。

microscopic医学意思

microscopic医学意思微观意味着非常小的尺寸。

在医学中，微观通常用于描述细胞、组织和器官的结构和功能。

因此，"microscopic医学"可以解释为研究微观尺度下医学方面的科学和技术。

在医学中，研究微观尺度的重要性不言而喻。

通过观察和研究细胞和组织的微观结构，医生和科学家可以更好地理解疾病的发生和发展机制，并找到相应的治疗方法。

以下是一些与微观医学相关的重要领域。

1. 细胞生物学：细胞是生命的基本单位，通过研究细胞的形态、结构和功能，可以了解细胞在生物体内的作用和相互作用。

微观观察细胞的形态和组织，可以帮助医生诊断疾病，并确定治疗方法。

2. 组织学：组织学研究组织的结构和功能。

通过使用显微镜观察和分析组织切片，可以了解组织中细胞的排列和组织的特征。

组织学在病理学中起着重要作用，通过观察组织切片的变化，可以诊断疾病，如肿瘤、炎症和感染等。

3. 免疫学：免疫学研究机体的免疫系统，以及免疫系统对疾病的反应。

微观观察免疫细胞如淋巴细胞、巨噬细胞和粒细胞等的形态和功能，可以了解免疫系统的工作原理，以及免疫反应与疾病发展之间的关系。

4. 病理学：病理学研究疾病的本质和发展过程。

通过观察和分析病理标本，可以了解疾病对细胞和组织的影响。

微观观察病理标本的异常细胞和组织结构，有助于医生诊断疾病，并制定相应的治疗方案。

5. 分子生物学：分子生物学研究生物分子的结构和功能。

通过观察和分析DNA、RNA和蛋白质等生物分子的微观结构和功能，可以了解基因表达和细胞信号传导的机制。

微观分析分子水平的细胞过程，有助于理解疾病的分子机制，并开发相应的药物和治疗方法。

在微观医学研究中，显微镜是不可或缺的工具。

显微镜可以放大细胞和组织的结构，使其可见并进行观察和分析。

随着科技的进步，显微镜的种类也日益增多，包括光学显微镜、电子显微镜、荧光显微镜和共聚焦显微镜等。

这些显微镜不仅可以放大细胞和组织的结构，还可以观察细胞内的分子过程和信号传导。

Confocal Microscopy

Confocal Microscopy Denis SemwogerereEric R.WeeksEmory University,Atlanta,Georgia,U.S.A.INTRODUCTIONA confocal microscope creates sharp images of a speci-men that would otherwise appear blurred when viewed with a conventional microscope.This is achieved by excluding most of the light from the specimen that is not from the microscope’s focal plane.The image has less haze and better contrast than that of a conven-tional microscope and represents a thin cross-section of the specimen.Thus,apart from allowing better observation ofﬁne details it is possible to build three-dimensional(3D)reconstructions of a volume of the specimen by assembling a series of thin slices taken along the vertical axis.BACKGROUNDConfocal microscopy was pioneered by Marvin Minsky in1955while he was a Junior Fellow at Harvard University.[1]Minsky’s invention would perform a point-by-point image construction by focusing a point of light sequentially across a specimen and then collect-ing some of the returning rays.By illuminating a single point at a time Minsky avoided most of the unwanted scattered light that obscures an image when the entire specimen is illuminated at the same time.Additionally, the light returning from the specimen would pass through a second pinhole aperture that would reject rays that were not directly from the focal point.The remaining‘‘desirable’’light rays would then be col-lected by a photomultiplier and the image gradually reconstructed using a long-persistence screen.To build the image,Minsky scanned the specimen by moving the stage rather than the light rays.This was to avoid the challenge of trying to maintain sensitive alignment of moving ing a60Hz solenoid to move the plat-form vertically and a lower-frequency solenoid to move it horizontally,Minsky managed to obtain a frame rate of approximately one image every10sec.MODERN CONFOCAL MICROSCOPYModern confocal microscopes have kept the key ele-ments of Minsky’s design:the pinhole apertures and point-by-point illumination of the specimen.Advances in optics and electronics have been incorporated into current designs and provide improvements in speed, image quality,and storage of the generated images. Although there are a number of different confocal microscope designs,this entry will discuss one general type—the other designs are not markedly different.[2] The majority of confocal microscopes image either by reﬂecting light off the specimen or by stimulating ﬂuorescence from dyes(ﬂuorophores)applied to the specimen.The focus of this entry will be onﬂuores-cence confocal microscopy as it is the mode that is most commonly used in biological applications.The difference between the two techniques is small.There are methods that involve transmission of light through the specimen,but these are much less common.[3] FluorescenceIf light is incident on a molecule,it may absorb the light and then emit light of a different color,a process known asﬂuorescence.At ordinary temperatures most molecules are in their lowest energy state,the ground state.However,they may absorb a photon of light (for example,blue light)that increases their energy causing an electron to jump to a discrete singlet excited state.[4]In Fig.1,this is represented by the top black line.Typically,the molecule quickly(within10À8sec) dissipates some of the absorbed energy through colli-sions with surrounding molecules causing the electron to drop to a lower energy level(the second black line). If the surrounding molecules are not able to accept the larger energy difference needed to further lower the molecule to its ground state,it may undergo sponta-neous emission,thereby losing the remaining energy, by emitting light of a longer wavelength(for example, green light).