FluorCam叶绿素荧光成像一
Fluorcam多光谱荧光成像技术及其应用
FluorCam多光谱荧光成像技术（Multi-color FluorCam）自上世纪90s年代PSI公司首席科学家Nedbal教授与公司总裁Trtilek博士等首次将PAM脉冲调制叶绿素荧光技术与CCD技术结合在一起，成功研制生产FluorCam叶绿素荧光成像系统（Nedbal等，2000）以来，FluorCam叶绿素荧光成像技术得到长足发展和广泛应用，先后有封闭式、开放式（包括标准版和大型版）、便携式叶绿素荧光成像系统，及显微叶绿素荧光成像系统、大型叶绿素荧光成像平台（包括移动式、样带式、XYZ三维扫描式等）等，近些年还进一步发展了PlantScreen植物表型成像分析平台（Phenotyping）（有传送带版、XYZ三维扫描版及野外版等）及多光谱荧光成像技术。
Multi-color FluorCam多光谱荧光成像技术包括多激发光－多光谱荧光成像技术和UV 紫外光激发多光谱荧光成像技术：1.多激发光－多光谱荧光成像技术：通过光学滤波器技术，仅使特定波长的光（激发光）到达样品以激发荧光，同时仅使特定波长的激发荧光到达检测器。
不同的荧光发色团（如叶绿素或GFP绿色荧光蛋白等）对不同波长的激发光“敏感”并吸收后激发出不同波长的荧光，根据此原理可以选配2个或2个以上的激发光源、绿波轮及相应滤波器，对不同波长荧光（多光谱荧光）进行成像分析。
如FluorCam便携式GFP/Chl.荧光成像仪及FluorCam封闭式GFP／Chl.荧光成像系统具备红光和兰光及相应滤波器，可以对GFP和叶绿素荧光成像分析；FluorCam开放式多光谱荧光成像系统可以进一步选配不同颜色的激发光，如除红光、蓝光外，还可选配绿色光源及相应滤波器，以对YFP进行荧光成像分析等；2.UV紫外光激发多光谱荧光成像技术：长波段UV紫外光（320nm－400nm）对植物叶片激发，可以产生具有4个特征性波峰的荧光光谱，4个波峰的波长为兰光440nm（F440）、绿光520nm（F520）、红光690nm（F690）和远红外740nm（F740），其中F440和F520统称为BGF，由表皮及叶肉细胞壁和叶脉发出，F690和F740为叶绿素荧光Chl-F。
植物表型组学研究技术(一)FluorCam 叶绿素荧光成像技术
植物表型组学研究技术（一） ——FluorCam叶绿素荧光成像技术FluorCam叶绿素荧光成像技术Rousseau等（High throughput quantitative phenotyping of plant resistance using chlorophyll fluorescence image analysis.Plant Methods, 2013, 9:17），利用FluorCam开放式叶绿素荧光成像系统作为高通量表型分析平台，采用图像阈值分割等分析方法，对植物病原体感染进行了定量分析检测，根据Fv/Fm将感染分为不同阶段／等级，特别是可以将用其它方法难以分辨出来的感染前期加以分辨，并对5个品种的菜豆对普通细菌性疫病的抗性进行了定量分析评价。
PSI公司首席科学家Nedbal教授与公司总裁Trtilek博士等首次将PAM叶绿素荧光技术（Pulse Amplitude Modulated technique——脉冲调制技术）与CCD技术结合在一起，于1996年在世界上成功研制生产出FluorCam叶绿素荧光成像系统（Heck等，1999；Nedbal等，2000；Govindjee and Nedbal, 2000）。
FluorCam叶绿素荧光成像技术成为上世纪90年代叶绿素荧光技术的重要突破，使科学家对光合作用与叶绿素荧光的研究一下子进入二维世界和显微世界，广泛应用于植物生理生态、植物胁迫与抗性监测、作物育种、植物表型分析等。
不同于其它成像分析技术，FluorCam叶绿素荧光成像只对叶绿素荧光波段敏感，可以有效避免环境光的干扰，特异性、高灵敏度反映植物生理生态状况。
主要功能特点如下：1)高灵敏度CCD，时间分辨率可达50帧／秒，有效抓取叶绿素荧光瞬变；可选配高分辨率CCD，分辨率1392x1040像素，用于气孔功能成像分析、稳态荧光如GFP荧光测量等2)具备完备的自动测量程序（protocol），可自由对自动测量程序进行编辑：a)Fv/Fm：测量参数包括Fo，Fm，Fv，QY等b)Kautsky诱导效应：Fo，Fp，Fv，Ft_Lss，QY，Rfd等荧光参数c)荧光淬灭分析：Fo，，Fm，Fp，Fs，Fv，QY，ΦIINPQ，Qp，Rfd，qL等50多个参数d)光响应曲线LC：Fo，Fm，QY，QY_Ln等荧光参数e)PAR吸收f)GFP等静态荧光测量g)OJIP与JIP-test（FKM与封闭式荧光成像系统）：Fo，Fj，Fi，P 或Fm，Mo（OJIP曲线初始斜率）、OJIP固定面积、Sm（对关闭所有光反应中心所需能量的量度）、QY、PI等26个参数3)自动重复实验功能，可无人值守自动循环完成选定的实验程序，重复次数及间隔时间客户自定义，成像测量数据自动按时间日期存入计算机4)FluorCam成像分析软件：具在线功能（Live）、实验程序选配功能（Protocols）、成像预处理功能（Pre-processing）及成像分析结果展示报告功能（Result）四大功能模块a)在线功能（live）：可对仪器和样品进行在线测试调试、快照、显示实验进度、在线显示荧光瞬变动态视频等b)实验程序选配功能（protocols）：可选配不同的实验程序，并可对实验程序进行编辑、设置、储存（以备以后使用同样的实验程序）等c)成像预处理功能：可浏览整个测量视频及任何点、任何区域的荧光动态变化曲线，可进行“选区操作”或“分级操作”（图像阈值分割功能）；选区操作可对成像进行自动或手动选区（ROI），还可使用“模具”包括多孔板模具、培养皿模具、桌面模具进行模具选区；分级操作具备荧光强度刻度标尺和四个“游标”，通过移动4个游标可以将成像按不同强度划分成不同的荧光范围组进行分析处理，可设置不同的阈值进行图像阈值分割d)结果展示报告功能：可展示所有选区（ROI）的叶绿素荧光参数值及其图像、每个参数的频率直方图及每个ROI的荧光动态图等，可对原数据（kinetic）、叶绿素荧光参数等导出到excel表，还可对每个参数成像图存储成位图5)数据分析具备“信号计算再平均”模式（算数平均值）和“信号平均再计算模式”两种功能模式，在高信噪比的情况下选用“信号计算再平均”模式，在低信噪比的情况下选择“信号平均再计算”模式以过滤掉噪音带来的误差FluorCam叶绿素荧光参数：参数符号概念描述Size 面积（像素值），经校准可测量实际面积Fo 暗适应后的最小荧光Fo_Dn 暗松弛最小荧光，红外光诱导PSIFo_Ln 光适应后的最小荧光，红外光诱导PSIFo_Lss 光适应后稳态最小荧光，红外光诱导PSIFm 暗适应后最大荧光Fm_Dn 暗松弛最大荧光Fm_Ln 光适应最大荧光Fm_Lss 光适应稳态最大荧光Fp Kautsky诱导效应最大荧光Ft_Dn 暗松弛即时荧光Ft_Ln 光适应即时荧光Ft_Lss 光适应稳态荧光Fv Fm-FoNPQ_Dn 暗松弛非光化荧光淬灭，＝(Fm-Fm_Dn)/Fm_DnNPQ_Ln 光适应非光化荧光淬灭，＝(Fm-Fm_Ln)/Fm_LnNPQ_Lss 稳态非光化荧光淬灭，＝(Fm-Fm_Lss)/Fm_LssqP_Dn 暗松弛光化学荧光淬灭，＝(Fm_Dn−Ft_Dn)/Fm_Dn−Fo_DnqP_Ln 光适应光化学淬灭，＝(Fm_Ln−Ft_Ln)/(Fm_Ln−Fo_Ln)qP_Lss 稳态光适应光化学淬灭，＝(Fm_Lss−Ft_Lss)/(Fm_Lss−Fo_Lss)qL_Ln 基于“Lake”模型的光适应光化学淬灭qL_Lss 基于“Lake”模型的稳态光适应光化学淬灭QY_Dn 暗松弛光量子效率，＝(Fm_Dn−Ft_Dn)/Fm_DnQY_Ln或ΔF/Fm 光适应光量子效率，＝(Fm_Ln−Ft_Ln)/Fm_LnQY_Lss 稳态光量子效率，＝(Fm_Lss−Ft_Lss)/Fm_LssFv/Fm或QY_max 最大光量子效率Fv/Fm_Ln 光适应光量子效率，＝(Fm_Ln−Fo_Lss)/Fm_LnFv/Fm_Lss 稳态光量子效率，＝(Fm_Lss−Fo_Lss)/Fm_LssRfd_Ln 光适应荧光衰减率，用于评估植物活力，＝(Fp−Ft_Ln)/Ft_LnRfd_Lss 稳态荧光衰减率，用于评估植物活力，＝(Fp−Ft_Lss)/Ft_Lss除上述叶绿素荧光参数外，还可以成像测量PAR吸收、植物光谱反射指数NDVI等，叶片大小（或植物大小）可以反映植物的生长等。
FluorCam叶绿素荧光成像文献 2012 Genetic Analysis of the Hox Hydrogenase
Genetic Analysis of the Hox Hydrogenase in theCyanobacterium Synechocystis sp.PCC 6803Reveals Subunit Roles in Association,Assembly,Maturation,and Function *□SReceived for publication,June 15,2012,and in revised form,November 7,2012Published,JBC Papers in Press,November 8,2012,DOI 10.1074/jbc.M112.392407Carrie Eckert ‡1,Marko Boehm ‡§,Damian Carrieri ‡,Jianping Yu ‡,Alexandra Dubini ‡,Peter J.Nixon §,and Pin-Ching Maness ‡From the ‡Biosciences Center,National Renewable Energy Laboratory,Golden,Colorado 80401and the §Department of Life Sciences,Imperial College London,South Kensington Campus,London SW72AZ,United KingdomHydrogenases are metalloenzymes that catalyze 2H ؉؉2e ؊7H 2.A multisubunit,bidirectional [NiFe]-hydrogenase has been identified and characterized in a number of bacteria,including cyanobacteria,where it is hypothesized to function as an electron valve,balancing reductant in the cell.In cyanobac-teria,this Hox hydrogenase consists of five proteins in two func-tional moieties:a hydrogenase moiety (HoxYH)with homology to heterodimeric [NiFe]-hydrogenases and a diaphorase moiety (HoxEFU)with homology to NuoEFG of respiratory Complex I,linking NAD(P)H 7NAD(P)؉as a source/sink for electrons.Here,we present an extensive study of Hox hydrogenase in the cyanobacterium Synechocystis sp.PCC 6803.We identify the presence of HoxEFUYH,HoxFUYH,HoxEFU,HoxFU,and HoxYH subcomplexes as well as association of the immature,unprocessed large subunit (HoxH)with other Hox subunits and unidentified factors,providing a basis for understanding Hox maturation and assembly.The analysis of mutants containing individual and combined hox gene deletions in a common parental strain reveals apparent alterations in subunit abun-dance and highlights an essential role for HoxF and HoxU in complex/subcomplex association.In addition,analysis of indi-vidual and combined hox mutant phenotypes in a single strain background provides a clear view of the function of each subunit in hydrogenase activity and presents evidence that its physiolog-ical function is more complicated than previously reported,with no outward defects apparent in growth or photosynthesis under various growth conditions.Hydrogenase enzymes are a unique and diverse family of metalloenzymes widely distributed throughout Archaea,Pro-karyotes,and some unicellular Eukaryotes that catalyze the reduction/oxidation of H ϩ/H 2.These enzymes are classified by their metal-containing active sites and include [Fe]-hydroge-nases,[FeFe]-hydrogenases,and [NiFe]-hydrogenases (1).The [NiFe]-hydrogenases are minimally heterodimeric,consisting of a large catalytic subunit and a small subunit containing at least one [FeS]cluster that functions in electron transfer to and from the large subunit (1).The maturation of [NiFe]-hydroge-nases involves at least six maturation proteins (HypABCDEF)essential for the assembly of the [NiFe]active site.Some [NiFe]-hydrogenases additionally require a specialized protease that cleaves the large subunit’s C terminus,a step required for hydrogenase function (2).Research in Escherichia coli suggests that the hydrogenase small subunit only associates with the large subunit after it is fully processed (3).Among the diverse hydrogenases,the bidirectional [NiFe]-hydrogenase (Hox)in cyanobacteria is of great interest for basic biological study as well as for the development of solar hydrogen production technologies (4–6).NAD(P)-linked Hox hydrogenases have been identified and characterized in cyano-bacteria (4,5),the Gram-positive bacterium Rhodococcus opacus (7,8),the Gram-negative bacterium Ralstonia eutropha (6),and the purple sulfur photosynthetic bacteria Thiocapsa roseopersicina and Allochromatium vinosum (9–12).These Hox hydrogenases are multimeric with at least four related sub-units expressed from a single operon.HoxH and HoxY form the hydrogenase moiety and are homologous to the large and small subunits of prototypical heterodimeric [NiFe]-hydrogenases,respectively (1).The [FeS]cluster-containing subunits HoxF,HoxU,and,in some organisms,HoxE form the diaphorase moi-ety (13,14)that catalyzes the oxidation/reduction of NAD(P)H/NAD(P)ϩ(via FMN and NAD binding sites in HoxF)coupled to the hydrogenase moiety (HoxYH)(10,12–15).The R.eutropha Hox hydrogenase does not contain HoxE and instead harbors an unrelated fifth subunit,HoxI,which functions in linkage to NADPH (16).*This work was supported by the National Renewable Energy Laboratory’sLaboratory Directed Research and Development Program (to P.M.,J.Y.,C.E.,and D.C.),the United States Department of Energy Fuel Cell Technol-ogies Program (contract number DE-AC36-08-GO28308)(to P.M.and J.Y.),the United States Department of Energy Biological and Environmental Research Program (contract KP160103)(to M.B.and A.D.),and Engineer-ing and Physical Sciences Research Council Grant (EP/F002070X/1)(to M.B.and P.N.).□SThis article contains supplemental Table 1and Figs.1–5.1To whom correspondence should be addressed:Biosciences Center,NREL,15013Denver West Pkwy.,Golden,CO 80401.Tel.:303-384-6891;Fax:303-384-7836;E-mail:carrie.eckert@.THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL.287,NO.52,pp.43502–43515,December 21,2012Published in the U.S.A.at NATL RENEWABLE ENERGY, on January 16, 2013 Downloaded from /content/suppl/2012/11/08/M112.392407.DC1.htmlSupplemental Material can be found at:Purification of the Hox hydrogenase has been performed in some of the organisms it has been characterized in with varied results.In R.eutropha,the purified Hox complex is Hox-FUYHI2,although HoxI(not present in other characterized Hox hydrogenases)dissociates easily upon more stringent puri-fication conditions(which is why the complex was initially characterized as HoxFUYH)(16).Other purifications in R.eutropha utilizing gene knockouts and ectopic expression demonstrate that HoxYH and HoxFU subcomplexes can be iso-lated,although these subcomplexes were reportedly unstable (13).In A.vinosum(12)and the cyanobacterium Gleocapsa alpicola CALU743(17),attempted purifications of native com-plex resulted in isolation of only HoxYH,whereas in T.roseop-ersicina(18)and Synechocystis(19,20),intact HoxEFUYH complexes were purified.Purification of the Synechocystis Hox hydrogenase by Schmitz et al.(19)resulted in a dimer of a 1:1:1:1:1HoxE/F/U/Y/H complex via a series of column chro-matography steps,whereas Germer et al.