FluorescenceMicroscopyinPharmacology熒光顯微鏡在藥理學課件

1、Fluorescence MicroscopyWolfgang Graier (wolfgang.graierkfunigraz.ac.at)F-actinNFkB (activation by H2O2)Pictures: W.F Graier, MBC & MB, Graz, Austria第1頁，共42頁。NOTE:This Powerpoint presentation also includes so far not published pictures and results. It has been released only for teaching the principle

2、s and possibilities of high resolution micrsocopy to graduate and post-graduate students. - Thank you very much for your fairness.If any other use is planed please contact:Prof. Wolfgang F. GraierDepartment of Medical Biochemistry and Medical Molecular BiologyKarl-Franzens University of GrazHarrachg

3、asse 21/IIIA-8010 GrazTel. +43-316-380-7560Fax. +43-316-380-9615E- mail: wolfgang.graierkfunigraz.ac.at 第2頁，共42頁。Basics and IntroductionFluorescence/TransmissionmicroscopyAdvantage/Drawback of light microscopyFluorescence DyesGFPsInstrumental DevicesConfocal laser scan microscopy (CLSM) Imaging in l

4、iving cellsDeconvolution microscopyComparison of techniques available第3頁，共42頁。Fluorescence MicroscopyIntroductionFluorescence microscopyAdvantages/disadvantages, limitationsFluorescence dyesVital dyes, GFP and derivativesImmunofluorescenceTechnology2 photon excitationFRAP and FRETFluorescence life t

5、ime imagingConfocal laser scanningDeconvolution and imagingExamples第4頁，共42頁。Limitation of light microscopy5 mmlimit ofresolutionlbluePicture: S. Kohlwein, B & FB, Graz, Austria第5頁，共42頁。Fluorescencemicroscopylight sourceobjectobjective lens1st barrier filter(lex)2nd barrier filter(lem)beam-splittingm

6、irroreyepiecePicture: S. Kohlwein, B & FB, Graz, Austria第6頁，共42頁。Fluorescence Microscopy Life Cell and Immuno Fluorescence Applications - dyesOrganelle-specific, pH, membrane potential, ionConcentration Caged compounds GFP, BFP, RFP, YFP; Aequorin; GFP and FRET Sample Preparation 第7頁，共42頁。Life Cell

7、Microscopy +dynamics !sample preparation !3d reconstruction - multi-dimensional“(3d + time, multiple wavelengths, reaction kinetics.) limits of resolution (wavelength of light)viability, temperature, oxygen, phototoxicity,bleachingdynamics of structures (loss of resolution)第8頁，共42頁。Immunofluorescenc

8、e Microscopy +protein localization3d reconstructionresolution life cells (no dynamics) limits of resolution (wave length of light)sample preparation, preparation artifacts (fixation, Ab specificity)dead cells !bleaching第9頁，共42頁。Applications - dyesOrganelle-specificpHmembrane potentialion selective.

9、(Molecular Probes) 第10頁，共42頁。Microscopic analysis of yeast organelles in vivomitochondria(DASPMI , Mito-Traker Mi)lipid particles(Nile Red)nucleus(DAPI, SYTO) endoplasmic reticulum (DiOC6, Mito-Traker ER)vacuoles(FM4-64, CDCFDA)endocyt. vesicles(FM4-64)membranesCholesterol: filipinpotential-sensitiv

10、e dyes: bis-oxonol第11頁，共42頁。Cholesterol distribution in 3T3 cells (fillipin)Pictures: W.F Graier, MBC & MB, Graz, Austria第12頁，共42頁。Pictures: W.F Graier, MBC & MB, Graz, AustriaDiOC6deconvoluteddeconvoluted第13頁，共42頁。Pictures: W.F Graier, MBC & MB, Graz, Austria第14頁，共42頁。Blue/Green/Yellow/Red fluoresc