[5]Fluorescein is a commonﬂuorophore that acts this way,emitting green light when stimulated with blue excitation light.The wavelengths of the exci-tation light and the color of the emitted light are mate-rial dependent.Microscopy in theﬂuorescence mode has several advantages over the reﬂected or transmitted modes. It can be more sensitive.Often,it is possible to attach ﬂuorescent molecules to speciﬁc parts of the specimen, making them the only visible ones in the microscopeEncyclopedia of Biomaterials and Biomedical Engineering DOI:10.1081/E-EBBE-120024153Copyright#2005by Taylor&Francis.All rights reserved.1and it is also possible to use more than one type of ﬂuorophore.[6]Thus,by switching the excitation light different parts of the specimen can be distinguished.FLUORESCENCE MICROSCOPYIn conventional ﬂuorescence microscopy a dyed speci-men is illuminated with light of an appropriate wave-length and an image is formed from the resulting ﬂuorescent light.In Fig.2the excitation light is blue and the emitted light is green.The microscope uses a dichroic mirror (also called a ‘‘dichromatic mirror’’)that reﬂects light shorter than a certain wavelength but transmits light of longer wavelength.Thus,the light from the main source is reﬂected and passes through the objective to the sample,while the longer-wavelengthlight from the ﬂuorescing specimen passes through both the objective and the dichroic mirror.This particular type of ﬂuorescence microscopy,in which the objective used by the illuminating light is also used by the ﬂuor-escing light in conjunction with a dichroic mirror,is called epiﬂuorescence.In the case of reﬂected light microscopy,a beamsplitter is used in place of the dichroic mirror.CONFOCAL MICROSCOPYTo understand confocal microscopy it is instructive to imagine a pair of lenses that focuses light from the focal point of one lens to the focal point of the other.This is illustrated by the dark blue rays in Fig.3.The light blue rays represent light from another point inFig.2Basic setup of a ﬂuorescence microscope.Light from the source is reﬂected off the dichroic mirror toward the specimen.Returning ﬂuorescence of a longer wavelength is allowed to pass through thedichroic mirror to the eye-piece.(View this art in color at .)high Fig.1Mechanism of ﬂuorescence.The horizon-tal lines indicate quantum energy levels of the molecule.A ﬂuorescent dye molecule is raised to an excited energy state by a high-energy photon.It loses a little energy to other molecules and drops to a lower excited state.It loses the rest of the energy by emitting light of a lower energy.(View this art in color at .)the specimen,which is not at the focal point of the left-hand-side lens.(Note that the colors of the rays are purely for purposes of distinguishing the two sets—they do not represent different wavelengths of light.)Clearly,the image of the light blue point is not at the same location as the image of the dark blue point.(Recall from introductory optics that points do not need to be at the focal point of the lens for the system of lenses to form an image.)In confocal microscopy,the aim is to see only the image of the dark blue point.[1]Accordingly,if a screen with a pinhole is placed at the other side of the lens sys-tem,then all of the light from the dark point will pass through the pinhole.a Note that at the location of the screen the light blue point is out of focus.Moreover,most of the light will get blocked by the screen,resulting in an image of the light blue point that is signiﬁcantly attenuated compared to the image of the dark blue point.To further reduce the amount of light emanating from ‘‘light blue’’points,the confocal microscope setup minimizes how much of the specimen is illuminated.Normally,in ﬂuorescence microscopy the entire ﬁeld of view of the specimen is completely illuminated,making the whole region ﬂuoresce at the same time.Of course,the highest intensity of the excitation light is at the focal point of the lens,but the other parts of the specimen do get some of this light and they do ﬂuoresce.Thus,light at a ‘‘dark blue’’point may include light that has been scattered from other ‘‘light blue’’points,thereby obscur-ing its ﬂuorescence.To reduce this effect the confocal microscope focuses a point of light at the in-focus dark blue point by imaging a pinhole aperture placed in front of the light source.[1]Thus,the only regions that are illu-minated are a cone of light above and below the focal (dark blue)point (Fig.9A).Together the confocal microscope’s two pinholes sig-niﬁcantly reduce the background haze that is typical of a conventional ﬂuorescence image,as shown in Fig.5.Because the focal point of the objective lens forms an image where the pinhole =screen is,those two points are known as ‘‘conjugate points’’(or alternatively,the specimen plane and the pinhole =screen are conjugate planes).The pinhole is conjugate to the focal point of the lens,hence the name ‘‘confocal’’pinhole.HOW DOES A CONFOCAL MICROSCOPE WORK?The confocal microscope incorporates the ideas of point-by-point illumination of the specimen and rejec-tion of out-of-focus light.One drawback with imaging a point onto the speci-men is that there are fewer emitted photons to collect at any given instant.Thus,to avoid building a noisy image each point must be illuminated for a long time to collect enough light to make an accurate measure-ment.