(20)purified a com-plex with a0.2:2:2:1:1HoxE/F/U/Y/H ratio using a StrepII-tagged HoxF,raising questions about Hox complex assembly and association in vivo.The physiological role of Hox hydrogenase varies,depending on the organism in which it is present,but it generally functions in anoxic/micro-oxic conditions due to a reversible inactivationby O2(1).In T.roseopersicina,Hox hydrogenase catalyzes H2production under dark,fermentative conditions and in lightwhen thiosulfate is present and alternatively functions in H2 uptake in light when O2is absent(11).The Hox hydrogenase ofR.eutropha(soluble hydrogenase)functions in H2oxidation linked to the regeneration of NADH in support of carbon fixa-tion(6).In cyanobacteria,Hox hydrogenase is expressed under both anaerobic and aerobic conditions(4,21)but is only active under dark,fermentative conditions and in the transition fromdark to light prior to inhibition by O2generated during photo-synthesis(22–24).Respiration and nitrate assimilation mutantsexhibit increased H2photoevolution rates under low O2condi-tions,whereas defects in photosynthetic reactions/ratios have been reported in Hox hydrogenase mutants(15,22–26).There-fore,it is hypothesized that the Hox hydrogenase functions as an electron valve for cells by H2production/oxidation in response to changes in redox states(25,26).HoxEFU are the only homologs for NuoEFG of respiratory complex I in cyano-bacteria,but there is no evidence to date that these diaphorase subunits play any role in respiration(14,27–30).In this work,we generated a series of Hox hydrogenase mutants in a common parental strain,deleting individual hox genes or combinations of hox genes in the unicellular cyanobac-terium Synechocystis sp.PCC6803.This comprehensive collec-tion of hox mutants enabled us to perform systematic studies of mutation effects on complex/subcomplex formation and com-position,subunit abundance,and hydrogenase activity.In addi-tion,we also provide data for growth,photosynthesis,and fer-mentation in a hox operon deletion mutant compared with wild type(WT),revealing that some previously reported hox mutant phenotypes may have been due to differences in strain back-grounds(31,32).EXPERIMENTAL PROCEDURESStrain Background/Construction—All hox mutants weregenerated in a Synechocystis sp.PCC6803glucose-tolerant strain from Teruo Ogawa,who also provided the whole operon deletion,hoxϪ::hygromycin resistance.The hoxHϪdeletion was constructed in a pSMART LC-Amp vector(Lucigen,Inc.) containing a3.6-kb region spanning hoxY and hoxH cut with SspI(removing816bp of hoxH)to insert the kanamycin resist-ance cassette from pUC4K.All remaining hox deletion strains were created by two-step fusion PCR(33)for open reading frame(ORF)replacement,combiningϳ600-bp5Ј-and3Ј-flanking regions for each hox gene ORF replaced with the fol-lowing antibiotic resistance gene ORFs:hoxEϪ::aac3ia(gen-tamicin resistance),hoxFϪ::ermC(erythromycin resistance), hoxUϪ::aph(kanamycin resistance),and hoxYϪ::dfra17(spec-tinomycin resistance).An slr0168neutral site integration vec-tor containing a plastocyanin(petE)promoter for ectopic gene expression(34)was altered by the addition of a225-bp frag-ment from pETDuet containing the T7terminator into the PpuMI restriction site.PCR primers to amplify HoxYH or HoxH from genomic DNA were constructed with sequence encoding His6/HRV3C protease site at the N terminus and5Ј-and3Ј-specific SapI sites for insertion behind the petE pro-moter following restriction digestion and ligation.The pPSBA2KS vector for the integration behind the psbAII pro-moter(35)was altered by removal of a SalI site via partial digest and blunting to allow for retention of the kanamycin resistance gene.PCR primers were designed for amplification and inser-tion of an N-terminal His6-tagged hoxE gene between NdeI and SalI sites.Transformations were conducted by incubatingϳ1g of linear purified fusion PCR products or targeted integra-tion vectors with200l of cells(adjusted to OD730ϭ2.5fromcell cultures at OD730ϭ0.2–0.5)for6h,followed by the addi-tion of2ml of BG11,24-h outgrowth in culture tubes under standard growth conditions,and plating of200l on BG11 plates with antibiotics as required for selection(200g/ml hygromycin or kanamycin,100g/ml gentamicin,erythromy-cin,spectinomycin,or chloramphenicol).Strain Growth—Cultures were inoculated into BG11medium(ATCC medium616)supplemented with3M NiCl2,20m M TES(unbuffered),100m M NaHCO3,and antibiotics as required(one-half concentrations as used on plates listedabove)at an initial OD730ϭ0.05and were grown by shaking inculture flasks with5%CO2under50microeinsteins2(E)mϪ2 sϪ1continuous light from cool white fluorescent bulbs.Cul-tures were generally grown to logarithmic/linear growth phase(OD730ϭ0.2–0.8)for analysis.FPLC Analysis of Synechocystis sp.PCC6803WT Soluble Extract—Soluble extract was prepared from a WT50-ml liquidculture at an OD730Ͼ1(stationary phase).Cells were har-vested,resuspended,and washed twice in ACA buffer(750m M ⑀-amino caproic acid,50m M BisTris/HCl,pH7.0,0.5m M EDTA).Approximately200l of glass beads(150–212m;2The abbreviations used are:E,microeinsteins;MV,methyl viologen;BN,blue native;PSI and PSII,photosystem I and II,respectively;ETR,electron transport rate;BisTris,2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol.Cyanobacterial Hox Hydrogenase Complex Association and Functionat NATL RENEWABLE ENERGY, on January 16, Downloaded fromSigma-Aldrich)were added to the cell suspension(ϳ500l), and cells were broken using a Disruptor Genie digital multi-place vortexer(Scientific Industries)at4°C for5min,setting 3,000,followed by centrifugation for10min at maximum speed in a microcentrifuge.Subsequently,2l of DNase I(10,000 units/ml,Thermo Scientific)was added,and the sample was spun again in an ultracentrifuge at100,000ϫg for30min at 4°C.For FPLC,the resulting supernatant was normalized toOD650ϭ10(typically,the OD650of a1:20dilution was mea-sured).To solubilize any remaining thylakoid membrane frag-ments,-dodecyl-D-maltoside was added to a final concentra-tion of0.5%(w/v)from a10%(w/v)stock solution.100l of this soluble extract was loaded onto a Superdex20010/300size exclusion column(GE Healthcare)connected to an Akta Puri-fier FPLC system(GE Healthcare).The sample was run at a flow rate of0.5ml minϪ1with50m M Tris,pH7.5,100m M NaCl,and 0.03%(w/v)-dodecyl-D-maltoside as the buffer.0.5-ml frac-tions were collected by a Frac-950fraction collector(GE Healthcare).A high molecular weight marker mix(GE Health-care)was run as a control for relative protein sizes in the frac-tions(supplemental Fig.1).Protein Preparations,Two-dimensional Blue Native/SDS-PAGE,Affinity Purification,and Western Blotting—Soluble extracts for two-dimensional blue native/SDS-PAGE(BN/ SDS-PAGE)were prepared as described for FPLC analysis above for WT and hox mutant strains,and the extracts werenormalized to an A650ϭ0.6.-Dodecyl-D-maltoside was addedto a final concentration of0.5%(w/v),and the same volume ofCoomassie loading solution(750m M⑀-amino caproic acid,5% (w/v)Coomassie-G)was added to the sample.Subsequently,20l of the sample was loaded per lane and run on a12%(w/v) polyacrylamide BN PAGE first dimension gel.The proteincomplexes in the first dimension gel strips were denatured for1h in solubilization buffer(66m M Na2CO3,2%(w/v)SDS,2%(v/v)-mercaptoethanol,and4M urea)prior to being layered on17.5%(w/v)polyacrylamide,6M urea two-dimensional SDS-polyacrylamide gels(36).The resultant two-dimensional gels were either Coomassie-stained,silver-stained(37),or electro-blotted onto nitrocellulose membranes.For WT and hox mutant one-dimensional Western blots and affinity purifications,cells were resuspended in20m M phos-phate buffer,pH7.4,plus protease inhibitors(Fermentas)and broken by bead disruption as described above.Protein concen-trations were determined by Bradford assay(Fermentas),and levels of each sample were adjusted toϳ100g/ml.For purifi-cation of His6-tagged proteins/complexes,Co2ϩbeads(Pierce)were added to cleared lysates and incubated for1h at4°C,and washes were performed using5times bead volume with20m M phosphate buffer,pH7.4,with or without0.5M NaCl through reloadable columns(Pierce).Beads were resuspended in a50% slurry in buffer.To rule out nonspecific binding of Hox proteins to beads,untagged WT samples were also subjected to the same regime,and a lack of Hox proteins in bead samples was verified by Western blot(data not shown).Samples for SDS-PAGE were boiled in1ϫLaemmli sample buffer(Bio-Rad),and10l of each was loaded onto precast TGX Stain-free Any kDa gels (Bio-Rad)and transferred onto a PVDF membrane(Bio-Rad). For Western blotting,the SnapID system(Millipore)was used according to the manufacturer’s instructions.Membranes were blocked in5%BSA,1ϫPBST,and primary and secondary anti-bodies were diluted in1%BSA,1ϫPBST.Polyclonal rabbit primary antibodies developed for this study were generated usingϳ20-amino acid C-terminal peptides(␣-HoxE,-F,-U,-Y,and-H(C-terminal);␣-HoxF and␣-HoxY from these prepara-tions are not shown but were used in quantitation of one-di-mensional Western blots)and a HoxH internal peptide(amino acids300–318)that allows recognition of both processed and unprocessed HoxH(YenZym,San Francisco,CA).␣-HoxY and ␣-HoxF antibodies were also generated in rabbit using full-length E.coli expressed protein(SeqLab).␣-Rps1(Agrisera;notshown)and␣-PsaD antibodies(38)were used as loading con-trols for normalization of band intensities for relative protein level quantitation.Secondary incubation was performed with Clean Blot HRP(Pierce),and Cell Biosciences ChemiWest or Pierce Dura Chemiluminescent reagents were used for signal development.Images and quantitation analysis were processed using a Cell Biosciences FluoroCam Q Gel imaging system. Hydrogenase Assays—Cells grown to similar logarithmicgrowth densities(OD730ϭ0.2–0.8)were concentrated toOD730ϭ2.5,and200l was added to600l of assay mixture containing Triton X-100(0.05%,w/v),phosphate buffer(50 m M,pH7),and either methyl viologen(MV;1m M final)or NADH(1m M final)in2-ml HPLC vials sealed with anaerobic septa.The mixture was sparged with argon for10min,100l of anaerobic sodium dithionite(10m M final)was added,and the mixture was incubated with shaking for30min at37°C.The reaction was stopped by the addition of100l of20%(w/v) trichloroacetic acid,and100l of the1-ml headspace was ana-lyzed using a gas chromatograph(Agilent1100)equipped with a Molecular Sieve5A column,a thermal conductivity detector,and argon as the carrier gas.To measure in vivo H2production,cells were spun down and resuspended to OD730ϭ2.5in2ml of BG11containing5m M glucose,placed in a15-ml culture tube, sealed with anaerobic septum,sparged with argon for30min, and incubated at30°C in the dark for24h.A100-l sample from the13-ml headspace was analyzed using a gas chromato-graph as above.Determination of Doubling Times—Log phase precultures (10ml)of WT and hoxϪstrains were used to inoculate400-ml capacity Photobioreactor culture vessels(FMT400,Photon System Instruments)without the addition of antibiotic to growth medium.Illumination parameters were programmed as listed in Table1.Optical density was measured every1–5min at wavelength maximum680nm and recorded digitally,alongwith pH and O2concentration(not shown).Culture vessels were maintained at30°C and continuously bubbled at a rate of ϳ250ml/min in air or argon supplemented with2%CO2.Opti-cal density values versus time were fitted to a single exponential function to determine doubling times.Fluorescence Measurements—Dark-adapted quantum effi-ciency(Fv/Fm)and effective quantum efficiency under illumi-nation(⌬F/FmЈ)of WT and hoxϪstrain suspensions from log phase cultures were measured in multiwell plates with a closed FluorCam(FC800-C/1010,Photon System Instruments)and used to calculate effective electron transport rates.