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1、欢迎各位老师参加！,-激光共聚焦扫描显微镜应用-,奥林巴斯（北京）销售服务有限公司上海分公司,光学显微镜技术的进展,生物用激光共焦扫描显微镜主要是对_显微观察方法的改进？,荧光,主要应用：生物荧光相关研究领域 几乎含盖整个生物领域的定位、定量、定时、定性相关的研究,WHAT CAN DO BY CONFOCAL,Applications of Fluorescence,生物检测Bioassays 细胞生物学 Cell Biology 核酸/蛋白定量DNA,RNA,and Protein Quantitation Systems 分子生物学 Genomics and Molecular Biol
2、ogy 离子检测Ion Indicators 微生物学Microbiology 微球/纳米粒子Microspheres and Nanospheres 蛋白组学Proteomics and Protein Biochemistry 活性染料Reactive Dyes and Chemicals 二级检测Secondary Detection 信号传导通路Signal Transduction 动物模型Small Animal In Vivo Imaging,Invitrogen Proprietary&Confidential,Toolbox of Labels,Alexa Fluor Dye
3、 SeriesUV to near IRSulfonated for increased H20 solubilityBright,photostable&High QYBODIPY Dye SeriesVisible to near IRZwitterionic High extinction coefficients&QYOregon Green,Pacific dyes,Texas Red and other generic dyes(FITC,TRITC)Includes positive,zwitterions,and negatively charged moleculesUV t
4、o near IRIncluding environmentally sensitive dyes(pH,polarity),Organic Dyes,Quantum Dot Nanotechnologies,Qdot nanocrystals Ability to archive samplesSingle excitationUniform sizeHigh extinction coefficient,Alexa Fluor 488,BODIPY FL,Biotin,Desthiobiotin,Color Selection Brightness Photostability,Alexa
5、 Fluor Dyes across the spectrum,Quantum Dots（量子点）,655 605 585 565 525 nm,Size of the nanocrystal determines the colorSize is tunable from 2-10 nm(3%)Size distribution determines the spectral width,Highly fluorescent,nanometer-size,single crystals of semiconductor materials,Most reliable and consiste
6、nt labeling methodYields the highest level of labeling:1 dye for 12-20 basesSame protocol regardless of the dye usedNo worries about dye interference with enzyme,Indirect Labeling Gives Highest and Most Consistent Level of Labeling,“Indirect”Labeling,核酸标记,Covalent Protein Labeling Chemistry,蛋白标记,Dye
7、,Dye,Dye,Dye,Indicator Response Types,Ca2+,Ca2+,Ion-dependent spectral shift,Ion-dependentfluorescence increase,no spectral shift,ExamplesFura-2Indo-1Premo Cameleon,ExamplesFluo-3Rhod-2Calcium GreenOregon Green 488 BAPTA,离子指示剂,Invitrogen Proprietary&Confidential,The Illuminated Cell Product Guide fo
8、r Fluorescence Imaging,荧光蛋白（B/C/G/Y/RFP）非破坏性融合表达：共转染示踪活体中蛋白表达及运输等低/无光毒性优秀的衍生产品：PA-GFP/Keade/FRET等,绿色荧光蛋白家族,共聚焦专用器材,可以与电动载物台共用，不是普通的多孔板,共聚焦专用器材,IBIDI培养皿,WHAT IS CONFOCAL MICROSCOPY,Olympus FV1000 configuration,1、活细胞或组织切片观察，进行细胞精细结构观察 和三维重建,2、多维图象获得，X、Y、Z、T、（光谱波长）、（旋转角度）I（光强度）等多维组合观察,4、细胞或组织内离子荧光标记，细胞
9、内Ph值和钙、镁离子浓度比例 和动态变化测定,3、荧光探针标记的细胞或组织，膜标记、免疫物质、受体或配体，核酸观察，多重荧光标记观察,5、利用电生理技术检测细胞或组织的离子通道信号,同时结合图象判断,6、蛋白质动力学、生物化学等等,Application,WHAT CAN DO BY CONFOCAL,多重标记、免疫荧光,3 channel acquisition:Nuclei(TO-PRO-3),Microtubules(FITC),Mitochondria(Mito tracker),3 channel acquisition:Green(Alex fluo488)and Blue(DRA