[1]In turn,this increases the length of time needed to create a point-by-point image.The solution is to use a light source of very high intensity,which Minsky did with a zirconium arc lamp.The modern choice is a laser light source,which has the additional beneﬁt of being available in a wide range of wavelengths.In Fig.4A the laser provides the intense blue excitation light.The light reﬂects off a dichroic mirror,which directs it to an assembly of vertically and hori-zontally scanning mirrors.These motor-driven mirrors scan the laser across the specimen.Recall that Minsky’s invention kept the optics stationary and instead scanned the specimen by moving the stage back and forth in the vertical and horizontal directions.As awkward (and slow)as that method seems it does have among others the following two major advantages:[7] The specimen is everywhere illuminated axially,rather than at different angles as in the case of the scanning mirror conﬁguration,thereby avoiding optical aberrations.Thus,the entire ﬁeld of view is illuminated uniformly.The ﬁeld of view can be made larger than that of the static objective by controlling the amplitude of the stage movements.aActually not all the light from the focal point reaches the pinhole.Some of it is reﬂected and absorbed by the optics in between.Fig.3Rejection of light not incident from the focal plane.All light from the focal point that reaches the screen is allowed through.Light away from the focal point is mostly rejected.(View this art in color at .)In Fig.4the dye in the specimen is excited by the laser light and ﬂuoresces.The ﬂuorescent (green)light is descanned by the same mirrors that are used to scan the excitation light (blue)from the laser and then passes through the dichroic mirror.Thereafter,it is focused onto the pinhole.The light that makes it through the pinhole is measured by a detector such as a photomultiplier tube.In confocal microscopy,there is never a complete image of the specimen because at any instant only one point is observed.Thus,for visualization the detec-tor is attached to a computer,which builds up the image one pixel at a time.For a 512Â512-pixel image this is typically done at a frame rate of 0.1–30Hz.The large range in frame rates depends on anumber of factors,the most important of which will be discussed below.The image created by the confocal microscope is of a thin planar region of the specimen—an effect referred to as optical sectioning.Out-of-plane unfocused light has been rejected,resulting in a sharper,better-resolved image.Fig.5shows an image created with and without optical sectioning.THREE-DIMENSIONAL VISUALIZATIONThe ability of a confocal microscope to create sharp optical sections makes it possible to build 3D rendi-tions of the specimen.Data gathered from a series ofFig.4Basic setup of a confocal microscope.Light from the laser is scanned across the specimen by the scanning mirrors.Opti-cal sectioning occurs as the light passes through a pinhole on its way to the detector.(View this art in color at .)Fig.5Images of cells of spirogyra generated with and without optical sectioning.The image in (B)was created using a slit rather than a pinhole for out-of-focus light rejection.Most of the haze asso-ciated with the cell walls of the ﬁlamentous algae is absent,allowing clearer distinction of the differ-ent parts.optical sections imaged at short and regular intervals along the optical axis are used to create the 3D recon-struction.Software can combine the 2D images to create a 3D rendition.Representing 3D information in a meaningful way out of 2D data is nontrivial,and a number of different schemes have been devel-oped.Fig.6shows a 3D reconstruction,from slices of a suspension of 2m m diameter colloidal particles using ‘‘alpha blending’’—a technique that combines images by ﬁrst making each of their individual pixels less or more transparent according to a computed weight called the ‘‘alpha’’value.[8]The result is a 3D-like structure.OTHER CONSIDERATIONSA confocal microscope,as with every instrument,has some limitations and often compromises must be made to optimize performance.The following is an outline of some of the most important of them.ResolutionAs with conventional microscopy,confocal microscopy has inherent resolution limitations due to diffraction.In the discussion above it is assumed that the point source used produces a point of light on the specimen.In fact it appears in the focal plane as an Airy disk,whose size depends on the wavelength of the light source and the numerical aperture of the objective lens.