⌬F/FmЈwas determined for cells under continuous illumination for at leastCyanobacterial Hox Hydrogenase Complex Association and Functionat NATL RENEWABLE ENERGY, on January 16, Downloaded from3min at each light intensity with dark adaption between exper-iments.Cells were dark-adapted for at least 5min prior to measurement and were measured in biological and technical triplicate (minimum number of samples per type ϭ9).RESULTSComposition of Hox Hydrogenase Protein Complexes —Re-ported subunit composition and stoichiometry of purified Hox hydrogenase varies in the literature (12,16–20),raising ques-tions about its in vivo structure.Therefore,we assessed frac-tionation of the Hox hydrogenase complex by performing size exclusion FPLC analysis of Synechocystis WT soluble extracts followed by Western blotting with Hox subunit-specific anti-bodies (Fig.1).As expected,all five subunits,HoxE (18.8kDa),HoxF (57.8kDa),HoxU (26.2kDa),HoxY (20.0kDa),and HoxH (52.9kDa),clearly co-fractionate (fractions 10–14).The band intensities of the diaphorase (HoxEFU)and hydrogenase (HoxYH)subunits fluctuate in a related manner,consistent with HoxYH and HoxEFU subcomplexes observed in purifica-tions of other Hox hydrogenases (12,16,17).The presence of what is probably monomeric HoxE (fractions 20–22)suggests that HoxE may dissociate relatively easily from the complex.Interestingly,unprocessed,immature HoxH co-fractionates with other Hox proteins,including processed HoxH (fractions 10and 16–18),implying association of unprocessed HoxH withother complex subunits prior to maturation and full complex assembly (Fig.1,HoxH (open arrows )and HoxH (C-terminal)blots).In addition,unprocessed HoxH is clearly present in higher molecular weight complexes (fractions 8and 9),reveal-ing its association with other unknown proteins (possibly mat-uration factors)prior to its cleavage.Results from size exclusion FPLC imply the presence of Hox complexes and possible subcomplexes,but co-fractionation does not provide enough evidence to confirm subunit associa-tion.To verify Hox protein complex/subcomplex associations in soluble extracts,we performed two-dimensional BN/SDS-PAGE followed by Western blotting (Fig.2).Using this approach,we were able to identify a complex containing all five Hox subunits,again consistent with the composition of the purified pentameric complex reported in the literature (19,20).The apparent molecular mass of this complex is ϳ160kDa,close to the calculated mass based on the predicted sizes of the individual subunits (175kDa).We note that no dimer of this pentameric hydrogenase protein complex could be reliably detected in our analysis despite previous reports (19).However,we were able to detect the presence of HoxFUYH,HoxEFU,and HoxFU subcomplexes.We also failed to reliably detect a HoxYH subcomplex,yet there was a weak antibody signal for the HoxH subunit with a higher apparent molecular massthanFIGURE 1.FPLC and Western blot analysis of WT Synechocystis sp.PCC 6803Hox hydrogenase.The crude soluble fraction of WT Synechocystis sp.PCC 6803was separated by FPLC,and the resulting fractions were analyzed by Western blotting.A ,silver-stained TGX Any kDa gradient SDS-polyacrylamide gel (Bio-Rad)of starting material (Sol .Extr .)and fractions 1–22(for the chromatogram at A 280and A 420see supplemental Fig.1).B–G ,Western blotting of FPLC fractions with the indicated Hox subunit-specific antibodies.Arrowheads in F indicate the signal for unprocessed HoxH.Cyanobacterial Hox Hydrogenase Complex Association and Functionat NATL RENEWABLE ENERGY, on January 16, 2013 Downloaded fromthat of the free HoxH protein alone (Fig.2G ,asterisk ),suggest-ing the association of HoxH with another protein.Monomeric subunits were also detected in the low molecular mass region of the Western blots,with the HoxE,HoxH,and HoxU subunits all accumulating to significant levels when compared with lev-els of each associated with subcomplexes.We were unable to consistently detect unprocessed HoxH in any of these sub-complexes,suggesting that its possible association may only be transient.Nevertheless,these data reveal for the first time the presence of various subcomplexes of Synechocystis Hox hydrogenase in vivo .Subunit Abundance in Hox Hydrogenase Mutants —The detection of Hox subcomplexes in the WT strain by FPLC and two-dimensional BN/SDS-PAGE analyses prompted us to con-struct mutants of each hox gene alone and in combination in the same parental WT strain (supplemental Fig.2)to further examine any relationship between complex/subcomplex asso-ciations and relative subunit abundance by Western blotting.All of the mutants except for hox Ϫ(whole operon deletion con-structed by T.Ogawa)and hoxH Ϫ(see “Experimental Proce-dures”and supplemental Fig.2)were created by ORF replace-ment with similar size antibiotic resistance genes to avoid changes in operon transcription from additional promoters in antibiotic resistance cassettes or large alterations in sequencesize.As expected,no detectable Western signal for Hox sub-units was apparent when the respective gene was disrupted,confirming full segregation of gene deletions (cyanobacteria carry multiple genome copies)(Fig.3A ).Interestingly,deletion of hoxE ,the first gene in the operon,led to a 2–3-fold increase in the remaining Hox protein subunits (Fig.3B ).The increase in Hox protein levels in this mutant is probably linked to the first 190bp of the hoxE ORF,whose absence results in increased hox transcript levels and possible enhancement in the rate of trans-lation due to a decrease in minimum free energy in the hoxE Ϫmutant mRNA (see “Discussion”and supplemental Fig.3,A–D ).The overexpression of HoxH in the hoxE Ϫmutant leads to a discernible increase in levels of unprocessed HoxH com-pared with WT and other hox mutants (Fig.3A and supplemen-tal Fig.4),consistent with increased unprocessed large subunit when the hydrogenase is overexpressed without additional maturation factors in previous studies (our unpublished results 3for R.eutropha soluble hydrogenase and Refs.13and 20).All other hox mutants exhibited decreased levels of remain-ing complex subunits (Fig.3B ).Levels of HoxH and HoxY are decreased to 20and 10%of WT in the respective hoxY Ϫand hoxH Ϫmutants,whereas HoxE,HoxF,and HoxU levels are less than 5%of WT in hoxF Ϫand hoxU Ϫmutants,highlighting interdependencies for HoxYH and HoxEFU subcomplex pro-tein abundance.In addition,HoxY and HoxH levels are decreased to 15–90%of WT in HoxEFU subcomplex mutants (most notably in hoxU Ϫmutants),and HoxE,HoxF,and HoxU levels are decreased to 25–70%of WT in HoxYH subcomplex mutants,revealing interdependencies in subunit abundance across subcomplexes as well.Hox Subunit Association in Individual Subunit hox Mutants —Although changes in transcription could be responsible for altered subunit abundance observed in hox mutants,the absence of complex subunits could also lead to instability of the remaining subunits because of an inability to form stable Hox complexes/subcomplexes.To assess the association of the remaining Hox subunits when one of the five subunits is absent,we again performed two-dimensional BN/SDS-PAGE and Western blotting of soluble extracts from single-subunit hox mutants (Fig.4).No notable differences in total protein were observed between the WT and hox mutant strains by Coomass-ie-stained one-dimensional BN or silver-stained two-dimen-sional SDS-polyacrylamide gels (data not shown).In the hoxE Ϫstrain,HoxFUYH and HoxFU subcomplexes accumulated (Fig.4A ),consistent with the observation of those subcomplexes in WT (Fig.2).Despite the increased levels of HoxY and HoxH in our hoxE Ϫstrain (Fig.3A ),we still were not able to observe the expected HoxYH subcomplex (Fig.4A ).Interestingly,none of the Hox subcomplexes are detected in hoxF Ϫor hoxU Ϫstrains,with the remaining Hox proteins observed only as unassembled monomers,indicating that HoxF and HoxU are necessary for stable assembly/maintenance of the full Hox complex and Hox subcomplexes (Fig.4,B and C ).In contrast,the diaphorase sub-units are still able to assemble into HoxEFU and HoxFU sub-complexes in hoxY Ϫor hoxH Ϫstrains (Fig.4,D and E ),con-3C.Eckert,J.Yu,and P.-C.Maness,unpublishedresults.FIGURE 2.Two-dimensional BN/SDS-PAGE and Western blot analysis of WT Synechocystis sp.PCC 6803Hox hydrogenase.A ,the soluble fraction of WT Synechocystis sp.PCC 6803was separated on a 12%(w/v)polyacrylamide BN-polyacrylamide gel followed by two-dimensional separation on a 17.5%(w/v)polyacrylamide SDS-polyacrylamide gel (B )and analysis by Western blotting with the indicated Hox subunit-specific antibodies (C–G ).The first dimension BN-polyacrylamide gel was Coomassie-stained,whereas the sec-ond dimension SDS-polyacrylamide gel was silver-stained.Cyanobacterial Hox Hydrogenase Complex Association and Functionat NATL RENEWABLE ENERGY, on January 16, 2013 Downloaded fromfirming that HoxY and HoxH are not required for the stable association of diaphorase subcomplexes.Pull-down Analysis of Hox Subunit Associations in Hox Hydrogenase Subcomplexes —Additional mutant strains were constructed to ectopically express His 6-tagged Hox subunits to further probe subcomplex associations:1)HoxYH,tagged on HoxY (hox ϪϩHis 6-HoxYH);2)HoxEFUYH,tagged on HoxH(hoxH ϪϩHis 6-HoxH);and 3)HoxEFUYH,tagged on HoxE (hoxE ϪϩHis 6-HoxE).Overexpression of HoxH by ectopic expression (Fig.5,A and B )or in the hoxE Ϫstrain background (Fig.5C )results in clear unprocessed and processed HoxH sig-nal (Fig.5,A–C ,upper and lower bands ,respectively,in HoxH blots and in the HoxH (C-terminal)blot),increasing the likeli-hood that we would see unprocessed HoxH in bead fractions ifFIGURE 3.One-dimensional SDS-PAGE and Western blot analysis of Synechocystis sp.PCC 6803hox mutants.A ,whole cell lysates of WT and individual/combined hox mutants were run on TGX Any kDa gradient SDS-polyacrylamide gels (Bio-Rad),transferred to PVDF,and immunoblotted with Hox subunit-specific antibodies and PsaD and/or Rps1(not shown)as loading controls.B ,relative levels of Hox subunits in each mutant,presented as a percentage of WT.Data represent quantitation of multiple Western blot analyses with error bars depicting variation between separate analyses.Cyanobacterial Hox Hydrogenase Complex Association and Functionat NATL RENEWABLE ENERGY, on January 16, 2013 Downloaded from。
叶绿素荧光成像技术的原理与应用
叶绿素荧光成像技术的原理与应用一、引言叶绿素是植物中最重要的光合色素，是植物进行光合作用的基础。
溶剂化的叶绿素主要吸收蓝色和红色光，在500～600和650～700nm波长范围内，具有两个吸收峰。
叶绿素荧光成像技术是基于叶绿素发出的荧光信号来进行影像测量的一种实时、无创的模拟测量方法。
本文将介绍叶绿素荧光成像技术的原理、实验流程及其应用。
二、原理叶绿素荧光成像技术是基于叶绿素荧光的成像，叶绿素荧光受光强度和环境因素的影响而变化，可以反映植物的生长状态、光合作用效率和叶片生理变化等信息。
叶绿素荧光成像系统具有高时间分辨率、高空间分辨率的特点，可以获取全景、彩色、实时和定量信息。
叶绿素荧光成像技术主要是利用荧光成像仪和其他仪器支持，通过蓝/绿或红/绿激发光、荧光图像采集和分析等步骤，可以获得叶绿素的分布信息。
三、实验叶绿素荧光成像技术的实验主要分为两个步骤：激发和成像。
首先是激发，将叶片放入光合器中，用荧光成像仪对植物叶片进行光激发，根据荧光成像仪的激光幅度，可以调整植物叶片的荧光强度。
之后，进行成像，将植物叶片放到荧光成像仪中进行拍摄，获取叶绿素的发光信号。
最后，通过荧光照片的处理，可以计算叶片荧光强度和叶绿素荧光参数，如最大光化学利用率、植物光合作用效率等。
四、应用叶绿素荧光成像技术的应用非常广泛，主要涉及到生物学、生态学、农业、气象学，特别适用于植物生长状态监测、植物抗性研究、光合作用效率评估等。
一些具体的应用领域可以如下简要介绍：1.光合作用研究叶绿素荧光成像技术可用于研究植物的光合作用效率、光能利用和光保护机制。
典型的光合作用实验是通过比较光照和黑暗条件下植物的荧光变化来确定植物的光合反应和光保护机制。
2.气候变化影响研究在气候变化方面，叶绿素荧光成像技术可用于研究气候变化导致的植物响应和适应。
通过对多个季节的荧光成像分析可以确定气候变化对地上层和植物生长的影响。
3.生态环境研究叶绿素荧光成像技术可用于研究萎缩地区的植被恢复和生态系统的响应。
FlourCam荧光成像系统
仪器使用