10、Q5)Red(Alex fluo568),视丘下部,大脑皮层,第三脑室,丘脑,厚（脑片）组织,皮肤里层,MeasurementIntensity(accumulated,mean,max,min)Area and perimeter measurementTime-lapse measurement2D data histogram displayCo localization,多重标记、免疫荧光,Localization and Co-localization,Activity-Induced Rapid Synaptic Maturation Mediated by Presynaptic
11、 Cdc42 Signaling,中科院神经所 段树民老师,Neuron 50,401414,May 4,2006,Example of the image.Mouse brain slice）,Dye:YFPOB：SAPO10 xNumber：5X5=25 imagesEach point:XYZ image 512x512、15 Z-Slices,大视野多Z轴拼图,Sample provided by:Ms.Mikako Sakurai,Mr.Masayuki Sekiguchi(Section Chief)Department of DegenerativeNeurological Di
12、seases,National Institute of Neuroscience,National Center of Neurology andPsychiatry,三维重建、光谱扫描,三维重建分析模块,强大的3D功能,3D反卷积运算功能,灵活方便的3D控制软件1、鼠标任意拖动,任意切面、多种内嵌式切法2、3D与2D的任意转换3、XYZ三维任意投射控制,From Yasuhiro Kamei,光谱拆分（Unmixing）,GFP（细胞质内）,FITC（细胞膜）,GFP（细胞质内）,FITC（细胞膜）,The Intensity:ROI1(GFP)and ROI2(CFP),Intensit
13、y Normalized,XYlambda acquisition condition LaserPower 40%(457nm),HV about 500-600,C.A.150,and DM457/515,ScanSpeed:125kHzResolution 10nm of,Step2nm,46 images(465 to 565 nm),CFP(Nucleus)/GFP(Microtubules),Influence of DM 457/515.,The Marge image after Un-Mixing,自动化光谱扫描技术,Getting fluorescence spectral
14、 data,Lambda scanning by tilting of grating-2nm,高精度光谱扫描技术,离子浓度比例和动态测量,Fluo 3/Ca2+wave,50mS,快速钙信号采集线扫描功能,Ca 2+Sparks,XYt4000 line/sec(512size)16 images/sec(256x256 pixel image size)16 frames/second at(256 x 256 pixels)XtXYZtup to 4D temporal detection procedures XYt/and XYZt image stacks(4D),culture
15、neuron cell are injenction with IP3 and calusume level is stained by Flow3,钙离子动态分析,Control of the experimentsPrecisionMicro second order High temporal precisionFlexibilityInteractively changing conditions while scanningProgrammableFlow controlSpecial program languageMulti point time laps,Multi chann
16、el acquicision,活体长时间的观察,共聚焦在长时间动态观察中的应用：,Calcium,Tomographic view of the specimen(microscopic CT),3-D reconstruction from z-section series,Thick specimen,Cross-talk correction,Far red emission which can not be detected by your eyes and CCD.,Green Fluorescent Protein(GFP),Multiphoton Excitation Laser
17、 Scanning Microscopy,More Application,FRET,FRAP,FLIP,FLIM,UNCAGING,photoactived,FRET backgroud,How FRET works:FRET occurs when 1.If the emission spectrum of a“donor”molecule overlaps the excitation spectrum of an“acceptor”molecule2.The distance between these two molecules are within 10-100,3.the don
18、or and acceptor fluoro-phores must be in favorable mutual orientation.then the excited donor molecule will transfer energy non-radiatively(without emitting of photon)to the acceptor.That is,when a double-labeled sample is excited in the excitation range of the donor,emission can be read in the emiss
19、ion range of the acceptor.,Two FRET model,Interaction-sensitive model allows the detection of molecular interaction by the resulting increase in FRET.Single-distance model permits the absolute measurement of the distance between the covalently bound donor and acceptor,FRET backgroud,Fluorescence Res
