Fluorescence Microscopy Wolfgang Graier F-actin NFkB (activation by H 2 O 2 ) 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. Graier Department of Medical Biochemistry and Medical Molecular Biology Karl-Franzens University of Graz Harrachgasse 21/III A-8010 Graz Tel Fax E- mail:

Basics and Introduction Fluorescence/Transmissionmicroscopy Advantage/Drawback of light microscopy Fluorescence Dyes GFPs Instrumental Devices Confocal laser scan microscopy (CLSM) Imaging in living cells Deconvolution microscopy Comparison of techniques available

Fluorescence Microscopy Introduction Fluorescence microscopy Advantages/disadvantages, limitations Fluorescence dyes Vital dyes, GFP and derivatives Immunofluorescence Technology 2 photon excitation FRAP and FRET Fluorescence life time imaging Confocal laser scanning Deconvolution and imaging Examples

Limitation of light microscopy 5  m limit of resolution blue Picture: S. Kohlwein, B & FB, Graz, Austria

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

Fluorescence Microscopy Life Cell and Immuno Fluorescence Applications - dyes Organelle-specific, pH, membrane potential, ion Concentration 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, bleaching dynamics of structures (loss of resolution)

Immunofluorescence Microscopy + protein localization 3d reconstruction resolution > life cells (no dynamics) – limits of resolution (wave length of light) sample preparation, preparation artifacts (fixation, Ab specificity) dead cells ! bleaching

Applications - dyes Organelle-specific pH membrane potential ion selective (Molecular Probes)

Microscopic analysis of yeast organelles in vivo mitochondria (DASPMI, Mito-Traker Mi) lipid particles (Nile Red) nucleus (DAPI, SYTO) endoplasmic reticulum (DiOC 6, Mito-Traker ER) vacuoles (FM4-64, CDCFDA) endocyt. vesicles (FM4-64) membranes Cholesterol: filipin potential-sensitive dyes: bis-oxonol

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

DiOC 6 deconvoluted

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

Blue/Green/Yellow/Red fluorescent proteins

Green Fluorescent Protein Cloning Strategies N, C-terminal fusions targeting signals ! endogenous heterologous promoter ! steady state-distribution "pulse-chase" ! function !

Chromosome GFP kanMX6 Plasmid ERGChromosome PCR YFG GFP kanMX YFGGFP kanMX PCR transformation G418 selection GFP C-terminal chromosomal fusion pUG plasmid template

Fluorescence Dyes Conjugates Substrates Agonists Chelators

Conjugates Principles: primary antibody secondary antibody (dye coupled) Samples: Alexa, Cy-X Immunfluorescence Pictures: Molecular Probes

4,5-Diaminofluorescein (DAF) Substrates

Agonists BODIPY- Ryanodine Pictures: W.F Graier, MBC & MB, Graz, Austria

Chelators Targeting of chelators by specific groups (e.g. fatty acids) Ca 2+ Na + H + K + Cl -.. Fura-2

Fluorescence Microscopy Technology Deconvolution Microscopy Confocal Laser Scanning Microscopy 2 Photon Microscopy; time-resolved FM FRAP fluorescence recovery after photo bleaching FRET fluorescence resonance energy transfer

Fluorescence Microscopy 2 Photon Excitation Microscopy 1 Photon 2 Photon

Fluorescence Microscopy Time-resolved fluorescence microscopy f l u o r e s c e n c e time (nsec) dye 1 (e.g. background) dye 2 time window

Fluorescence Microscopy FRAP FRET (Cameleon) B F P G F P Ca ++ o c a l m d u l i n exBFP emBFP emGFP BFPGFP Calmodulin/M13 Ca ++ emBFP exBFP

ER-tagged-Cameleons Mi-tagged-Cameleons Pictures: W.F Graier, MBC & MB, Graz, Austria Organell-specific expression of an Ca 2+ -sensitive proteine Cameleons (developed by R.Y. Tsien)

(local concentration !) sensitivity resolutionrec. speed 100 x 100 x 300 nmmsec – sec Electronic Light Microscopy cell viability, structure dynamics

The Confocal Principle optical resolution: >100 nm (x/y) >300 nm (z) Point source Objective lens Focal plane Specimen Dichroic mirror Illuminating aperture Confocal detector aperture Photomultiplier in-focus rays out of focus rays

The Confocal Principle Single optical section multiple optical sections 3d reconstruction z z x y picture element (pixel; e.g. 60x60 nm)

The Confocal Principle cover slide depth ‘‘focal spot‘‘ Picture: S. Kohlwein, B & FB, Graz, Austria

Yeast Light Microscopy 100 x

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

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

Microfluorometry: Simultaneously recordings of Ca 2+ and ion currents Pictures: W.F Graier, MBC & MB, Graz, Austria

Fluorescence Imaging

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

Point spread function

Focus Out-of-focus fluorescence

3D reconstruction pixel (x-y plane) voxel (x-y-z plane) 2D reconstruction Deconvolution microscopy allows time resolved two dimensional fluorescence recordings in high x-y resolution and app. 200 to 300 µm thick slices (pixel)

Confocal vs Deconvolution Out-of-focus light Signal to noise ratio Serial lines (time scan) Image acquisition Imaging quality Thick samples Excitation Costs pinhole low (10 p/px) √ slow == f(object) > 100 µm # laser lines PSF & comput. high (10 4 p/px) n.a. fast == f(object) << 100 µm Spectral lamp