• 连接线路 • 连接电脑
1。Usb 2.0 2。部分电脑可能需要专门的usb驱动(可下 载) • 软件安装 可解压直接执行，也可安装使用。
PSI公司软件与相关资料下载地址http://www.psi.cz/ftp/
科研应用
1.光系统II反应中心光化学效率的表征
光化学效率（ FV’/Fm’）的变化，反映PSII
反应中心色素
光反应过程
特定波长光能
原初电 接收的光能
子受体
P·A→ P*·A → P+·A-
后 续
原初电子分离
反 应
PSII（700nm）&PSI（680nm）
基态捕光色素
激发态
热能
产生荧光
叶绿素的激发与退激
叶绿素荧光现象
光合作用示意图
蓝光
热
光合
热
红光
荧光
NADP
NADPH
光系统Ⅰ（PSI）能被波长700nm的 光激发，又称P700 光系统Ⅱ（PSⅡ）吸收高峰波长为680nm，又称P680
250 FM
200
150 FV
100
50 F0
0
FM’ FS
-10 0 10 20 30 40 50 60 70 80
TIME, seconds
运用叶绿素荧光技术来表征光 能的利用、传递、耗散，
能够很好地研究植物的光合作 用过程.
Thanks for your interesting and
questions!
光化学淬灭（qp）：反映PSII天线色素分子 吸收光能后，用于光化学电子传递的份 额， 因此也反映了色素天线吸收的光能用 于光合电子传递的变化；同时，qp 也反映 PSII初级电子受体(QA)氧化还原状态的变化。 要保持高的光化学淬灭，就要使PSII反应中 心处于开放状态
叶绿素荧光成像技术在植物生长中的应用
叶绿素荧光成像技术在植物生长中的应用叶绿素荧光成像技术，是一种非侵入式的植物生长观测方法。
它可以在不对植物造成任何伤害的情况下，实时地观测植物的光合作用和植物生长状态。
叶绿素荧光成像技术的应用范围十分广泛，包括植物生长研究、环境监测、农业生产等方面。
叶绿素荧光成像技术的基本原理是，利用叶绿素分子在光合作用中产生的荧光信号，来反映叶片的光合效率。
这种荧光信号可以通过特殊的摄像设备，即叶绿素荧光成像仪来采集。
通过对采集到的荧光图像进行处理，可以得到植物的光合作用效率、光能利用率等多项指标，从而揭示植物生长状态和环境条件对植物生长的影响。
在植物生长方面，叶绿素荧光成像技术的应用主要集中在三个方面：一、对不同生长环境下的植物进行光合作用效率观测。
利用叶绿素荧光成像仪可以在植物生长中实时地观测其光合作用的运作情况。
通过在不同环境和条件下对植物进行观测，可以更加准确地了解植物生长的条件和需求，为生产和研究提供参考。
二、对不同植物的生长状态进行监测。
叶绿素荧光成像技术还可以用于对不同植物的生长状态进行监测，从而判断不同的生长阶段、生长速度等。
这对于农业生产和植物育种方面都具有很大的意义，可以指导地面管理、育种选材等方面的工作。
三、对不同生物模型进行生长动态分析。
除了对植物进行观测之外，叶绿素荧光成像技术还可以用于对其他生物模型的生长状态进行监测。
例如，可以将该技术应用于对微生物、食品发酵过程等生物模型进行生长动态分析，从而更好地了解生物系统的生成规律和规律变化，为相关研究提供参考。
总之，叶绿素荧光成像技术的应用具有非常广泛、多样化的特点。
通过该技术可以实时地观测不同生境下植物的生长状态，从而更好地了解植物的光合作用效率、生长阶段等内容。
这对于农业生产、生物育种和环境监测都具有很大的实用价值。
因此，该技术的发展和应用前景十分广阔。
植物叶绿素荧光成像技术在国内的应用
植物叶绿素荧光成像技术在国内的应用（第四期）植物叶绿素荧光成像技术作为最早实用化的叶绿素荧光成像技术，是目前世界上最权威、使用范围最广、种类最全面、发表论文最多的叶绿素荧光成像技术。
涵盖了从叶绿体、单个细胞、微藻到叶片、果实、花朵，乃至整株植物和植物灌层，几乎可以测量所有的植物样品，甚至包括含有叶绿素的微生物和动物。
叶绿素荧光成像技术最早在21世纪初引进到国内，但一直到2010年后国内的科学家才在国际交流中逐渐发现这项技术的巨大价值，在短短数年中也利用这一技术发表了几十篇高水平SCI 文献。
本期主要介绍目前叶绿素荧光成像技术在国内的应用情况。
一、 植物光合生理研究叶绿素荧光可以直接反应植物光系统的生理状况，因此从叶绿素荧光技术发明之初，就被用于各种植物光合生理研究。
山东农科院使用FluorCam 叶绿素荧光成像技术研究了小麦旗叶与外露花梗光合能力的差异[1]。
研究中发现在小麦生长前中期，旗叶与外露花梗的最大光化学效率Fv/Fm 和量子产额ΦPSII 基本相同。
但在生长后期，旗叶的光合能力显著下降，而花梗光合能力的下降幅度要小于旗叶（图1）。
这证明了在生长后期的灌浆期，花梗对维持籽粒的生长更为重要。
之后，他们又研究了小麦叶片和颖片季节衰老过程中以及颖果发育过程中光合特性的变化[2；3，图2]。
图2. 不同生长期小麦叶片和颖片的最大光化学效率Fv/Fm （A ）、量子产额ΦPSII （B ）和非光化学淬灭NPQ （C ）的变化图1. 不同生长阶段的旗叶（A ，C ）和外露花梗（B ，D ）的Fv/Fm （A ，B ）和ΦPSII （C ，D ）典型叶绿素荧光成像图二、植物生物/非生物逆境胁迫与抗逆性研究由于几乎所有种类的生物/非生物逆境胁迫都会影响到植物光合系统的正常生理功能，而叶绿素荧光技术是公认的植物逆境光合功能研究最灵敏的无损探针。
因此通过叶绿素荧光成像技术不但能反映植物受胁迫程度和抗逆能力的差异，而且能指明胁迫影响光合系统的具体机理过程。
FluorCam叶绿素荧光成像一
FluorCam
荧 光 成 像 ： 荧 光 淬 灭 分 析
JIP-‐test（电子传递抑制剂敌草隆对OJIP的影响
叶绿素荧光技术著名厂商
• PSI：捷克Brno（孟德尔在此实验并发现著名的孟德 尔遗传定律），Ladislav Nedbal为首席科学家和主要 股东（另一股东为David Kramer，美国密执根州立 大学教授），1997年为美国华盛顿大学H. Pakrasi教 授研制成了第一台FluorCam荧光成像系统。主要产 品有
Kautsky effect
（资料源自Ecolab实验室荧光成像数据库）
荧光淬灭分析（Quenching Analysis）
1. Fv/Fm, 最大光量子产量 2. Fq’/Fm’，Genty参数、ϕPSII，又称光量子效率，用于光化学过程分析评估， 表示PSII吸收的光辐射用于光化学过程的比率，不需要暗适应，但受自然 光照影响大 3. Fv’/Fm’，开放PSII反应中心的光量子效率，不需暗适应 4. Fq’/Fv’，光化学淬灭qP，大致相当于初级受体QA的再氧化状态 5. (Fm-‐Fm’)/Fm’，非光化荧光淬灭
显微叶绿素荧光成像系统
• 可对植物组织、藻类等细胞或亚亚细 胞结构进行荧光成像测量 • 便携式标准版显微荧光成像系统， Olympus CX31显微镜 • 便携式增强版显微荧光成像系统， Olympus BX40 显微镜及可扩展配件 （通过扩展可测量GFP等） • 多功能显微荧光成像系统，除具备 Olympus BX40 显微镜及可扩展配件外， 还具备6位滤波轮（有机械调节和电 子调节两种模式供选配），因而除可 以成像测量叶绿素荧光外，还可测量 GFP、CY3、CY5等多种稳态荧光或生 物荧光
FluorCam荧光成像系统Protocols（实验程序）
叶绿素荧光成像技术及其在光合作用研究中的应用
Fluorcam荧光成像技术及其在光合作用研究中的应用Eco‐lab生态实验室北京易科泰生态技术有限公司info@eco‐目录1、叶绿素荧光成像技术发展过程2、荧光参数及其生理意义3、PSI介绍（荧光成像的发明者）4、PSI产品介绍5、应用案例叶绿素荧光技术发展历程•Kautsky effect: Kautsky and Hirsch(1931)首次用肉眼发现叶绿素荧光现象并发表论文“CO2同化新实验”，后被称作“Kautsky effect”•PAM(Pulse Amplitude Modulated Fluorometer): Schreiber（1986）等发明了PAM脉冲调制技术测量叶绿素荧光。
•FluorCam：KineKc imaging of chlorophyll fluorescence: Ladislav Nedbal（2000）等于上世纪90年代末期发明了与PAM技术相结合的叶绿素荧光成像技术成像测量局部放大荧光参数及其意义•Fo、Fm与QY，此外还有PAR_Abs及ETR•Kautsky诱导效应：Fo，Fp，Fv，Ft_Lss，QY，Rfd•荧光淬灭分析：Fo，Fm，Fp，Fs，Fv，QY，NPQ，Qp，Rfd 等50多个参数•OJIP曲线：快速荧光诱导曲线。
Fo，Fj，Fi，P或Fm，Mo（OJIP曲线初始斜率）、FixArea固定面积、Sm（对关闭所有光反应中心所需能量的量度）、QY、PI等•LC光响应曲线：Fo，Fm，QY，QY_Ln叶绿素荧光仪著名厂商•PSI：捷克布尔诺Brno（孟德尔在此发现著名的孟德尔遗传定律），Ladislav Nedbal为首席科学家和主要股东（另一股东为David Kramer，美国密执根州立大学教授），1997年为美国华盛顿大学H.Pakrasi教授研制成了第一台FluorCam荧光成像系统。
主要产品有：–FluorCam叶绿素荧光成像系列产品–FL3500/FL5000双调制荧光仪系列产品–FluorPen及AquaPen等手持式荧光仪产品–光养生物反应器等藻类培养与在线监测产品–光源与植物培养室•Optics：美国，主要产品为OS5p‐PAM叶绿素荧光仪等•Walz：德国，主要产品为PAM2500叶绿素荧光仪等PSI厂家介绍PSI厂家剪影laboratoryFluorCam叶绿素荧光成像：1. Handy FC——FluorCam便携式叶绿素荧光成像系统2. Handy GFPCam——FluorCam便携式荧光蛋白成像系统3. Handy Leaf chamber——便携式光合联用叶绿素荧光成像系统4.Closed FC——封闭式叶绿素荧光成像系统5. Closed GFPCam——封闭式多光谱荧光蛋白成像系统6. Open FC——开放式叶绿素荧光成像系统‐Rover FluorCam——移动式大型植物荧光成像系统‐Transect FluorCam——样带扫描式植物荧光成像系统‐XY‐Plane FluorCam——多光谱XY‐平台式大型植物荧光成像系统‐Arch FluorCam——拱形三维植物荧光扫描成像系统7. Micro‐FluorCam——显微叶绿素荧光成像系统，又分标准版、增强版（可选配GFP FilterCube Set）及滤波轮版8. Conveyor and RoboKc PlantScan System——PlantScan全自动植物光谱成像分析系统9. Fluorescence KineKc Microscope——FKM荧光动态显微光谱成像系统Fluorcam荧光成像技术特点◆对叶片无损伤、测量迅速◆测量对象多样，包括叶片、果实、藻类、地衣、苔藓、拟南芥等◆具备自动重复测量功能，从而实现无人职守自动成像实验◆结果以图片或视频形式输出，直观、易于观察◆应用领域广泛，如光合作用、植物胁迫生理学、水生生物学、海洋学和遥感等◆实验室、野外均可使用◆测量面积范围广，小至微米，大至整块草坪◆用户可根据实验需要，自定义测量参数FlourCam叶绿素荧光成像技术应用领域•植物光合特性和代谢紊乱植株的筛选•生物和非生物胁迫的检测•植物抗胁迫能力或者易感性研究•气孔非均一性研究•长势与产量评估•植物——微生物交互作用研究•植物——原生动物交互作用研究Kautsky effect in a diuron‐inhibited leaf（敌草隆抑制电子传递实验）OJI PScreen mutants by NPQ parameters （通过荧光淬灭分析筛选变异植株）水分对沙漠中苔藓的光合特性的影响加水0.5 h后高光胁迫获得的衣藻突变体重金属胁迫条件下的烟叶荧光成像左图为对照烟叶，中图为通过叶脉浸泡硫酸铜30分钟后的荧光成像，右图为经硫酸铜浸泡处理60分钟后的荧光成像。
FluorCam样带扫瞄式叶绿素荧光成像系统
PlantScreen叶绿素荧光与RGB自动扫描成像分析系统PlantScreen叶绿素荧光与RGB自动扫描成像分析系统集成了自动化控制系统、FluorCam大型叶绿素荧光成像测量分析、RGB植物真彩成像分析等先进技术，实现对各种培养植物——从拟南芥、水稻到各种其它植物的生理生态与形态结构成像分析，用于高通量植物表型成像分析测量、植物胁迫响应成像分析测量、生态毒理学与污染生态学研究、性状识别及植物生理生态分析研究、作物育种与抗性检测、生物多样性／遗传多样性表型检测分析及土壤种子库研究等。
成像平台可在主机箱内XYZ三维自动化移动，自动扫瞄成像范围为60cm x 129cm，植物最大高度约50cm。
系统配置与工作原理：系统由XYZ三维自动控制箱、XYZ三维移动成像平台及自动控制与分析软件等组成，LED光源、FluorCam叶绿素荧光成像、RGB成像集成于一个可XYZ三维自动化移动的成像平台上，程序控制XYZ三维精确定位和定时，在线数据分析。
采用世界上单幅成像面积最大的叶绿素荧光成像系统，成像面积达35×35cm。
技术指标：1.XYZ三轴机械臂可自由移动至植物上方成像分析，成像扫瞄面积范围60cm x 129cm（可选配其它大型系统），植物高度49cm，镜物距25cm，Z轴最大负重30kg2.标准配置X轴活动范围0－101cm，精确度±1mm；Y轴活动范围0－72cm，精确度±1mm；Z轴活动范围0-49cm，精确度±5mm；3.叶绿素荧光成像：镜头分辨率1392x1040像素，单幅成像面积35x35cm，测量光橙色618nm，橙色和白色双波长光化学光，饱和光闪为白色，最大光强3600μmol/m2/s,具735nm红外光源4.叶绿素荧光成像测量参数包括Fo, Fm, Fv,Fo’, Fm’,Fv’, Ft,Fv/Fm, Fv’/Fm’,Phi_PSII, NPQ, qN,qP, Rfd等几十个叶绿素荧光参数5.RGB成像分析测量参数包括：1)叶面积（Leaf Area: Useful for monitoring growth rate）2)植物紧实度／紧密度（Solidity/Compactness. Ratio between the area covered by theplant’s convex hull and the a rea covered by the actual plant）3)叶片周长（Leaf Perimeter: Particularly useful for the basic leaf shape and widthevaluation (combined with leaf area)）4)偏心率（Eccentricity: Plant shapeestimation, scalar number, eccentricityof the ellipse with same secondmoments as the plant (0...circle, 1...linesegment)）5)叶圆度（Roundness: Based on evaluatingthe ratio between leaf area andperimeter. Gives information about leafroundness）6)叶宽指数（Medium Leaf Width Index: Leafarea proportional to the plant skeleton(i.e. reduction of the leaf to linesegment)）7)叶片细长度SOL (Slenderness of Leaves)8)植物圆直径（Circle Diameter. Diameter of a circle with the same area as the plant）9)凸包面积（Convex Hull Area. Useful for compactness evaluation）10)植物质心（Centroid. Center of the plant mass position (particularly useful for theeccentricity evaluation)）11)节间距（Internodal Distances）12)生长高度（Growth Height）13)植物三维最大高度和宽度（Maximum Height and Width of Plant in 3 Dimensions）14)相对生长速率（Relative growth rate）15)叶倾角（Leaf Angle）16)节叶片数量（Leaf Number at Nodes）17)其它参数如用于植物适合度估算的颜色定量分级、绿度指数（Other parameters suchas color segmentation for plant fitness evaluation, greening index and others）6.高灵敏度RGB成像传感器，CMOS 1/2”,分辨率2560x1920像素，像素大小2.2μm，四个LEDs高强白色光源，成像信息包括时间和位置，纪录格式为日期－月份－年度－小时－分钟－秒－Pos_X_Y_Z.bmp7.系统控制与数据采集分析系统：用户友好的图形界面，用户定义、可编辑自动测量程序（protocols），控制单元有主电源开关、紧急关闭、XYZ三维轴启动开关、暂停键、移动键等，用户名和密码保护8.程序控制XYZ三维精确定位和定时，并纪录带时间和三维空间位置的数据（四维信息数据）9.三相电源供电，3x230/220VAC，50/60Hz10.大小规格200cm(长)x150cm(宽)x230(高)，重量约400kg产地：欧洲。
西北农林科技大学园艺学院 FluorCam大型版开放式多光谱荧光成像系统
西北农林科技大学园艺学院FluorCam大型版开放式多光谱荧光成像系统案例报告用户单位：西北农林科技大学园艺学院研究方向：农作物逆境生理、育种及拟南芥荧光蛋白筛选用户情况：客户既做黄瓜、西红柿之类的农作物，也做拟南芥。
虽然水平一般，但对仪器的兴趣还是很大。
一方面想开展植物逆境研究，一方面想做各种荧光蛋白筛选。
用得好的话，可以成为西农一个示范点。
仪器配置：叶绿素荧光+PAR吸收+GFP/YFP/DsRED/CFP/DAPI+MCF，配红、蓝、绿、青、白光源板仪器图片：测试数据：1.叶绿素荧光首先用一般的红蓝光源进行测试。
饱和脉冲（蓝光）30%，1200µE；光化光（红光）56%，约150µE。
测试样品为室外的冬青（实际学名应为大叶黄杨）。
左侧为有病害叶片，中间为较绿叶片，右侧为较为衰老的叶片，因为在室外生长，3个叶片实际都都是受到一定的低温胁迫。
Fv/Fm QY_Lss NPQ_Lss三者Fv/Fm分别为0.50,0.65,0.62。
QY_Lss分别为0.08,0.16,0.15。
NPQ为1.15,1.31,1.21。
三项参数都是绿叶>衰老叶>病害叶。
再更换青色光源板进行测试。
饱和脉冲（青光）60%，约1360µE；光化光（青光）分别使用20%（200µE）、40%（450µE）和60%（700µE）。
每次测量之间进行大约5分钟暗20% 40% 60%Fv/Fm20%和40%的Fv/Fm基本没有变化，而且与之前红蓝光测量结果近似。
表明1360µE的青色饱和脉冲也可以满足样品测量的需要。
但60%的Fv/Fm确有显著下降，怀疑是之前多次测量，尤其青光关闭气孔的效应造成了一定的损伤，5分钟的暗适应没有恢复。
QY_LssNejad（2006）研究紫露草时就是用QY作为气孔开闭分布的表征指数。
我们可以看到随着青光的增强，QY快速下降。
FluorCam叶绿素荧光成像技术应用案例(第二期)
FluorCam叶绿素荧光成像技术应用案例（第二期）——藻类研究中的应用PSI公司首席科学家Nedbal教授与公司总裁Trtilek博士等首次将PAM叶绿素荧光技术与CCD技术结合在一起，研制成功了FluorCam叶绿素荧光成像技术（Nedbal等，2000），并于1997年为美国华盛顿大学提供了第一台商业FluorCam系统。
FluorCam叶绿素荧光成像技术成为上世纪90年代叶绿素荧光技术的重要突破，使科学家们对光合作用与叶绿素荧光的研究正式进入了二维世界和显微世界。
目前FluorCam叶绿素荧光成像技术已成为世界上最权威、使用最广、种类最全面、发表论文最多的叶绿素荧光成像技术，已经发展出FluorCam便携式叶绿素荧光成像仪、FluorCam便携式Chl/GFP荧光成像仪（Handy GFPCam）、FluorCam便携式光合联用型叶绿素荧光成像系统、FluorCam封闭式叶绿素荧光成像系统、FluorCam封闭式Chl/GFP荧光成像系统（Closed GFPCam）、FluorCam封闭式多光谱荧光成像系统、FluorCam开放式标准版叶绿素荧光成像系统、FluorCam开放式大型版叶绿素荧光成像系统、FluorCam开放式多光谱荧光成像系统、FKM多光谱荧光动态显微成像系统、FluorCam移动式大型版叶绿素荧光成像系统、FluorCam样带扫瞄叶绿素荧光成像系统等一系列仪器系统。
现有SCI发表参考文献近400篇，可联系Ecolab生态实验室索取或点击以下链接下载FluorCam 叶绿素荧光成像部分参考文献名录链接本期应用案例主要介绍使用FluorCam叶绿素荧光成像技术进行藻类研究的各种案例。
一、蓝藻固氮与光合放氧的关系（论文发表于Science）文献：I Berman-Frank, et al. 2001. Segregation of Nitrogen Fixation and Oxygenic Photosynthesis in the Marine Cyanobacterium Trichodesmium. science, 294: 1534-1537使用仪器技术：FKM多光谱荧光动态显微成像技术和FRRF快速重复荧光测量技术（FRRF技术介绍链接）FKM技术详细介绍链接研究内容：现代海洋中，有相当一部分固氮作用是由Trichodesmium属蓝藻完成的。
2.FluorCam叶绿素荧光成像技术
Fluorcams荧光成像技术
（一）适用性 （二）测量技术 （三）硬件配置 （四）功能特点 （五）Fluorcam软件功能（下午演示） （六）Fluorcams系列产品 （七）Fluorcams在各领域的应用
（一）Fluorcam的适用性
适用于各种复杂情况 • 测量面积（显微视野到样带成像） • 适用范围（叶绿体——群落） • 从二维成像到三维成像 • 样品大量筛选（条码识别） • 可用于荧光蛋白与荧光素成像 • 弥补普通荧光成像对荧光瞬变过程在时间分辨上
蓝（能量高&气孔）或白光（贴近自然光照）
光化光强度
标准配置：2,500 µmol(photons)/m².s
升级配置：5,000 µmol(photons)/m².s （光胁迫）
饱和光强度
标准配置：3,000 µmol(photons)/m².s（多次翻转） 升级配置：10,000 µmol(photons)/m².s（单次翻转）
叶绿素荧光成像+ 荧光标记发光成像
现在多为GFP成像或叶绿素荧光成像，无功能重叠仪器
配置&功能
标准配置：
• 高分辨率CCD镜头 • 光源：4组 LED 发光板：
4 X 470 nm
2 X 447 nm + 2 X 470 nm
2 X 627 nm + 2 X 470 nm • 8位滤波轮 • 条形码识别
QY局部放大图
分辨率512X512 成像面积3X3cm 单像素代表面积：60X60um
理想的仪器是什么样子？
• 适用性（宏观&微观，室内&室外，光合生 理&基因工程）
• 测量结果（可靠，时间分辨率&空间分辨率） • 功能（越强越好） • 软件（人性化、便于操作） • 携带（方便） • 耐用（维护量低） • 科研界认可度（文献）
FluorCam野外移动式叶绿素荧光成像系统
产地：欧洲 PSI
技术指标： 移动式大型植物荧光成像，成像面积可达 35× 35cm，具移动轮和暗适应屏幕 镜头及光源高度 20cm–150cm 可调，从而适于不同生长类型不同高度植物的原位 非损伤荧光成像测量 测量参数包括 F0，F0’，Fs，Fm，Fm’，Fp，FtDn，FtLn，Fv，NPQ_Dn，NPQ_Ln， Qp_Dn，Qp_Ln，qN，QY，QY_Ln，PARabs，Rfd 等 50 多个叶绿素荧光参数 高分辨率、 高灵敏度 2/3”CCD， 10.2x8.3mm 阵列， 最高可达 1392x1040 像素 （15fps） ， 像素大小 6.45 微米；可 2x2、3x3、4x4 像素叠加以提高灵敏度和时间分辨率；高速 USB2.0 图像传输， 480Mbits／秒 自动测量分析功能：可预设 1 个试验程序，系统可自动测量储存，数据文件自动按 时间命名
FluorCam 野外移动式大型叶绿素荧光成像系统
FluorCam 野外移动式大型叶绿素荧光成像系统专为原位植物表型成像分析及生理生态 研究而设计，成像面积可达 35× 35cm，系统装配在一个可以自由移动的支架上，高度可调， 适用于温室乃至野外植物的光合生理研究、植物 Phenotyping、植物胁迫生理及抗性筛选、 植物优良品种选育、植物生态毒理学研究等。 功能特点： 是世界上单幅成像面积最大的叶绿素荧光 成像系统，达 35× 35cm，可对整株植物及 多株植物同时进行叶绿素荧光成像分析 整套系统装配在具备 4 个轮子的支架上， 可在野外自由移动，非损伤原位对植物进 行叶绿素荧光成像研究 可进行自动重复成像测量，可设置两个实 验程序（Protocols）自动循环成像测量， 成像测量数据自动按时间日期存入计算机 （带时间戳），从而实现无人职守自动监 测功能 可选配 RGB 成像分析模块， 用于植物形态 测量分析等 在没有交流电的情况下，可选配直流供电单元供电