20、onance Energy Transfer(FRET),Single-distance model,FRET backgroud,Olympus FV1000的FRET模块是非常强大的，提供三种FRET方法，并且带有WIZARD功能，提供方便的实验向导，内嵌有多种计算公式（从FRET系数、效率到距离都一步可以得到）,方便的FRET使用向导,FRET Detection by Direct Measurement of Intensity Changes,Step1:Cells expressing YFP construct only-measure CFPex/YFPem,YFPex/YF
21、PemIn this case there is a real YFP signal but no CFP signal.Any signal in the FRET channel(CFPex/YFPem)is therefore due to crosstalk of the YFP signal into this channel.This crosstalk(measured as a ratio of the CFPex/YFPem signal to the YFPex/YFPem signal)will be subtracted from the FRET signal mea
22、sured in experimental cells.Call this value a.Usually a=20-30%.,Step2:Cells expressing CFP construct only-measure CFPex/YFPem,CFPex/CFPemIn this case there is a real CFP signal but no YFP signal.Any signal in the FRET channel(CFPex/YFPem)is therefore due to crosstalk of the CFP signal into this chan
23、nel.This crosstalk(measure as a ratio of the CFPex/YFPem signal to the CFPex/CFPem signal)will be subtracted from the FRET signal measured in experimental cells.Call this value b.Usually b=50-70%.,Step3:Cells expressing both constructs-measure CFPex/YFPem,YFPex/YFPem,CFPex/CFPemNet FRET=FRET signal(
24、a*YFP signal)(b*CFP signal)In this case there are both YFP and CFP signals in the cell,and any signal measured in the FRET channel must have the appropriate percentages of these two signals subtracted from it.Remember:Its important to let cells express the constructs in the appropriate relative leve
25、ls.It works best if the YFP signal is equal to or slightly higher than the equivalent CFP signal.,J Biol Chem.2005 Jul 1;280(26):24923-30.Epub 2005 May 10 Subunit assembly of N-methyl-d-aspartate receptors analyzed by fluorescence resonance energy transfer.Qiu S,Hua YL,Yang F,Chen YZ,Luo JH.Departme
26、nt of Neurobiology,Zhejiang University School of Medicine,Hangzhou,Zhejiang 310031,China,With Olympus FV1000 confocal micorscope,FRET Detection by Direct Measurement of Intensity Changes,FRET Detection by Acceptor Photobleaching,EF=(Iafter Ibefore)/Iafter,Donor before Acceptor bleaching,Donor after
27、Acceptor bleaching,FRET,0.553,0.428,0.002,0.002,0.533,0.428,Hela cell stain with cameleon by Olympus FV1000 confocal 60 xO(1.42)ob.,FRET Detection by Acceptor Photobleaching,光刺激与取图同时进行!,FV1000 安装了第二个扫描头“SIMS”,取图的同时可以任何时间、任何位置进行光刺激,Stimulation laser 405nm,Imaging laser488nm&543nm,Kaede蛋白简介：A new fluo
28、rescence protein cloned from a coral.特性：UV light turn Kaedes color from green to red.(Photo conversion),KAEDE,KAEDE,KAEDE,Kaede蛋白的应用：-Marking any cells for identification.,Photoactivatable Kaede的应用,光刺激Anytime,anywhereand many times同时进行取图.,Data from;Dr.Atsushi Miyawaki and Ms.Ryoko Ando in RIKEN BSI,
29、Photoactivatable Kaede的应用,Keade蛋白,Keade蛋白在神经学中的应用,Sato T,Takahoko M,Okamoto H.(2006)HuC:Kaede,a useful tool to label neuralmorphologies in networks in vivo.Genesis.44:136-42.,PA-GFP简介：A GFP mutant from wild type GFP.特性：UV light increase fluorescence intensity(Photo activation),PA-GFP,PA-GFP,PA-GFP,P
