Fluorescence Microscopy Wolfgang Graier ([email protected]) F-actin NFkB (activation by H 2 O 2 ) Pictures: W.F Graier, MBC & MB, Graz, Austria

Fluorescence Microscopy

Wolfgang Graier ([email protected])

0́ 23´ 47´

92´ 117´ 147´

F-actin NFkB (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. Graier

Department of Medical Biochemistry and Medical Molecular BiologyKarl-Franzens University of Graz

Harrachgasse 21/IIIA-8010 Graz

Tel. +43-316-380-7560Fax. +43-316-380-9615

E- mail: [email protected]

Basics and IntroductionFluorescence/TransmissionmicroscopyAdvantage/Drawback of light microscopyFluorescence DyesGFPs

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

Comparison of techniques available


•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 ofresolution

blue

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

Fluorescencemicroscopy

light source

object

objective lens

1st barrier filter(ex) 2nd barrier filter

(em)

beam-splittingmirror

eyepiece

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


• 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,

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 - dyesOrganelle-specificpHmembrane potentialion selective....

• http://www.probes.com (Molecular Probes)

Microscopic analysis of yeast organelles in vivo

mitochondria(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)



DiOC6

deconvoluted

deconvoluted

C D

X

YZ

A B

10 µm

10 µm

10 µm

10 µm

X

Y


Blue/Green/Yellow/Red fluorescent proteins

http://www.clontech.com/

Green Fluorescent Protein Cloning Strategies

N, C-terminal fusions <–> targeting signals !

endogenous <–> heterologous promoter !

steady state-distribution <–> "pulse-chase" !

function !

GFP kanMX6ERG

Chromosomal fusionvia homologous recombination

GFP kanMX6

Chromosome

GFP

kanMX6

Plasmid

ERG Chromosome

PCR

GFP kanMX6ERG


GFP kanMX6

Chromosome

GFP

kanMX6

Plasmid

ERG Chromosome

PCR

GFP kanMX6ERG


GFP kanMX6

Chromosome

GFP

kanMX6

Plasmid

ERG Chromosome

PCR

YFG

GFP kanMX

YFG GFP kanMX

PCRtransformationG418 selection

YFG

GFP kanMX

YFG GFP kanMX

PCRtransformationG418 selection

GFP C-terminal chromosomal fusion

pUG plasmid template

Fluorescence DyesFluorescence Dyes

ConjugatesConjugates SubstratesSubstrates

AgonistsAgonists ChelatorsChelators

ConjugatesConjugates

Principles:primary antibodysecondary antibody

(dye coupled)

Samples:Alexa, Cy-X

Immunfluorescence

Pictures: Molecular Probes

0120240360480600720840960100

110

120

L-ArginineBradykinin+ L-NA

Time (min)

4,5-Diaminofluorescein(DAF)

SubstratesSubstrates

A B

D E F

CAgonistsAgonists BODIPY- Ryanodine


ChelatorsChelators

Targeting of chelators by specific groups (e.g. fatty acids)

F.I.

Wavelenghts (nm)

Ca2+

340 360 380 EX

EM510

Ca2+

Na+

H+

K+

Cl-

..

..

..

Fura-2


•Technology

Deconvolution Microscopy

Confocal Laser Scanning Microscopy

2 Photon Microscopy; time-resolved FM

FRAP fluorescence recovery after photo bleaching

FRET fluorescence resonance energy transfer


2 Photon Excitation Microscopy

ex λem

1 Photon

ex λem

2 Photon


Time-resolved fluorescence microscopyflu

ore

scen

ce

time (nsec)

dye 1 (e.g. background)

dye 2

time window


FRAP

FRET (Cameleon)

BFP GFP

Ca++

o

calm

dulin

exBFP

emBFP emGFP

BFP GFPCalmodulin/M13

Ca++

emBFP

exBFP

ER-tagged-Cameleons Mi-tagged-Cameleons


Organell-specific expression of an Ca2+-sensitive proteineCameleons (developed by R.Y. Tsien)

(local concentration !)

sensitivity

resolution rec. speed100 x 100 x 300 nm msec – 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

Dichroicmirror

Illuminating aperture

Confocal detectoraperture

Photomultiplier

in-focus raysout of focus rays


Single optical section

multiple optical sections

3d reconstruction

z

z x

y

picture element (pixel; e.g. 60x60 nm)


cover slide

dep

th

‘‘focal spot‘‘Picture: S. Kohlwein, B & FB, Graz, Austria

Yeast Light Microscopy

100 x

Microfluorometry


Computer

A/ D-Wandler

OptischerProzessor

Videorecorder

Camera

Xe-LampeMonitor

Filterrad

PM

Microskop

Zelle

Zelle

Blende

BlendeLampe

510 nm Filter

>600 nm Filter

Pipette

Verstärker

Microfluorometry


Microfluorometry:Simultaneously recordings of Ca2+ and ion currents

0

500

1000

1500

2000

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

30 32 34 36 38 40 42 44 46 48 50

Current: I (pA) [Ca2+]

bulk

: Ratio (F

360

/F380

)

Elapsed Time (s)

1st spikeB

Histamine


Fluorescence Imaging

Deconvolution microscopy


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 resolvedtwo 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 (104 p/px)

n.a.

fast

== f(object)

<< 100 µm

Spectral lamp

Documents

Fluorescence Microscopy Wolfgang Graier ([email protected]) F-actin NFkB (activation by H 2 O 2 ) Pictures: W.F Graier, MBC & MB, Graz, Austria