FluorCam叶绿素荧光成像文献 2009 Characterization of soldat8 , a Suppressor
N. S. Coll et al.
பைடு நூலகம்
Downloaded from / by guest on March 15, 2012
Keywords: Acclimation • flu mutant • Light stress • Singlet oxygen • SIGMA6 • soldat8.
Abbreviations: CAPS, cleaved amplified polymorphic sequence; LHC, light-harvesting complex; NEP, nuclearencoded RNA polymerase; NPQ, non-photochemical quenching; Pchlide, protochlorophyllide; PEP, plastidencoded RNA polymerase; ROS, reactive oxygen species; SSLP, simple sequence length polymorphism.
Noctor 2000). At the same time, excited triplet Chl in PSII interacts with O2 to generate singlet oxygen (1O2) by energy transfer (Macpherson et al. 1993, Ledford and Niyogi 2005).
The flu mutant of Arabidopsis thaliana overaccumulates in the dark the immediate precursor of chlorophyllide, protochlorophyllide (Pchlide), a potent photosensitizer, that upon illumination generates singlet oxygen (1O2). Once 1O2 has been released in plastids of the flu mutant, mature plants stop growing, while seedlings die. Several suppressor mutations, dubbed singlet oxygen-linked death activator (soldat), were identified that specifically abrogate 1O2-mediated stress responses in young flu seedlings without grossly affecting 1O2-mediated stress responses of mature flu plants. One of the soldat mutations, soldat8, was shown to impair a gene encoding the SIGMA6 factor of the plastid RNA polymerase. Reintroduction of a wildtype copy of the SOLDAT8 gene into the soldat8/flu mutant restored the phenotype of the flu parental line. In contrast to flu, seedlings of soldat8/flu did not bleach when grown under non-permissive dark/light conditions, despite their continuous overaccumulation of the photosensitizer Pchlide in the dark. The activity of SIGMA6 is confined primarily to the very early stage of seedling development. Inactivation of SIGMA6 in soldat8 mutants disturbed plastid homeostasis, drastically reduced the nonphotochemical quenching capacity and enhanced the light sensitivity of young soldat8 seedlings. Surprisingly, after being grown under very low light, soldat8 seedlings showed an enhanced resistance against a subsequent severe light stress that was significantly higher than in wild-type seedlings. In order to reach a similar enhanced stress resistance, wild-type seedlings had to be exposed to a brief higher light treatment that triggered an acclimatory response. Such a mild pre-stress treatment did not further enhance the stress resistance of soldat8 seedlings. Suppression of 1O2-mediated cell death in young
叶绿素荧光成像实验报告
叶绿素荧光成像实验报告实验目的本实验旨在通过叶绿素荧光成像技术，探究不同水稻品种在逆境条件下叶绿素荧光特性的变化，为水稻逆境抗性的研究提供依据。
实验设备和试剂- 叶绿素荧光成像仪- 水稻品种样本- 逆境处理设备和试剂（例如高温、干旱等）- 显微镜实验过程实验准备1. 使用叶绿素荧光成像仪对水稻品种样本进行预处理，包括根据仪器要求调整参数、对样本进行调整和定位等。
2. 准备逆境处理设备和试剂，例如设置高温处理，将水稻样本暴露在高温环境中。
实验操作1. 将水稻样本分为对照组和处理组，其中对照组不接受任何逆境处理，处理组进行高温处理。
2. 使用叶绿素荧光成像仪对对照组和处理组的水稻样本进行荧光成像。
3. 记录下荧光成像结果中的各项参数，例如叶绿素荧光强度、叶绿素荧光最大光子量子产率、叶绿素荧光平均光子量子产率等。
4. 使用显微镜观察水稻叶片的形态特征，记录下叶片的颜色、形状等变化。
实验结果与分析通过对比对照组和处理组的荧光成像结果，可以得到以下结论：1. 在逆境条件下，处理组的叶绿素荧光强度明显低于对照组，表明高温处理抑制了水稻叶片的光合作用活性。
2. 处理组的叶绿素荧光最大光子量子产率和平均光子量子产率也显著降低，说明高温处理导致水稻叶片的光能吸收和利用效率降低。
3. 处理组的叶片颜色变得苍白，形状也出现变化，与对照组相比较明显。
结论通过叶绿素荧光成像实验，我们可以初步推断出高温处理对水稻叶片的影响。
高温处理抑制了水稻叶片的光合作用活性，降低了叶绿素荧光最大光子量子产率和平均光子量子产率。
同时，高温处理还引起了水稻叶片的颜色苍白和形状变化。
这些结果表明水稻在逆境环境下光合作用和光能利用能力受到了极大的影响。
讨论与展望本实验只研究了高温处理对水稻叶片的影响，还可以进一步研究其他逆境条件，例如干旱和盐碱等，以全面了解水稻在逆境环境下的光合作用特性。
同时，可以结合其他分子生物学和生物化学实验手段，深入研究逆境对水稻光合作用和叶绿素荧光特性的影响机制，为培育抗逆性水稻品种提供科学依据。
叶绿素荧光成像技术及其应用
土壤呼吸研究监测技术方案
• 便携式： – Soilbox 343 – SRS1000/2000 – Soilbox-FMS/FGA • 实验室： – SoilLab – EGA60 • 自动监测系统： – ACE与ACE-Net – OTC-Auto – SCG-N • 扩展配置：＋BTC-100，＋土壤水分，＋PTM48
智能手持式荧光仪—FP100&AP100
叶绿素荧光仪的局限性
• 仅能通过光纤测量一个点的总值
• 只能获得数据和动力学曲线图
• 一般只能测量叶片
• 在进行光合相关突变体筛选时工作量极大
叶绿素荧光成像技术的发展
• Ladislav Nedbal（2000）等于上 世纪90年代末期发明了与PAM 技术相结合的叶绿素荧光成像 技术，研制成功了第一台 FluorCam调制式荧光成像仪。 从此正式开启了叶绿素荧光研 究的二维时代。
易科泰生态技术公司Ecolab生态实验室 / eco-lab@ info@
ห้องสมุดไป่ตู้加利福尼亚大学保护生物学研究中心（Vargas，2008） 原位研究监测土壤表面与剖面CO2浓度、根系及土壤过 程的时空动态变化
• BTC-100微根窗根系 观测系统、 • 3层土壤剖面CO2、 水分、温度观测系 统 • Li8100和ACE土壤呼 吸监测系统
上图：细根长度（上图空心蓝点）、菌根长度（上图实心红点）及 土壤呼吸动态变化； 下图：土壤温度与土壤体积含水量的动态变化（DOY为Day of year）
Ouyang，2010
Fan，2015
Screen mutants by NPQ parameters （通过荧光淬灭分析筛选变异植株）
• 在筛选对除草剂抗性强的玉米品种与基因型时， FluorCam叶绿素荧光成像可以直观并且定量地反映 其抗性及产生毒害部位的差异（Kopsell，2011）
FluorCam叶绿素荧光成像文献 2007 Low-temperature limitation of primary photosynthetic
Polar BiolDOI 10.1007/s00300-007-0331-x123ORIGINAL PAPERLow-temperature limitation of primary photosynthetic processes in Antarctic lichens Umbilicaria antarctica and Xanthoria elegansMilo n Barták · Peter Váczi · Josef Hájek · Jerzy SmyklaReceived: 7 May 2007 / Revised: 13 June 2007 / Accepted: 25 June 2007© Springer-Verlag 2007Abstract Temperature response curves of chlorophyll a X uorescence parameters were used to assess minimum sub-zero temperature assuring functioning of photosynthetic photochemical processes in photosystem II (PS II) of Antarctic lichens. Umbilicaria Antarctica and Xanthoria elegans were measured within the temperature range from ¡20 to +10°C by a X uorometric imaging system. For potential (F V /F M ) and actual ( II ) quantum yields of photo-chemical processes the minimum temperature was found to be between ¡10 and ¡20°C. Non-photochemical quench-ing (NPQ) of absorbed excitation energy increased with temperature drop reaching maximum NPQ at ¡15°C.Image analysis revealed intrathalline heterogeneity of chlo-rophyll a X uorescence parameters with temperature drop.Temperature response of II exhibited an S-curve with pro-nounced intrathalline di V erences in X. elegans. The same relation was linear with only limited intrathalline di V erence in U. antarctica . The results showed that Antarctic lichen species were well adapted to sub-zero temperatures and capable of performing primary photosynthesis at ¡15°C.Keywords Chlorophyll X uorescence imaging · Extreme temperature · Freezing · Non-photochemical quenchingIntroductionLichens are symbiotic organisms capable of surviving in extreme environments, including sub-zero temperatures.Lichens’ high tolerance to freezing temperature is reported e.g. for Xanthoria candelaria and Rhizoplaca melanophth-alma (Lange and Kappen 1972; Kappen 1989, 1993).Those species tolerated gradual or rapid freezing to ¡196°C, even after being stored up for several years,almost immediately resumed normal photosynthetic rates when warmed and wetted. After 5–7 months of cold and continuous darkness, they remain green with intact photo-synthetic pigments. In spite of the fact that optimum tem-perature for lichen photosynthesis lies above zero temperature (Uchida et al. 2006), it is well established that the majority of poikilohydric autotrophs abundant in alpine and polar environments photosynthesize at temperatures below 0°C (Reiter and Turk 2000; Kappen 2000). How-ever, it is still under debate regarding the minimum freez-ing temperature for photochemical and biochemical processes of photosynthesis for individual lichen species.Schroeter et al. (1994) and Kappen and Schroeter (1997)measured photosynthetic rates gasometrically in Antarctic lichens and reported net CO 2 uptake in Umbilicaria aprina at ¡17°C. Another study (Reiter and Turk 2000) that used gasometric measurements of CO 2 exchange brought the evidence that Flavocetraria nivalis exhibited positive net photosynthesis at ¡8°C. It is therefore believed that for majority of lichens well adapted to low temperature, net photosynthesis has temperature minimum within the range of ¡5 to ¡20°C. For ecophysiological studies in lichens, it is essential to understand photosynthetic performance of lichens at freezing temperatures so that the mechanisms of lichen productivity can be analyzed in long-term studies performed in alpine (e.g. Reiter and Turk 2000), tundraM. Barták (&) · P. Váczi · J. HájekDepartment of Plant Physiology and Anatomy, Institute of Experimental Biology, Masaryk University, Kotlál ská 2, 61137 Brno, Czech Republic e-mail: mbartak@sci.muni.czJ. SmyklaDepartment of Biodiversity, Institute of Nature Conservation, Polish Academy of Sciences,A. Mickiewicza 33, Krakow 31-120, Poland e-mail: smykla@iop.krakow.plPolar Biol123(Uchida et al. 2006), and polar regions (Schroeter et al.2000; Pannewitz et al. 2003).In lichens, primary photochemical processes of photo-synthesis in photosystem II (PS II) and thylakoid mem-brane of a chloroplast are e V ective at low temperatures. For lichens, measured both in the W eld (Schlensog and Schro-eter 2001) and in a laboratory (Hájek et al. 2001), either constant or slightly decreasing F V /F M is reported for the range of 0 to ¡5°C. E V ective yield of photochemical processes in PS II ( II ) is another characteristic investi-gated in low-temperature studies of lichen photosynthesis.Our last studies of central European lichen species mea-sured under optimum hydration indicated that the minimum temperature for primary photochemical processes of photo-synthesis, quantum yield of PS II ( II ) in particular, is about ¡10°C for control (Hájek et al. 2001) and certainly below ¡10°C for thalli treated with the addition of osmoti-cally active sugar alcohol, ribitol (Hájek et al. 2006). The aim of the present study was to quantify temperature response curves for selected chlorophyll X uorescence (Chl X uorescence) parameters (F V /F M , II , NPQ) and W nd mini-mum temperatures for lichens collected in Antarctica using Chl X uorescence technique.Materials and methodsLichen thalli collection and handlingUmbilicaria antarctica and Xanthoria elegans were collected in the Maritime Antarctic at Galindez Island in a close vicinity of Ukrainian station Vernadsky (65°14Ј43ЈЈS, 64°15Ј24ЈЈW). The thalli in a naturally dry state were transferred to the laboratory in Brno, Czech Republic, stored under a dim light (10 mol m ¡2s ¡1) at 5°C. Before measurements, the thalli were gradually hydrated by regular spraying with demineralized water for 48h. Then, their vitality and physiological status were tested X uorometrically (OS1-FL X uorometer, OptiScience,USA) using F V /F M as a marker. When maximum F V /F M ratio was reached (typically 0.55–0.60), the thalli were con-sidered optimally hydrated. For both species, it represented water potential of ¡3 to ¡1MPa (WP-4T water potential meter, Decagon, USA, data not shown) checked before and after measurements. For measurements at manipulated tem-perature, we selected the specimens that exhibited highest F V /F M and II . To study the e V ect of freezing temperatures on primary photosynthetic processes in lichens, U. antarc-tica and X. elegans were chosen as experimental species.The two species are quite abundant along the Antarctic Peninsula (Krzewicka and Smykla 2004; Olech 2001, 2004;Øvstedal and Lewis Smith 2001) and typical for vegetation oases in the maritime and continental Antarctic.Chlorophyll X uorescence measurementsResponse of primary photosynthetic processes to the tem-perature range from ¡20 to +10°C was monitored by Chl X uorescence parameters measured by a PC-linked X uorom-eter FluorCam 700 MF (Photon Systems Instruments,Czech Republic) equipped with a CCD camera and image analysis software (FluorCam v. 6.0). During measure-ments, temperature within the measuring chamber was maintained constant by a thermo-regulator controlled cool-ing unit (ConBrio, Czech Republic) consisting of two Peltier coolers, one adjacent to the metal plate on which an experimental lichen thallus was placed, the other maintain-ing air temperature inside the measuring chamber. The Peltier coolers were connected to a water loop with circu-lating water (thermostat Labio, Czech Republic) ensuring e V ective cooling. During the low-temperature experiments,both chamber and lichen thallus temperatures were moni-tored with a set of Cu–Co thermocouples linked to a data-logger (VV/VX Minicube, Environmental Monitoring Systems, Czech Republic). Optimally hydrated lichen thalli were placed in the measuring chamber and exposed consequently to a temperature decreasing in a 5°C step within the range of 10 to ¡25°C. At each temperature,thalli equilibrated for 0.5h at moderate irradiance of 30 mol m ¡2s ¡1. After the equilibration, a set of Chl X uo-rescence parameters was measured using Chl X uorescence imaging. For detailed description of the method and its application in lichen photosynthetic studies see, e.g. Barták et al. (2000, 2004).In our experiments, Chl X uorescence imaging technique provided high resolution (512£512pixels) false-colour images of Chl X uorescence distribution over a lichen thal-lus. For each pixel of the images taken, the kinetics of Chl X uorescence induced by the below-speci W ed light treat-ment was recorded and basic Chl X uorescence parameters determined. In dark-adapted state, lichen thalli were exposed to experimental protocol using actinic light and