11、ent proteins /第15頁，共42頁。Green Fluorescent Protein Cloning StrategiesN, C-terminal fusions targeting signals !endogenous heterologous promoter !steady state-distribution pulse-chase !function !第16頁，共42頁。 GFPkanMX6ERGChromosomal fusionvia homologous recombinationGFPkanMX6ChromosomeGFPkanMX6PlasmidERGC

12、hromosomePCRGFPkanMX6ERGChromosomal fusionvia homologous recombinationGFPkanMX6ChromosomeGFPkanMX6PlasmidERGChromosomePCRGFPkanMX6ERGChromosomal fusionvia homologous recombinationGFPkanMX6ChromosomeGFPkanMX6PlasmidERGChromosomePCRYFGGFPkanMXYFGGFPkanMXPCRtransformationG418 selectionGFP C-terminal ch

13、romosomal fusionpUG plasmid template第17頁，共42頁。Fluorescence DyesConjugatesSubstratesAgonistsChelators第18頁，共42頁。ConjugatesPrinciples:primary antibodysecondary antibody(dye coupled)Samples:Alexa, Cy-XImmunfluorescencePictures: Molecular Probes第19頁，共42頁。4,5-Diaminofluorescein(DAF)Substrates第20頁，共42頁。Ago

14、nistsBODIPY- RyanodinePictures: W.F Graier, MBC & MB, Graz, Austria第21頁，共42頁。ChelatorsTargeting of chelators by specific groups (e.g. fatty acids)Ca2+Na+H+K+Cl-.Fura-2第22頁，共42頁。Fluorescence MicroscopyTechnologyDeconvolution MicroscopyConfocal Laser Scanning Microscopy2 Photon Microscopy; time-resolv

15、ed FMFRAP fluorescence recovery after photo bleachingFRET fluorescence resonance energy transfer第23頁，共42頁。Fluorescence Microscopy2 Photon Excitation Microscopy1 Photon2 Photon第24頁，共42頁。Fluorescence MicroscopyTime-resolved fluorescence microscopyfluorescencetime (nsec)dye 1 (e.g. background)dye 2time

16、 window第25頁，共42頁。Fluorescence MicroscopyFRAPFRET (Cameleon)BFPGFPCa+ocalmdulinlexBFPlemBFPlemGFPBFPGFPCalmodulin/M13Ca+lemBFPlexBFP第26頁，共42頁。ER-tagged-CameleonsMi-tagged-CameleonsPictures: W.F Graier, MBC & MB, Graz, AustriaOrganell-specific expression of an Ca2+-sensitive proteineCameleons (develop

17、ed by R.Y. Tsien)第27頁，共42頁。(local concentration !)sensitivity resolutionrec. speed100 x 100 x 300 nmmsec secElectronic Light Microscopycell viability, structure dynamics第28頁，共42頁。The Confocal Principleoptical resolution:100 nm (x/y)300 nm (z)Point sourceObjective lensFocal planeSpecimenDichroicmirro

18、rIlluminating apertureConfocal detectoraperturePhotomultiplierin-focus raysout of focus rays第29頁，共42頁。The Confocal PrincipleSingle optical sectionmultiple optical sections3d reconstructionzzxypicture element (pixel; e.g. 60 x60 nm)第30頁，共42頁。The Confocal Principlecover slidedepthfocal spotPicture: S.

19、 Kohlwein, B & FB, Graz, Austria第31頁，共42頁。Yeast Light Microscopy100 x第32頁，共42頁。MicrofluorometryPictures: W.F Graier, MBC & MB, Graz, Austria第33頁，共42頁。MicrofluorometryPictures: W.F Graier, MBC & MB, Graz, Austria第34頁，共42頁。Microfluorometry:Simultaneously recordings of Ca2+ and ion currentsPictures: W.F Graier, MBC & MB, Graz, Austria第35頁，共42頁。Fluorescence Imaging第36頁，共42頁。Deconvolution microscopyPictures: W.F Graier, MBC & MB, Graz, Austria第37頁，共42頁。Point spread