[9](The numerical aperture of a lens is a measure of how well it gathers light.)The graph of Fig.7B shows a plot of the intensity of light as a function of radius of an Airy disk—the image is circularly symmetric,as shown in Fig.7A.The Airy disk limits the maximum resolution that can be attained with the confocal microscope—the best reso-lution is typically about 200nm.Ideally,the image of a point would just be a single intense point right at radius ¼0.However,the ﬁnite size of the Airy disk sets the scale for which details can be resolved.According to the Rayleigh criterion,the minimum separation between two Airy disks for which they are distinguishable is equal to their radius.This corresponds to the maximum of one Airy disk superimposed on the minimum of the other.Resolution along the optical axis is also limited by diffraction effects.As in the lateral direction there is a periodic,but elliptical distribution of intensity in the shape of an Airy disk.[10]Pinhole SizeThe optical sectioning capability of a confocal micro-scope derives from having a pinhole to reject out-of-focus light rays.In turn,the strength of the optical sectioning (the rate at which the detected intensity drops off in the axial direction)depends strongly on the size of the pinhole.[11]It is tempting to assume that making the pinhole as small as possible is the best choice.However,as the pinhole size is reduced,so too are the number of photons that arrive at the detector from the specimen.This may lead to a reduced signal-to-noise ratio.To offset the weaker signal more ﬂuorescence is needed from the specimen.This usually can be done,to a limit,by raising the intensity of the excitation light.But high intensities can damage the specimen,and in the case of ﬂuorescence,also degrade the ﬂuorophore.Moreover,it has been shown that optical sectioning does not improve considerably with the pinhole size below a limit that approximates the radius of the ﬁrst zero of the Airy disk.[2,11](Note that the study considered imaging in coherent mode;however,the result still qualitatively applies to inherently incoherent confocal ﬂuorescence microscopy.)Thus,a good approximation is to make the pinhole about the size of the Airy disk.Intensity of Incident LightAn important component of a confocal microscope is the photodetector that captures light from the speci-men.In confocal ﬂuorescence imaging the pinhole along with the optics preceding it signiﬁcantly reduce the intensity of the emission that reaches the detector.Thus,the detector’s sensitivity and noise behavior are vitally important.[3]The sensitivity is characterized by the quantum efﬁciency,which,as in any measurement involving quantum interactions,is limited by Poisson statistics.[12]That is,the accuracy of the measurement is improved by increasing the number ofphotonsFig.6Three-dimensional reconstruction of a series of 2D images of PMMA spheres suspended in a cyclohexyl-bromide and decalin solution.The image was created using ‘‘alpha blending.’’arriving at the detector.In practical terms this can be done by averaging data from many frames—which has the drawback of slowing down the effective frame rate of the microscope.Or,it can be done by increasing the intensity of the ﬂuorescence signal.Fluorescence can be increased by dyeing the speci-men with a larger concentration of ﬂuorophore mole-cules or by raising the intensity of the excitation light.However,each of these methods increases excita-tion up to some limit.For high ﬂuorophore concentra-tions,the individual molecules can quench each other.[5]They may also reduce the amount of ﬂuores-cence deep inside the specimen—ﬂuorophores nearest the light source can absorb enough light to signiﬁ-cantly reduce the portion available to the rest of the specimen.[3]Increasing ﬂuorescence by increasing the excitation light intensity leads eventually to saturation of the ﬂuorophore.Higher intensities drive a larger fraction of ﬂuorophore molecules into excited states,which in turn leads to a smaller fraction of ground state molecules.Ultimately,the rate at which ﬂuoro-phores are excited matches their decay rate,causing ground state depopulation.Fluorescence then ceases to increase with excitation intensity.[13]FluorophoresAmong the most important aspects of ﬂuorescence confocal microscopy is the choice of ﬂuorophore.It is typically inﬂuenced by several factors.The ﬂuorophore should tag the correct part of the specimen.It must be sensitive enough for the given excitation wavelength.For living specimens it should not signiﬁcantly alter the dynamics of the organism;and an extra considera-tion is the effect of the specimen on the ﬂuorophore—its chemical environment can affect the position of the peaks of the excitation and emission spectra.