saturation light pulses that allowed calculating and visual-izing F V /F M and II distribution over a thallus. The mea-surements started with F 0 determination at low light (5 mol m ¡2s ¡1), followed by a maximum Chl X uores-cence (F M ) determination at (1,500 mol m ¡2s ¡1). Then,after 5min exposure to a constant moderate irradiance (100 mol m ¡2s ¡1, su Y cient for lichens, tested before),the lichen thallus exhibited steady-state Chl X uorescence (F S ). In this state, maximum Chl X uorescence (F M Ј) in light-adapted state was determined and quantum yield of photochemical processes in photosystem II ( II ) calcu-lated as (F M Ј¡F S )/F M Ј. Non-photochemical quenching was calculated as NPQ =(F Minit -F M Ј)/F M Ј (see e.g.Vráblíková et al. 2005). After switching o V the actinic light, F M ЈЈ Chl X uorescence level was determined andPolar Biol123used for qE determination. The images were taken for typically W ve lichen thalli at each experimental tempera-ture. The images were then analyzed using a FluorWin software and the above-speci W ed Chl X uorescence param-eters were evaluated either for the whole thallus or selected tiny spots (typically eight, each of which 5mm in diameter) representing thallus area exhibiting high/low photosynthetic activity.ResultsWhen calculated for whole thallus, mean Chl X uorescence parameters of X. elegans and U. antarctica showed a simi-lar response to decreasing temperature (Fig.1), however,some species-speci W c di V erences were found. It was partic-ularly apparent for F V /F M which was signi W cantly higher in X. elegans than U. antarctica within the temperature range of 0–10°C. For both species, critical low temperature at which F V /F M started to decline was ¡5°C. F V /F M further decreased with temperature reaching their minimum values at about ¡15°C. Temperature-induced change in II was similar in both species; however, the II decline withtemperature decrease was steeper in X. elegans than U.antarctica .Within a single thallus, signi W cant di V erences in F V /F M , II , and NPQ found between di V erent thallus parts proved heterogeneity in distribution of the parameters. Maxima of the two parameters showed species-speci W c patchiness. In X. elegans , maximum F V /F M was predominantly found in close-to-central parts of the thalli where fruiting bodies, if formed, were located. In U. antarctica , areas of maximum F V /F M were located in irregularly distributed patches within the thalli.In the images of F V /F M and II distribution over lichen thalli (data not shown), some apparently demarked local maxima were apparent. In thalli of U. antarctica , few areas exhibiting F V /F M higher than mean were located between thallus centre and margin, apparently distant from umbili-cus. Areas with low photochemical activity were located to marginal parts. The di V erence between thallus parts exhib-iting high and low photosynthetic photochemical activity was apparent mainly at the temperature range of 0–10°C (See Fig.2). Gradual decrease in thallus temperature led to inhibition of photosynthetic activity both in the areas exhib-iting high and low F V /F M . First signs of the inhibition werePolar Biol123manifested, similarly to the whole thalli, at ¡5°C. In X. ele-gans , maxima of F V /F M were located to the youngest grow-ing parts of individual thalli (see also Discussion ). Minima were found either in the oldest central parts where thallus was dropped o V (sometimes with missing lichen biomass)or close to the margin. Distribution of areas exhibiting high/low II was, irrespective of experimental temperature,similar to F V /F M (data not shown). However, contrastingly to F V /F M , the decrease in II was found within the whole temperature range, more apparently in U. antarctica .Another di V erence was that II did not di V er in the thallus parts exhibiting high and low capacity of photosynthetic photochemical processes (F V /F M ). Non-photochemical quenching exhibited typical response with temperature decrease. In both species, it increased at freezing tempera-tures showing di V erences between thalli parts typical by di V erent F V /F M .DiscussionThe response of F V /F M , II and NPQ showed for both spe-cies that photochemical processes in PS II of both studied lichen species still have detectable activity at least at the temperature of ¡10°C. Below that point, freezing tempera-ture brings a substantial inhibition of photochemical pro-cesses of photosynthesis. Incident light might be an interacting factor limiting primary photosynthetic pro-cesses. The phenomenon of low-temperature photoinhibi-tion was reported, e.g. for poikilohydric mosses (Lovelock et al. 1995). For Antarctic lichens, either no (Kappen et al.1998) or moderate photoinhibition (Barták et al. 2003) was found in W eld and laboratory studies, respectively. On chlo-roplast level, net photosynthesis decreases due to low sup-ply of ATP and NADPH from temperature-dependent limitation of photochemical processes related to electron transport through thylakoid membrane. However, in spite of extreme low values of II , found at sub-zero tempera-ture, positive net photosynthesis need not be necessarily diminished. Lichens possess several mechanisms assuring cell functioning at low temperature. At freezing tempera-tures from 0 to ¡10°C, due to the presence of osmotically active compounds, cells of mycobiont are increasingly cav-itated and their protoplast closely attached to cell wall while photobiont cells are still in function (Schroeter and Scheidegger 1995). Another protective mechanism is e.g.ice nucleation activity (Worland et al. 1996) that reduces intracellular formation of ice crystals with sharp edges. At extremely low freezing temperature, however, negative e V ects of intra- and extracellular ice formation in lichens cannot be excluded. At the temperatures below critical, ice formation may cause decline of functioning of cells of symbiotic algae and thus a V ect primary photochemicalprocesses. Our samples were exposed to decreasing tem-perature under optimum hydration. Therefore, negative e V ects of extra- and/or intracellular freezing of water on photosynthesis might be higher than expected in partly or fully desiccated thalli. Such alleviation of the negative e V ect by a progressive thalli desiccation during freezing was documented for Antarctic lichens (Schroeter et al.1997) and moss (Kennedy 1993). Moreover, the most apparent decrease in F V /F M and II was found at about ¡5°C which corresponds to the temperature reported as critical for ice-nucleation activity in fully hydrated lichen thalli (Umbilicaria aprina , Schroeter and Scheidegger 1995) during gradual freezing. Haranczyk et al. (2003)showed for several lichens with contrasting anatomy that ¡5°C is an edge temperature for freezing of water in their thalli.Non-photochemical quenching increased at sub-zero temperature similarly as found in an earlier study (Hájek et al. 2001). This indicated low temperature-induced involvement of quenching mechanism scavenging excita-tion energy absorbed by light harvesting complexes of PS II. The NPQ increase might be attributed both to zeaxan-thin-dependent component manifested as an increase in DEPS (Lovelock et al. 1995) and qE (our data, not shown),and PS II quenchers-dependent thermal energy dissipation (Heber et al. 2006).At each experimental temperature, primary photosyn-thetic processes exhibited spatial heterogeneity over examined thalli, similarly to previous studies on lichens under diverse environmental conditions such as, e.g. high light (Barták et al 2000, 2004), osmotic (Hájek et al.2006), and chemical stress (Jensen and Siebke 1997). In the present study, the heterogeneity of F V /F M , II and NPQ might be attributed to thallus anatomy, thallus thick-ness, distribution of growing pseudomeristems, and age of photobiont cells present in particular thallus zones. It is clear that the investigated X. elegans has been formed by several formerly independent thalli growing for decades before they reach each other. Finally, they formed a semi-uniform cover over a stone. The heterogeneity in F V /F M and II of investigated thallus, therefore, showed a certain regularity corresponding to the distribution of round-shaped objects forming the respective independent thalli.The highest F V /F M and II were found close to the object edges. This is the same type of F V /F M heterogeneity observed earlier in X. elegans in the W eld (Barták et al.2005) during gradual desiccation.Acknowledgments The research of stress physiology of Antarctic lichen photosynthesis was supported by projects No. 522/03/0754(Grant Agency of the Czech Republic), KONTAKT No. ME 945 pro-vided by the Czech Ministry of Education, Sport and Youth and partly also from grant No. 2P04F00127 provided by the Polish State Commit-tee for Scienti W c Research.Polar Biol123ReferencesBarták M, Hájek J, Gloser J (2000) Heterogeneity of chlorophyll X uo-rescence over thalli of several foliose macrolichens exposed to adverse environmental factors: interspeci W c di V erences as related to thallus hydration and high irradiance. Photosynthetica 38:531–537Barták M, Vráblíková H, Hájek J (2003) Sensitivity of photosystem 2of Antarctic lichens to high irradiance stress: Fluorometric study of fruticose (Usnea antarctica ) and foliose (Umbilicaria decuss-ata ) species. Photosynthetica 41:497–504Barták M, Hájek J, Vráblíková H, Dubová J (2004) High-light stressand photoprotection in Umbilicaria antarctica monitored by chlorophyll X uorescence imaging and changes in zeaxanthin and glutathione. Plant Biol 6:333–341Barták M, Gloser J, Hájek J (2005) Visualized photosynthetic charac-teristics of the lichen Xanthoria elegans related to daily sources of light, temperature and hydration: a W eld study from Galindez Is-land, maritime Antarctica. Lichenologist 37:433–443Hájek J, Barták M, Gloser J (2001) E V ects of thallus temperature andhydration on photosynthetic parameters of Cetraria islandica from contrasting habitats. Photosynthetica 39:427–435Hájek J, Barták M, Dubová J, Váczi P (2006) Inhibition of photosyn-thetic processes in foliose lichens induced by temperature and os-motic stress. Biol Plant 50:624–634Haranczyk H, Grandjean J, Olech M (2003) Freezing of water boundin lichen thallus as observed by H-1 NMR. II. Freezing protection mechanisms in a cosmopolitan lichen Cladonia mitis and in Ant-arctic lichen species at di V erent hydration levels. Colloid Surf B 28:251–260Heber U, Bilger W, Shuvalov VA (2006) Thermal energy dissipationin reaction centres and in the antenna of photosystem II protects desiccated poikilohydric mosses against photo-oxidation. J Exp Bot 57:2993–3006Jensen M, Siebke K (1997) Fluorescence imaging of lichens in themacro scale. Symbiosis 23:183–195Kappen L (1989) Field measurements of carbon dioxide exchange ofthe Antarctic lichen Usnea sphacelata in the frozen state. Antarct Sci 1:31–34Kappen L (1993) Lichens in the Antarctic region. In: Friedmann EI(ed) Antarctic microbiology, Wiley-Liss, New York, pp 433–490Kappen L (2000) Some aspects of the great success of lichens inAntarctica. Antarct Sci 12:314–324Kappen L, Schroeter B (1997) Activity of lichens under the in X uenceof snow and ice. Proc NIPR Symp. Polar Biol 10:163–168Kappen L, Schroeter B, Green TGA, Seppelt RD (1998) Chlorophyll aX uorescence and CO 2 exchange of Umbilicaria aprina under ex-treme light stress in the cold. Oecologia 113:325–331Kennedy AD (1993) Photosynthetic response of the Antarctic mossPolytrichum alpestre Hoppe to low temperatures and freeze–thaw stress. Polar Biol 13:271–279Krzewicka B, Smykla J (2004) The lichen genus Umbilicaria from theneighbourhood of Admiralty Bay (King George Island, maritimeAntarctic), with a proposed new key to all Antarctic taxa. Polar Biol 28:15–25Lange OL, Kappen L (1972) Photosynthesis of lichens from Antarc-tica. In: Llano GA (ed) Antarctic terrestrial biology. Antarctic Res Series 20:83–95Lovelock CE, Jackson EJ, Melick DR, Seppelt RD (1995) ReversiblePhotoinhibition in Antarctic moss during freezing and thawing.Plant Physiol 109:955–961Olech M (2001) Annotated chcklist of Antarctic lichens and lichenic-olous fungi. Jagiellonian University, Kraków, pp 1–145Olech M (2004) Lichens of King George Island, Antarctica. Instituteof Botany of Jagiellonian University, Kraków, pp 1–385Øvstedal DO, Lewis Smith RI (2001) Lichens of Antarctica and SouthGeorgia. Cambridge University Press, England, p 424Pannewitz S, Schlensog M, Green TGA, Sancho LG, Schroeter B(2003) Are lichens active under snow in continental Antarctica?Oecologia 135:30–38Reiter R, Turk R (2000) Investigations on the CO 2 exchange of lichensin the alpine belt. II. Comparative patterns of net CO 2 exchange in Cetraria islandica and Flavocetraria nivalis . Phyton 40:161–177Schlensog M, Schroeter B (2001) A new method for the accuratein situ monitoring of chlorophyll a X uorescence in lichens and bryophytes. Lichenologist 33:443–452Schroeter B, Scheidegger C (1995) Water relations in lichens at subze-ro temperatures—structural changes and carbon dioxide ex-change in the lichen Umbilicaria aprina from continental Antarctica. New Phytol 131:273–285Schroeter B, Green TGA, Kappen L, Seppelt RA (1994) Carbon diox-ide exchange at subzero temperatures: W eld measurements on Umbilicaria aprina in Antarctica. Cryptogram Bot 4:233–241Schroeter B, Schulz F, Kappen L (1997) Hydration-related spatial andtemporal variation of photosynthetic activity in Antarctic lichens.Antarctic communities: species, structure and survival: proceed-ings of the 6th SCAR Biology Symposium, Venice, 1994. Batta-glia B, Valencia J, Walton DW (eds) Cambridge University Press,Cambridge pp 221–225Schroeter B, Kappen L, Sancho LG (2000) Seasonal variation in thecarbon balance of lichens in the maritime Antarctic: long-term measurements of photosynthetic activity in Usnea aurantiaco-atra . In: Davison W, Howard-Williams C, Broady P (eds) Antarc-tic ecosystems: models for wider ecological understanding. 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FluorCam叶绿素荧光成像文献 2009 Identification of photosynthetic sacoglossans from Japan
