Fluorescence Microscopy

Wolfgang Graier (wolfgang.graier@kfunigraz.ac.at)F-actinNFkB (activation by H2O2)Pictures: W.F Graier, MBC & MB, Graz, Austria

NOTE:This Powerpoint presentation also includes so far not published pictures and results. It has been released only for teaching the principles 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 GrazHarrachgasse 21/IIIA-8010 GrazTel. +43-316-380-7560Fax. +43-316-380-9615E- mail: wolfgang.graier@kfunigraz.ac.at

Basics and IntroductionFluorescence/TransmissionmicroscopyAdvantage/Drawback of light microscopyFluorescence DyesGFPs

Instrumental DevicesConfocal laser scan microscopy (CLSM) Imaging in living cellsDeconvolution microscopy

Comparison of techniques available

Fluorescence Microscopy

IntroductionFluorescence microscopyAdvantages/disadvantages, limitationsFluorescence dyesVital dyes, GFP and derivativesImmunofluorescenceTechnology2 photon excitationFRAP and FRETFluorescence life time imagingConfocal laser scanningDeconvolution and imagingExamples

Limitation of light microscopy5 mmlimit ofresolution

Picture: S. Kohlwein, B & FB, Graz, Austria

FluorescencemicroscopyPicture: S. Kohlwein, B & FB, Graz, Austria

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

Life Cell 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)

Immunofluorescence 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

Applications - dyesOrganelle-specificpHmembrane potentialion selective.... http://www.probes.com (Molecular Probes)

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-sensitive dyes: bis-oxonol

Cholesterol distribution in 3T3 cells (fillipin)Pictures: W.F Graier, MBC & MB, Graz, Austria

Pictures: W.F Graier, MBC & MB, Graz, AustriaDiOC6deconvoluteddeconvoluted

Pictures: W.F Graier, MBC & MB, Graz, Austria

Blue/Green/Yellow/Red fluorescent proteins

Green Fluorescent Protein Cloning StrategiesN, C-terminal fusions targeting signals !

endogenous heterologous promoter !

steady state-distribution "pulse-chase" !

function !

ChromosomeGFPkanMX6PlasmidERGChromosomePCRYFGGFPkanMXYFGGFPkanMXPCRtransformationG418 selectionGFP C-terminal chromosomal fusionpUG plasmid template

Fluorescence DyesConjugatesSubstratesAgonistsChelators

ConjugatesPrinciples:primary antibodysecondary antibody(dye coupled)Samples:Alexa, Cy-XImmunfluorescencePictures: Molecular Probes

4,5-Diaminofluorescein(DAF)Substrates

AgonistsBODIPY- RyanodinePictures: W.F Graier, MBC & MB, Graz, Austria

ChelatorsTargeting of chelators by specific groups (e.g. fatty acids)Ca2+Na+H+K+Cl-......

Fura-2

Fluorescence Microscopy

TechnologyDeconvolution MicroscopyConfocal Laser Scanning Microscopy2 Photon Microscopy; time-resolved FMFRAP fluorescence recovery after photo bleachingFRET fluorescence resonance energy transfer

Fluorescence Microscopy2 Photon Excitation Microscopy

Fluorescence Microscopy

Time-resolved fluorescence microscopyfluorescencetime (nsec)dye 1 (e.g. background)dye 2time window

Fluorescence MicroscopyFRAPFRET (Cameleon)

ER-tagged-CameleonsMi-tagged-CameleonsPictures: W.F Graier, MBC & MB, Graz, AustriaOrganell-specific expression of an Ca2+-sensitive proteineCameleons (developed by R.Y. Tsien)

(local concentration !)sensitivity resolutionrec. speed100 x 100 x 300 nmmsec sec

Electronic Light Microscopycell viability, structure dynamics

The Confocal Principleoptical

resolution:

>100 nm (x/y)

>300 nm (z)Point sourceObjective lensFocal planeSpecimenDichroicmirrorIlluminating apertureConfocal detectoraperturePhotomultiplierin-focus raysout of focus rays

The Confocal PrincipleSingle optical sectionmultiple optical sections3d reconstructionpicture element (pixel; e.g. 60x60 nm)

The Confocal Principlefocal spotPicture: S. Kohlwein, B & FB, Graz, Austria

Yeast Light Microscopy

MicrofluorometryPictures: W.F Graier, MBC & MB, Graz, Austria

MicrofluorometryPictures: W.F Graier, MBC & MB, Graz, Austria

Microfluorometry:Simultaneously recordings of Ca2+ and ion currentsPictures: W.F Graier, MBC & MB, Graz, Austria

Fluorescence Imaging

Deconvolution microscopyPictures: W.F Graier, MBC & MB, Graz, Austria

Point spread function

FocusOut-of-focus fluorescence

3D reconstructionpixel (x-y plane) .......voxel (x-y-z plane)2D reconstructionDeconvolution microscopy allows time resolvedtwo dimensional fluorescence recordings in high x-yresolution and app. 200 to 300 m thick slices (pixel)

Confocal vs DeconvolutionOut-of-focus lightSignal to noise ratio Serial lines (time scan)Image acquisitionImaging qualityThick samplesExcitation lCostspinholelow (10 p/px) slow== f(object) > 100 m# laser linesPSF & comput.high (104 p/px)n.a.fast== f(object)