[14]PhotobleachingA major problem with ﬂuorophores is that they fade (irreversibly)when exposed to excitation light (Fig.8).Although this process is not completely under-stood,it is believed in some instances to occur when ﬂuorophore molecules react with oxygen and =or oxy-gen radicals and become nonﬂuorescent.[13,15]The reaction can take place after a ﬂuorophore molecule transitions from the singlet excited state to the triplet excited state.Although the fraction of ﬂuorophores that transitions to the triplet state is small,its lifetime is typically much longer than that of the singlet state.This can lead to a signiﬁcant triplet state ﬂuorophore population and thus to signiﬁcant photobleaching.[5]Several strategies have been developed to reduce the rate of photobleaching.[5,13]One method is to simply reduce the amount of oxygen that would react with the triplet excited states.This can be done by displa-cing it using a different gas.[5]Another method is by the use of free-radical scavengers to reduce the oxygen radicals.Shortening the long lifetime of the triplet excited state has also been shown to be effective.[16]Other ways include using a high numerical aperture lens to collect more ﬂuorescence light and thus use less excitation light.[17]Also,keeping the magniﬁcation as0.30.250.20.150.10.05–10 –5 0 5 10RadiusA BI n t e n s i t yFig.7Airy disk similar to that of an image of a very small particle.(A)The image is ‘‘overexposed’’and in reverse color to allow distinction of the faint secondary peak.(B)A graph of the intensity change with radius.low as is permissible spreads the excitation light over a larger area,thereby reducing the local intensity.While photobleaching makes ﬂuorescence micro-scopy more difﬁcult,it is not always undesirable.One technique that takes advantage of it is ﬂuorescence photobleaching recovery (FPR)or ﬂuorescence recovery after photobleaching (FRAP).It involves exposing a small region of the specimen to a short and intense laser beam,which destroys the local ﬂuorescence,and then observing as the ﬂuorescence is recovered by transport of other ﬂuorophore molecules from the surrounding region.Quantities such as the diffusion coefﬁcient of the dyed structures can then be determined.[18]LIVING CELLSConfocal microscopy has been used effectively for the 3D study of dynamics in living cells.However,the ima-ging of living specimens adds the challenge of maintain-ing the life and normal function of the organism.[19]There are of course difﬁculties involved in preparing the sample for viewing as is the case in conventional microscopy.But on top of that the effect of photo-damage on the specimen caused by the focused high-intensity excitation light must be taken into account.This is compounded by the repeated exposure required for tracking the cellular dynamics—a problem that is worsened for 3D data collection.Fluorescence also introduces the problem of the ﬂuorophore inﬂuencing the cell behavior as well as the risk that oxygen mole-cules reacting with ﬂuorophores in triplet excited states may generate free radicals that damage the cell.[19]Despite the challenges,a wide variety of sophisticated ﬂuorophores have been developed to study different aspects of cell biology.They are designed to mark speci-ﬁc parts of the cell interior and often can simply be intro-duced to the cell wall.[19]The ﬂuorophores molecules make their way into the cell and attach to the intracellu-lar structures of interest such as the mitochondria and the Golgi apparatus.This is not always the case,how-ever,as some ﬂuorophores must be injected directly into the cell.[19]Labeling is even applied to the study of ‘‘nonphysical’’structures of the cell—some ﬂuoro-phores have been developed for the measurement of dynamic processes such as membrane potentials and ion concentrations.[5]Multicolor FluorescenceTo distinguish between small features such as proteins within a cell it is useful to tag them with different ﬂuor-ophores and image them as separate colors.There are two ways to do this:in one method ﬂuorophores are selected to correspond with the wavelengths of a multi-line laser and in the other their response to the same excitation wavelength causes emission at different wavelengths.[20]In both cases the resulting emission is separated with appropriate ﬁlters and directed to dif-ferent detectors.However,there can be cross talk between channels of the emitted light.[14]For most of the commonly used ﬂuorophores there is usually some overlap between their emission spectra,making perfect channel separation impossible by ﬁltering alone.To ﬁrst order this can be corrected by determining the level of overlap of emission of each individual ﬂuoro-phore into the channels of the other ﬂuorophores and subtracting it out mathematically.FAST CONFOCAL MICROSCOPYMost confocal microscopes generate a single image in 0.1–1sec.