112Encocytobiosis Cell Res. (2009) 19, 112-119Identification of photosynthetic sacoglossans from JapanYoshiharu Y. Yamamoto1,2, Yoichi Yusa3, Shoko Yamamoto3, Yayoi Hirano4, Yoshiaki Hirano4, Taizo Motomura5, Takanori Tanemura1, Junichi Obokata1,6,*Center for Gene Research, Nagoya University, Nagoya 464-8602, Japan; 2Faculty of Applied Biological Sciences, Gifu University, Yanagido 1-1, Gifu City, 501-1193, Japan; 3Department of Biological Sciences, Faculty of Science, Nara Women’s University, Kitauoyanishi, Nara, Nara 630-8506, Japan; 4Department of Biology, Faculty of Science, Chiba University, Yayoi-cho, Inage-ku, Chiba, Chiba 263-8522, Japan; 5Muroran Marine Station, Field Science Center for Northern Biosphere, Hokkaido University, Muroran 051-0003; 6 Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Kyoto 606-8552, Japan. * Corresponding author: obokata@gene.nagoya-u.ac.jp, obokata@kpu.ac.jp1AbstractSome sacoglossan molluscs, including several species of Elysiidae, are known to incorporate algal chloroplasts, and the incorporated chloroplasts are functional for days to months, depending on species. This incorporation and maintenance of foreign chloroplasts are known as kleptoplasty. In this article, we surveyed photosynthetic activity of several sacoglossans collected in Japan, by analysis of in vivo chlorophyll fluorescence. Our survey found 8 new species that have active chlorophylls, judged by the parameter Fv/Fm, an indicative of the functionality of photosystem II. Identified sacoglossans with active chlorophylls belong to the Bosella, Costasiella, Elysia, Julia, Placida, Stiliger, and Thuridilla genera. Our results strongly suggest that these species possess kleptoplasts. Possession of chloroplasts was confirmed for one of the identified sacoglossans, Elysia trisinuata, by transmission electron microscopy.IntroductionSome sacoglossans are known to have ability to photosynthesize using chloroplasts present in their cells. These chloroplasts are not inherited from the parents, but “stolen” from their food algae, such as Codium or Caulerpa. Acquired algal chloroplasts are maintained in cells of digestive glands of sacoglossans fordays to months, depending on species. This incorporation and maintenance is called as kleptoplasty (TRENCH and OHLHORST 1976; RUMPHO et al. 2007). After the first report of kleptoplasty by Elysia astroviridis (KAWAGUTI and YAMASU 1965), several species have been shown to have kleptoplasts by transmission electron microscopic (TEM) analysis (TRENCH et al. 1969). Functionality of kleptoplasts was demonstrated by light-driven CO2-incorporation using radioisotope (14C) (HINDE and SMITH 1974), light-dependent O2-emission detected by oxygen electrode (RUMPHO et al. 2000), and in vivo chlorophyll fluorescence analysis for detection of functional chlorophylls (EVERTSEN et al. 2007). Now, kleptoplasts have been found in more than 20 species of sacoglossans that belong to genera Alderia, Bosellia, Caliphylla, Elysia, Hermaea, Limapontia, Mourgona, Oxynoe, Plakobranchus, Tridachia, and Thuridilla (EVERTSEN et al. 2007). In this report, we screened for Japanese sacoglossans containing functional chloroplasts, judged by possession of active chlorophylls that can be detected by in vivo chlorophyll fluorescence analysis. We first report possession of active chlorophylls in eight species, including ones belonging to Costasiella, Julia, Placida, and Stiliger genera. Our results, together with previous studies in this field,Yamamoto et al. – Identification of photosynthetic sacoglossans from Japan113suggest that kleptoplasty occur in a wide range of sacoglossans.1963), and observed with a JEM-1011 electron microscope (JEOL, Tokyo, Japan).Materials and MethodsSea slugs were collected in 2007 and 2008 at Kominato (Kamogawa, Chiba, Japan), Misaki (Miura, Kanagawa, Japan), Shirahama (Nishimuro, Wakayama, Japan), Sesoko (Motobu, Okinawa (Ryukyu), Japan), and Zanpa (Yomitani-son, Okinawa, Japan). Collected sea slugs were sent to Nagoya University and subjected to in vivo chlorophyll fluorescence analysis using FluorCam (Photon Systems Instruments, Brno, Czech Republic) based on the pump and probe system with a CCD camera detector (NEDBAL and WHITMARSH 2004). Fv/Fm (BAKER 2008) was measured per individual using the camera system, and the average and standard deviation were calculated, if possible. Assays were done within a week after collection except for Costasiella cf. kuroshimae and Elysia ornata. These two species were assayed in a month after collection. Until assays, E. ornata, Placida sp., and Costasiella cf. kuroshimae were kept together with their food algae that are Codium, Codium, and Avrainvillea, respectively. Because we could not prepare food algae for the other sea slugs, it was impossible to adjust days before assay after start of starvation. This problem caused some difficulty in strict comparison of Fv/Fm among species. For transmission electron microscopy (TEM), samples were fixed with 3% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) containing 3% NaCl for 2 h at 4˚C. In this fixative, the samples were cut into small fragments. Then they were postfixed with 1% OsO4 in 0.1 M cacodylate buffer (pH 7.2) containing 3% NaCl for 2 h at room temperature. They were dehydrated with a graded acetone series and embedded in Spurr’s epoxy resin. Thin sections were cut by a diamond knife on a Ultracut (Reichert-Jung, Wien, Austria), mounted on formvar-coated slot grids, stained with 4% uranyl acetate and lead citrate (REYNOLDSResults and DiscussionsSacoglossans collected at several shores in Japan were subjected to in vivo chlorophyll fluorescence analysis. Plakobranchus ocellatus and Pteraeolidia ianthina were used as positive controls. P. ocellatus has kleptoplasts (GREENE 1970b; HIROSE 2005), whose functionality has been confirmed by detection of light-dependent CO2-fixation (GREENE 1970a) as well as Fv/Fm determined by in vivo chlorophyll fluorescence analysis (EVERTSEN et al. 2007). P. ianthina is known to be a host of endosymbiotic zooxanthella, rather than kleptoplasts. P. ianthina has also been reported to have photosynthetic activity due to the symbionts, revealed by light-dependent carbon fixation activity (HOEGH-GULDBERG and HINDE 1986). As shown in Table 1, both P. ocellatus and P. ianthina showed high Fv/Fm, as expected. These results confirmed possession of active chlorophylls in these organisms. It should be noted that this analysis cannot distinguish between kleptoplasts (P. ocellatus) and endosymbiotic algae (P. ianthina). We collected 12 species in sacoglossa in Japan, and subjected them to in vivo chlorophyll fluorescence analysis. Among them, 8 species showed measurable Fv/Fm values (Table 1). These results indicate that the positive species, that belong to genera of Costasiella, Bosellia, Elysia, Thuridilla, Julia, Placida, and Stiliger, possess active chlorophylls and thus photosynthetically functional chloroplasts. Considering that all these species belong to sacoglossa, these results strongly suggest that they have kleptoplasts. Photographs of positive species are shown in Figure 1. One of the positive species, Elysia trisinuata, was subjected to electron microscopic analysis (Figure 2). As expected, cells of digestive glands contained chloroplasts (Ct in the Figure 2B), judged by their ultrastructure. As shown in114Yamamoto et al. – Identification of photosynthetic sacoglossans from JapanTable 1: Sacoglossans with active chlorophylls identified in this study Species SACOGLOSSA Costasiellidae Costasiella roshimae Elysiidae Bosellia sp. Elysia ornata Ibid. Elysia trisinuata Thuridilla vatae Juliidae Julia zebra Limapontiidae Placida sp. (sensu (BABA 1986) Stiliger ornatus Plakobranchidae Plakobranchus ocellatus NUDIBRANCHIA Pteraeolidia ianthina1 Kominato 2008 Oct. 4 20-50 0.831 ± 0.023 Kominato: Kamogawa, Chiba; Misaki: Miura, Kanagawa; Shirahama: Nishimuro, Wakayama; Sesoko: Motobu, Okinawa; Zanpa: Zanpamisaki, Yomitani-son, Okinawa. 1 Known to retain Symbiodinium by endosymbiosis (BURGHARDT et al. 2008). Sesoko 2008 Jun. 1 22 0.811 Shirahama 2008 Oct. 3 7-10 0.595 ± 0.0795 Sesoko 2008 Apr. 9 4-8 0.475 ± 0.075 Sesoko 2008 Apr. 1 2 0.616 Sesoko Shirahama Misaki Misaki Sesoko 2008 Apr. 2007 Sep. 2007 Oct. 2007 Aug. 2008 Jun. 4 3 5 6 1 7-11 20-40 20-40 15-30 14 0.716 ± 0.052 0.815 ± 0.0201 0.844 ± 0.0190 0.853 ± 0.042 0.725 cf. kuZanpa 2008 Dec. 5 1-3 0.661 ± 0.0395 Sampling place Sampling date Number of individuals Size (mm) Fv/Fmthe figure, some chloroplasts possess starch grains (white ovals, Figure 2B), suggesting functionality of these kleptoplasts. As shown in Table 1, Fv/Fm varies from 0.85 (Elysia trisinuata) to 0.48 (Placida sp.) according to species, and variation within a species was much smaller than difference among species. As for Elysia ornata, we analyzed two batches, one from Shirahama collected in September and the other from Misaki caught in October. Although these two groups were caught at different places in different seasons, again they showed small difference in Fv/Fm (0.815 ± 0.0201 and 0.844 ± 0.0190, respectively).Fv/Fm is a parameter that reflects maximum efficiency of photosystem II, and thus it indicates intactness of photosystem II (BAKER 2008). Injury of photosystem II, that can be caused by environmental stresses, results in reduction of Fv/Fm (BAKER 2008). Although degree of injury against environmental stresses can vary among individuals, we found that there is little variation of Fv/Fm among the analyzed individuals of the same species. In the case of kleptoplasts, decrease of Fv/Fm was observed during starvation, corresponding to the loss of kleptoplasts (EVERTSEN et al. 2007). Periods until loss of kleptoplasts vary from days to months, depending on spe-Yamamoto et al. – Identification of photosynthetic sacoglossans from Japan115Thin bar: 0.5 mm, thick bar: 10 mmFigure 1: Sacoglossans with functional chlorophylls Thin and thick scale bars indicate 0.5 mm and 10 mm, respectively. A: Bosellia sp., B: Elysia trisinuata, C: Elysia ornata, D: Julia zebra, E: Stiliger ornatus, F: Costasiella cf. kuroshimae, G: Thuridilla vatae, H: Placida sp. (sensu BABA, 1986)cies. Sacoglossans at a higher level of kleptoplasty maintain chloroplasts for a longer period (CLARK et al. 1990). The reduction of Fv/Fm during starvation is related to the loss of kleptoplasts, but it is not due to the reduction of the chloroplast number, because Fv/Fm should not be affected by amount of chloroplasts or chlorophylls in an individual (BAKER 2008). Reason for the reduction of Fv/Fm duringstarvation is not clear. Theoretically, the reduction can be caused by either increase of injured photosytem II as mentioned above, or saturation of the electron transport system after photosystem II in the dark (BAKER 2008). Anyway, this reduction causes variations of Fv/Fm within a species. In spite of the above tendency, observed Fv/Fm was quite similar within a species (Table 1). This would be in116Yamamoto et al. – Identification of photosynthetic sacoglossans from JapanFigure 2: Transmission electron micrographs of Elysia trisinuata A. Section of digestive glands. B. part of a cell in digestive glands. Ct: chloroplast. Electron-dense black circles in chloroplasts are plastoglobules, and electron-sparse ovals in chloroplasts are starch grains.part due