30、A-GDP 的应用：Observing dynamic state of proteins anywhere and anytime.,PhotoactivatablePA-GFP的应用,Intensity at each ROIs,Data from;Dr.Atsushi Miyawaki,Dr.Kenji Nagai and Muneyuki Miyauchi in RIKEN BSI.,PhotoactivatablePA-GFP的应用,Uncaging的简介：Use a caged compound(e.g.caged ATP,caged glutamate etc.)特性：UV li
31、ght activate the chemical compound(e.g.ATP,glutamate etc.),Caged-ATP,ATP,Ca-(Fluo-3),Uncaging 的应用：Observing reaction caused by a chemical compound in selected area.,Uncaging的应用,Intensity at each ROIs,Data from:Dr.Atsushi Miyawaki and Dr.H.Hama in RIKEN BSI amd Dr.,Furuta in Toho Univ.,Caged-Glutamat
32、e on FV1000,Stimulation 405nm,Imaging laser 488nm,Fluo-3,Fluorescence Recovery After Photobleaching,FRAP 的应用：Observing moving state of fluorescence molecule in live cells.,FV1000在FRAP中的应用,Fluorescence Recovery After Photobleaching,Data from:Dr.Shigeo Okabe(Tokyo medical and dental univ.),Imaging are
33、a,Breaching area,Intensity at ROI,FRAP on FV1000,Imaging 100ms/f,Breaching time total 80ms,Diffusion rate of GFP and GFP chimaeras,Molecule GFP in waterGFP in cytoplasmGFP in endoplasmic-reticulum(ER)lumenGFP in mitochondrial matrixER membraneVSVG tsO45-GFP(in ER+BFA32)VSVG tsO45-GFP(in ER40)Signal
34、recognition particle-subunit-GFPGolgi apparatus membrane Galactosyltransferase-GFPNucleoplasmGFP ERCC1/XPFPlasma membraneE-cadherin-GFP,D(um2s-1)87255-1020-300.490.450.260.54150.03-0.04,D 10 um2/s is unexplored dynamic state,Continuous breaching point,FLIP application by SIMS,Cell:HelaFluorescence f
35、rom Free GFP,FLIP application has been not well-adapted to as an confocal application because of it requests fixed point breaching during data sampling.Therefore,before SIMS comes,no CLSM could make excellent FLIP data.To measure diffusion constant of free GFP in same case,FRAP is not suitable becau
36、se its limitation against the rapid recovery of free GFP.FLIP is more suitable method than FRAP if you need to take data of rapid diffusion constant in your experiment.,SIMS,全内反射荧光显微技术,一套系统实现 LSM 和 TIRFM两种观察方式FV10-MCOMB 的所有激光器可以TIRFM共享临界角的高重复性针对不同物镜自动进行光路优化 应用适用于细胞膜动力学研究（胞吞，胞吐等）化学分子运动或物理特殊结构研究,TIRFM
37、 unit INFor TIRFM illumination（no scanning,fixed beam）,TIRFM unit OUTFor LSM,系统示意图:,系统软件界面：,Adjustment of laser power,Adjustment of penetration ratio,Adjustment of FS,Button for TIRFM laser activation,Display of penetration ratio,EVA,通过TTL信号触发可以实现和共聚焦系统连用：,Output the TTL signal from printer port,Rec
38、eive the signal at FV10-PUS,Laser activate when the signal is active High,Bit 1 Use PSU D-sub 4pinBit0 Use PSU D-sub 15Pin,Active High is more than 2.4VActive Low is less than 0.9V,FV5-TCBNC cable(Olympus product),Cable for shutter control TTL control cable for parallel port(MD product),Dr.S.Simon,J.Schmoranzer/Rockefeller Univ.Dr.Axelrod/Univ.of Michigan,应用举例：,S.Simon et.al/Rockefeller Univ.,FV1000 best choice for image,hankyou！,