[21]For many dynamic processes this rate may be too slow,particularly if 3D stacks of images are required.Even for a single 2D image,slow frame rates translate into long exposure times of the specimen to intense laser light,which may damage it or cause photobleaching.Two commonly used designs that can capture images at high speed are the Nipkow disk confocal microscope and a confocal microscope that uses an acousto-optic deﬂector (AOD)for steering the excitation light.Acousto-Optic DeﬂectorSpeeding up the image acquisition rate can be achieved by making the excitation light beam scan morequicklyFig.8Dyed suspension of densely packed polymethyl-methacrylate beads with signiﬁcant photobleaching.The rec-tangular region near the center faded after about 30sec of exposure to excitation light.across the specimen.For most confocal microscopes, the limitation is the galvanometers that move the mir-rors back and forth in the characteristic saw-tooth pat-tern.The usual conﬁguration is a slow vertical scan combined with a rapid scan in the horizontal direction. For512Â512-pixel images at a frame rate of$30 frames per second the horizontal galvanometer would have to scan at a frequency of approximately 30Â512¼15kHz,which is beyond its normal cap-ability of several kilohertz.[21]Fast horizontal scans are achieved using an AOD. An AOD is a device that deﬂects light by creating a dif-fraction grating out of a crystal using sound waves. The sound waves are high-frequency pressure waves that locally alter the refractive index of the crystal. Thus,when monochromatic light shines through the crystal,it forms sharp fringes at an angle of deﬂection that depends on the wavelength of the acoustic pres-sure waves.Rapidly changing the frequency,and hence the wavelength,of the sound waves allows quick and accurate steering.[22]The major disadvantage of AODs is that they are wavelength sensitive.That is,different wavelengths experience different degrees of deﬂection.This presents a problem forﬂuorescence microscopy because the light fromﬂuorescence has a different wavelength from the excitation light and thus cannot be descanned by the AOD as is done in Fig.4with the mirrors.To get around this problem the confocal microscope is designed to descan only along the vertical direction that is controlled by the slow galvanometer and to then collect the light using a slit rather than a pinhole.[21]The penalty is a reduction in the amount of optical sectioning and a very slight distortion in the image caused by the loss of circular symmetry.Nevertheless,it is possible to obtain high-quality images(Fig.5B)using slits.[10]Note that descanning is not a problem for monochromatic reﬂected light microscopy because the incident and reﬂected light are of the same wavelength.Nipkow DiskAn even faster technique is the so-called Nipkow disk microscope.Instead of scanning a single point across the specimen the Nipkow disk microscope builds an image by passing light through a spinning mask of pin-holes,thereby simultaneously illuminating many dis-crete points.In the setup by Xiao,Corle,and Kino the mask is a disk with thousands of pinholes arranged in interleaved spirals.[23]At any given time only a small section of the disk with a few hundred pinholes is illu-minated.The light travels through the pinholes and onto the specimen and the returning light passes through the same pinholes for optical sectioning.As the disk spins,the entire specimen is covered several times in a single rotation.At a rotation of40revolu-tions per second Xiao,Corle,and Kino were able to generate over600frames per second.The disadvantage of the Nipkow disk microscope is that only a small fraction($1%)of the illuminating light makes it through the pinholes to the specimen.[24] While that is not a major problem when operating in reﬂected light mode,it can lead to a weak signal and poor imaging inﬂuorescence mode.However,with strongﬂuorophores an image as good as that of a con-focal laser scanning microscope can be obtained.[24] Increasing transmission would require an increase in pinhole size,which would lead to less effective optical sectioning and xy resolution.TWO-PHOTON MICROSCOPYA fast-growing technique that is related to confocal microscopy and also provides excellent optical section-ing is two-photon microscopy.It addresses a fundamen-tal drawback of confocal laser scanning microscopy: that the beam also excites the specimen above and below the focal-plane(Fig.9A).For each2D imagecreated Fig.9One-photon vs.two-photon emission from a solution ofﬂuorescein.(A)One-photon emission shows strongﬂuorescence at the waist,but also ﬂuorescence in a cone-like region above and below the focal volume.(B)In two-photon emissionﬂuorescence is limited to the focal volume. (From Ref.[25].)(View this art in color at www. .)。