to the same starvation period for a batch of assays. Among the sacogrossans shown in the table, Elysia ornata, E. trisinuata, and Plakobranchus ianthobaptus showed the highest Fv/Fm values. These species can survive for the longest periods (more than one month) in the list during starvation (GREENE 1970b) (data not shown). On the other hand, Placida sp. shows the lowest Fv/Fm value among the positive list, and this species survived for the shortest period during starvation among the positive species, that is, less than a week (data not shown). Therefore, there seems to be a rough correlation between Fv/Fm and survival period during starvation. As for molluscs with photosynthetic activity,there are two possible cytological situations: possession of kleptoplasts or of endosymbiotic algae (VENN et al. 2008). We suggest that the photosynthetic sacoglossans shown in Table 1 have kleptoplasts, because almost all of the known photosynthetic sacoglossans possess kleptoplasts and there is no report on endosymbiosis of algae in adult sacoglossans. Sacoglossans with kleptoplasts, including our results, are summarized in Table 2. As shown in this table, around 30 species are now known to have kleptoplasts. The largest group in the table is Elysiidae, and most of the families in Sacoglossa are included in the table, except for two families, Gascoignellidae and Volvatellidae. This demonstrates widespread distribution of kleptoplasty in Sacoglossa.Yamamoto et al. – Identification of photosynthetic sacoglossans from Japan Table 2: List of sacoglossans reported to possess functional chloroplasts117Species Method Reference Caliphyllidae 14 Caliphylla mediterranea C (CLARK et al. 1990) 14 Mourgona germainea C (CLARK et al. 1990) Costasiellidae Costasiella cf. kuroshimae Fv/Fm This work Elysiidae 14 Bosellia mimetica C (CLARK et al. 1990) Bosellia sp. Fv/Fm This work 14 Elysia australis C (HINDE 1980) Chla (CLARK and BUSACCA 1978) Elysia cauze (= E. subornata)1 Elysia chlorotica O2 (RUMPHO et al. 2000) 2 a Elysia crispata (= Tridachia crispata) Chl (CLARK and BUSACCA 1978) 14 C, Chl (GREENE 1970a) Elysia hedgpethi Elysia ornata Fv/Fm This work Elysia pusilla Fv/Fm (EVERTSEN et al. 2007) Elysia sp. Fv/Fm (EVERTSEN et al. 2007) Elysia timida Fv/Fm (EVERTSEN et al. 2007) Elysia tomentosa Fv/Fm (EVERTSEN et al. 2007) Elysia trisinuata Fv/Fm This work ibid. TEM This work Elysia tuca Chla (CLARK and BUSACCA 1978) 14 C (TRENCH et al. 1973; HINDE and SMITH 1974) Elysia viridis Thuridilla carloni Fv/Fm (EVERTSEN et al. 2007) Thuridilla lineolata Fv/Fm (EVERTSEN et al. 2007) Thuridilla vatae Fv/Fm This work Hermaeidae 14 Hermaea cruciata C (CLARK et al. 1990) Juliidae Julia zebra Fv/Fm This work Limapontiidae 14 C (CLARK et al. 1990) Alderia modesta Placida sp. (sensu (BABA 1986) Fv/Fm This work 14 Limapontia depressa C, Chla (HINDE and SMITH 1974) Stiliger ornatus Fv/Fm This work Oxynoidae Oxynoe antillabrum Chla (CLARK and BUSACCA 1978) Plakobranchidae Chla (GREENE 1970a) Plakobranchus ocellatus (=P. ianthobaptus)3 ibid. Fv/Fm (EVERTSEN et al. 2007), this work ibid. TEM (HIROSE 2005) The table shows sacoglossans containing functional chloroplasts (Level 4 and higher categories (CLARK et al. 1990)). Chl: extraction and measurement of chlorophyll content, Fv/Fm: PSII activity determined by in vivo chlorophyll fluorescence measurement (pulse amplitude modulation (PAM) or pump & probe methods), O2: light-dependent O2 emission, 14C: light-dependent incorporation of NaH14CO3. aPossession of chlorophylls does not directly mean functionality of chloroplasts, but stable maintenance of chlorophylls suggest it (EVERTSEN et al. 2007). References for synonymous species are: 1(CLARK 1984), 2(GOSLINER 1995), 3(JENSEN 1992).118Yamamoto et al. – Identification of photosynthetic sacoglossans from Japan GREENE, R.W. (1970b) Symbiosis in sacoglossan opisthobranchs: Symbiosis with algal chloroplasts. Malacologia 10, 357-368 HINDE, R. (1980) Chloroplast "symbiosis" in sacoglossans molluscs In Proceedings of the international colloquium on endosymbiosis and cell research. (Schwemmler, W and Schenk, HEA, eds). Tübingen: Walter de Gruyter, pp. 729-736 HINDE, R. and SMITH, D.C. (1974) "Chloroplast symbiosis" and the extent to which it occurs in Sacogrossa (Gastropoda: Mollusca). Biol. J. Linn. Soc. 6, 349-359 HIROSE, E. (2005) Digestive system of the sacoglossan Plakobranchus ocellatus (Gastropoda: Opisthobranchia): light- and electron-microscopic observations with remarks on chloroplast retention. Zool. Sci. 22, 905-916 HOEGH-GULDBERG, O. and HINDE, R. (1986) Studies on a nudibranch that contains zoozanthellae I. Photosynthesis, respiration and translocation of new fixed carbon by zooxanthellae in Pteraeolidia ianthina. Proc. R. Soc. Lond. B. 228, 493-509 JENSEN, K.R. (1992) Anatomy of some Indo-Pacific Elysiidae (Opisthobranchia: Sacoglossa (=Ascoglossa)), with a discussion of the generic division and phylogeny. J. Moll. Stud. 58, 257-296 KAWAGUTI, S. and YAMASU, T. (1965) Electron microscopy on the symbiosis between an elysioid gastropod and chloroplasts of a green alga. Biol. J. Okayama Univ. 11, 57-65 NEDBAL, L. and WHITMARSH, J. (2004) Chlorophyll fluorescence imaging of leaves and fruits. In: Chlorophyll a fluorescence. (C., PG and Govindjee, eds). Dordrecht: Springer, pp. 389-407 REYNOLDS, E.S. (1963) The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell Biol. 17, 208-211 RUMPHO, M.E., DASTOOR, F.P., MANHART, J.R. and LEE, J. (2007) The kleptoplast In The structure and function of plastid. (Wise, RR and Hoober, JK, eds). Dordrecht: Springer, pp. 451-473 RUMPHO, M.E., SUMMER, E.J. and MANHART, J.R. (2000) Solar-powered sea slugs. Mollusc/algal chloroplast symbiosis. Plant Physiol. 123, 29-38 TRENCH, R.K., BOYLE, J.E. and SMITH, D.C. (1973) The association between chloroplasts of Codium fragile and the mollusc Elysia viridis II. ChloroplastAcknowledgementsWe would like to thank Ms. Ayami Sekizawa of Osaka City University for collection of some sacogrossans. This study was supported in part by a grant from the Research Institute for Marine Invertebrates, Tokyo, and by Grants-In-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.ReferencesBABA, K. (1986) Anatomical information of Placida sp. = Hermaea dendritica of Baba, 1937 and 1955 from Japan. Shells Sea Life 18, 21-22 BAKER, N.R. (2008) Chlorophyll fluorescence: A probe of photosynthesis in vivo. Annu. Rev. Plant Biol. 59, 89-113 BURGHARDT, I., STEMMER, K. and WAGELE, H. (2008) Symbiosis between Symbiodinium (Dinophyceae) and various taxa of Nudibranchia (Mollusca: Gastropoda), with analyses of long-term retention. Org. Div. Evol. 8, 66-76 CLARK, K.B. (1984) New records and synonymies of Bermuda opisthobranchs (Gastropoda). Nautilus. 98, 85-97 CLARK, K.B. and BUSACCA, M. (1978) Feeding specificity and chloroplast retention in four tropical ascoglossa, with a discussion of the extent of chloroplast symbiosis and the evolution of the order. J. Moll. Stud. 44, 272-282 CLARK, K.B., JENSEN, K.R. and STIRTS, H.M. (1990) Survery for functional kleptoplasty among west Atrantic Ascoglossa (=Sacoglossa) (Mollusca: Opisthobranchia). Veliger 33, 339-345 EVERTSEN, J., BURGHARDT, I., JOHNSEN, G. and WAGELE, H. (2007) Retention of functional chloroplasts in some sacoglossans from the Indo-Pacific and Mediterranean. Mar. Biol. 151, 2159-2166 GOSLINER, T.M. (1995) The genus Thuridilla (Opisthobranchia: Elysiidae) from the tropical Indo-Pacific, with a revision of the phylogeny and systematics of the Elysiidae. Proc. Calif. Acad. Sci. 49, 1-54 GREENE, R.W. (1970a) Symbiosis in sacoglossan opisthobranchs: functional capacity of symbiotic chloroplasts. Mar. Biol. 7, 138-142Yamamoto et al. – Identification of photosynthetic sacoglossans from Japan ultrastructure and photosynthetic carbon fixation in E. viridis. Proceedings of the Royal Society of London. Series B, Biological SciencesProceedings of the Royal Society of London. Series B, Biological Sciences 184, 63-81 TRENCH, R.K., GREENE, R.W. and BYSTROM, B.G. (1969) Chloroplasts as functional organelles in animal tissues. J. Cell Biol. 42, 404-417119TRENCH, R.K. and OHLHORST, S. (1976) The stability of chloroplasts from siphonaceous algae in symbiosis with sacoglossan molluscs. New Phytol. 76, 99-109 VENN, A.A., LORAM, J.E. and DOUGLAS, A.E. (2008) Photosynthetic symbioses in animals. J. Exp. Bot. 59, 1069-1080。
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（2012年5月中科院植物所 叶绿素荧光技术及其应用研习班） 易科泰生态技术公司 Ecolab生态研究室
FluorCam叶绿素荧光成像技术及其应用
• • • • • 叶绿素荧光技术 FluorCam叶绿素荧光成像 FluorCam叶绿素荧光成像应用案例 Ecolab实验室叶绿素荧光成像实验 FluorPen手持式叶绿素荧光仪与AquaPen 藻类荧光仪
FluorCam荧光成像技术特点
• • • • • • • • • • • 高灵敏度CCD镜头，时间分辨率可达每秒50张，快速响应和实验程序分析，精 确反映叶绿素荧光瞬变 可选配高分辨率（高像素值）CCD镜头，适于需要高分辨率荧光成像分析如 GFP等稳态荧光 软件具备Fv/Fm、Kautsky 诱导效应、荧光淬灭分析、稳态荧光成像、PAR 吸 收指数、LC等自动实验程序，实验程序可设置更改，或创建新的实验程序 具备实验程序自动重复功能，从而实现无人职守自动成像实验 “4+1”LED光源（周边4个LED光源板，顶部一个LED光源板），可选配红色光 源、橙色光源、绿色光源、青色光源、蓝色光源、白色光源、红外光源、紫 外光源等各种波长光源，光强可达10000μmol/m2.s 可选配单周转光闪（STF），100μs光闪（光脉冲）强度达120000μmol/m2.s 可选配8位滤波轮 可选配QA再氧化动力学测量模块 可选配OJIP快速荧光动力学测量模块 可选配可见光真彩成像分析模块 可选配植物呼吸与光合作用测量模块
FluorCam开放式叶绿素荧光成像系统
标准版成像面积可达13x13cm，大型版可达20x20cm， 镜头光源可灵活调整
FluorCam移动式植物荧光成像系统
• 整套系统装在有4个轮子的 支架上，方便野外移动 • 镜头及光源高度可以在 20-‐150cm范围内调节 • 可选配暗适应箱 • 成像面积20x20cm • 可附加镜头用于真彩成像分 析
样带扫瞄式植物荧光成像系统与 三维立体植物荧光扫瞄成像系统
• 样带荧光成像系统可以对20-‐200cm或 100x200cm范围的样方植物荧光扫描成像，以 便于梯度胁迫研究等 • 立体荧光成像系统可以通过对整株植物叶绿 素荧光扫描成像，并通过软件形成3维立体荧 光成像
多光谱XY-‐平台式大型植物荧光成像系统
显微叶绿素荧光成像系统
• 可对植物组织、藻类等细胞或亚亚细 胞结构进行荧光成像测量 • 便携式标准版显微荧光成像系统， Olympus CX31显微镜 • 便携式增强版显微荧光成像系统， Olympus BX40 显微镜及可扩展配件 （通过扩展可测量GFP等） • 多功能显微荧光成像系统，除具备 Olympus BX40 显微镜及可扩展配件外， 还具备6位滤波轮（有机械调节和电 子调节两种模式供选配），因而除可 以成像测量叶绿素荧光外，还可测量 GFP、CY3、CY5等多种稳态荧光或生 物荧光
FluorCam便携式叶绿素荧光成像仪
最大成像面积3.5x3.5cm， 备选配件包括叶夹、三脚架、便携蓄电池等
FluorCam封闭式叶绿素荧光成像系统
成像面积可达13x13cm， 可选配QA再氧化测量模块、OJIP测量模块等
GFPCam封闭式多光谱荧光成像系统
• 配有8位滤波轮，除具有封闭 式叶绿素荧光成像系统的功能 外，还可以成像测量绿色荧光 蛋白（GFP）等其它稳态生物 荧光 • 成像面积可达13x13cm • 可选配QA再氧化动力学测量模 块，OJIP快速荧光动力学测量 模块
– FluorCam叶绿素荧光成像系列产品 – FL3500/FL5000双调制荧光仪系列产品 – FluorPen及AquaPen等手持式荧光仪产品 – 光养生物反应器等藻类培养与在线监测产品 – 光源与植物培养室
• Walz：德国，主要产品为PAM2500叶绿素荧光仪等 • OpKcs：美国，主要产品为OS5p-‐PAM叶绿素荧光仪 等
– Ulrich Schreiber, Pulse-‐Amplitude-‐ModulaKon(PAM) Fluorometry and SaturaKon Pulse Method, An Overview – Ladislav Nedbal, Chlorophyll Fluorescence Imaging of Leaves and Fruits
FluorCam叶绿素荧光成像
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. Handy FC——便携式叶绿素荧光成像仪 Handy GFPCam——便携式荧光成像仪 Handy Leaf-‐chamber——便携式光合联用叶绿素荧光成像系统 Open FC——开放式叶绿素荧光成像系统，有13x13cm和20x20cm两种配 置方案 Closed FC——封闭式叶绿素荧光成像系统 Closed GFPCam——封闭式多光谱荧光成像系统 Micro-‐FluorCam——显微叶绿素荧光成像系统，又分标准版、增强版 （可选配GFP Filter Cube Set）及滤波轮版 Rover FluorCam——移动式大型植物荧光成像系统 Transect FluorCam——样带扫描式植物荧光成像系统 XY-‐Plane FluorCam——多光谱XY-‐平台式大型植物荧光成像系统 Conveyor and RoboKc PlantScan System——PlantScan全自动植物光谱成像 分析系统 Arch FluorCam——拱形三维植物荧光扫描成像系统 Fluorescence KineKc Microscope——FKM荧光动态显微光谱成像系统
叶绿素荧光技术发展
• Kautsky effect: Kautsky and Hirsch (1931) 首次用肉眼发现 叶绿素荧光现象并发表论文“CO2同化新实验”，后被 称作“Kautsky effect” • PAM (Pulse Amplitude Modulated Fluorometer): Schreiber（1986）等发明了PAM脉冲调制技术测量叶绿 素荧光 • FluorCam：KineKc imaging of chlorophyll fluorescence: Ladislav Nedbal（2000）等于上世纪90年代末期发明了 与PAM技术相结合的叶绿素荧光成像技术 • Chlorophyll a Fluorescence, A Signature of Phptosynthesis（Papageorgiou and Govindjee, 2009）
Kautsky effect
（资料源自Ecolab实验室荧光成像数据库）
荧光淬灭分析（Quenching Analysis）
1. Fv/Fm, 最大光量子产量 2. Fq’/Fm’，Genty参数、ϕPSII，又称光量子效率，用于光化学过程分析评估， 表示PSII吸收的光辐射用于光化学过程的比率，不需要暗适应，但受自然 光照影响大 3. Fv’/Fm’，开放PSII反应中心的光量子效率，不需暗适应 4. Fq’/Fv’，光化学淬灭qP，大致相当于初级受体QA的再氧化状态 5. (Fm-‐Fm’)/Fm’，非光化荧光淬灭
பைடு நூலகம்
FluorCam
荧 光 成 像 ： 荧 光 淬 灭 分 析
JIP-‐test（电子传递抑制剂敌草隆对OJIP的影响
叶绿素荧光技术著名厂商
• PSI：捷克Brno（孟德尔在此实验并发现著名的孟德 尔遗传定律），Ladislav Nedbal为首席科学家和主要 股东（另一股东为David Kramer，美国密执根州立 大学教授），1997年为美国华盛顿大学H. Pakrasi教 授研制成了第一台FluorCam荧光成像系统。主要产 品有
• 测量系统在柜体内， 光源及摄像头高度和 位置可调 • 测量面积40x80cm或 40x100cm供选择 • 可用于培养箱样品多 光谱分析
PlantScan全自动植物光谱成像分析系统
• 高通量植物成像观测系统，具备植 物传送系统、植物称重系统、自动 浇灌系统等 • 可配备温湿度及光照自动控制系统 • 植物荧光成像与可见光真彩成像， 植物荧光成像面积30x35cm • 可选配植物热成像模块 • 可选配植物红外成像模块 • 可选配植物VNIR高光谱成像分析模 块，光谱范围400-‐1000nm，成像面 积30x35cm；或选配SWIR高光谱 （1000-‐2500nm）成像
FKM荧光动态显微光谱成像系统
• 由显微荧光成像、双调制荧光测量及光谱仪组成 • 可同步荧光成像和点快速荧光动力学测量 • 可通过选择不同的激活光谱激活不同部位的天线色素；可用于分析 哪些色素蛋白复合体对光化学荧光淬灭或非光化学荧光淬灭贡献最 多 • 可用于活体检测非叶绿素荧光动态，如生物自发光或荧光颜料等， 并与同一细胞的叶绿素荧光动态相比较 • 可用于检测分析QA再氧化、连通性、天线大小等快速过程
叶绿素荧光技术常用参数
• Fo、Fm与QY，此外还有PAR_Abs及ETR • Kautsky诱导效应：Fo，Fp，Fv，Ft_Lss，QY， Rfd • 荧光淬灭分析：Fo，Fm，Fp，Fs，Fv，QY， ΦII，NPQ，Qp，Rfd，qL等50多个参数 • OJIP与JIP-‐test：Fo，Fj，Fi，P或Fm，Mo（OJIP 曲线初始斜率）、OJIP固定面积、Sm（对关闭 所有光反应中心所需能量的量度）、QY、PI等 • 光响应曲线LC：Fo，Fm，QY，QY_Ln
FluorCam荧光成像系统Protocols（实验程序）
Quenching（荧光淬灭分析） Kautsky inducKon（Kautsky诱导效应） Light curve（光响应曲线） PAR absorpKvity 实验程序自动重复功能 QA reoxidaKon OJIP，Fast fluorescence inducKon with 1μs resoluKon Measured parameters: Fo, FM, Fv, Fo’, FM’, Fv’, QY(II)，more than 50 calculated parameters • GFP等静态荧光 • NDVI • • • • • • • •