超高分辨率显微镜推进纳米生物学研究说明书

第26卷第12期2014年12月V ol. 26, No. 12Dec., 2014生命科学Chinese Bulletin of Life Sciences文章编号：1004-0374(2014)12-1255-11DOI: 10.13376/j.cbls/2014181收稿日期：2014-12-01*通信作者：E-mail:**************.cn超高分辨率显微镜推进纳米生物学研究任煜轩*，于　洋，王　艳(中国科学院上海生命科学研究院生物化学与细胞生物学研究所国家蛋白质科学中心·上海(筹)，上海 201210)摘　要：超高分辨率显微镜是近年来生命科学领域重要的研究手段之一。

2014年诺贝尔化学奖颁发给超高分辨率显微技术领域的三位科学家，以表彰他们在该领域所作出的杰出贡献。

超高分辨率技术的典型代表有受激损耗、结构光照明以及单分子定位等。

这些技术的出现使得传统光学显微镜难以分辨的细胞器、分子等细节信息可以被观察到，帮助科学家从纳米尺度认识细胞内分子结构、定位以及相互作用。

关键词：超高分辨率；受激损耗；结构光照明；光敏定位；随机光学重构中图分类号：Q-336 文献标志码：A Super-resolution microscopy in NanobiologyREN Yu-Xuan*, YU Yang, WANG Yan(National Center for Protein Sciences Shanghai, Institute of Biochemistry and Cell Biology,Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201210, China )Abstract: Super-resolution microscopy is one of the advanced means for the study of life sciences. The 2014 Nobel prize in Chemistry was awarded to three scientists for their contributions to the super-resolution microscopy. Representative techniques achieving super-resolution are stimulated-emission, structured-illumination, and single-molecule localization. The advancement of these techniques enable the visualization of the detail in cell organelle, macromolecules, and the localization of molecules. Such kind of information was unresolvable under traditional optical microscopes and will help understand the structure, location, interaction of molecules within cells in nanometer scale.Key words: super-resolution; stimulated emission depletion; structured illumination; photo-activation localization; stochastic optical reconstruction生命科学处处充满着神奇。

纳米氧化锌对两种蔬菜种子发芽及幼苗生长的影响

材料[6]。ZnO NPs 一般通过农业化学品的施用、大气司。直径 9 cm 的玻璃培养皿与滤纸、30% H2O（2 优级
沉降、降水和灌溉等途径进入土壤[7-8]。ZnO NPs 在土纯）以及硫酸锌（ZnSO4·7H2O，优级纯）均购自天津海
壤中的迁移能力较小，最终导致其在土壤中的ቤተ መጻሕፍቲ ባይዱ度远斯凯尔科技发展有限公司；试验所用去离子水为实验
种子萌发是植物生命周期的一个关键时期，也是
drum sativum）光合色素含量[8]和莴苣（Lactuca sativa）的定纳米颗粒的比表面积。
生物量及净光合速率[14]。然而，相关研究证实 ZnO NPs 1.2.2 ZnO NPs 悬浮液的制备
及其释放出来的Zn2+对植物具有一定的毒性[15]。ZnO NPs
通过向超纯水中加入适量的 ZnO NPs 制备 NPs
农业资源与环境学报 2021, 38（1）: 72-78
Journal of Agricultural Resources and Environment
林茂宏，沈玫玫，吴佳妮，等 . 纳米氧化锌对两种蔬菜种子发芽及幼苗生长的影响[J]. 农业资源与环境学报, 2021, 38（1）: 72-78. LIN Mao-hong, SHEN Mei-mei, WU Jia-ni, et al. Effects of zinc oxide nanoparticles on germination and seedling growth of two vegetables [J]. Journal of Agricultural Resources and Environment, 2021, 38（1）: 72-78.

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