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Fluorescence Microscopy
Wolfgang Graier ([email protected])
F-actin
NFkB (activation by H2O2)
0´
92´
23´
47´
117´
147´
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. +43-316-380-7560
Fax. +43-316-380-9615
E- mail: [email protected]
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
lblue
limit of
resolution
5 mm
Picture: S. Kohlwein, B & FB, Graz, Austria
Fluorescence
microscopy
eyepiece
1st barrier filter
(lex)
2nd barrier filter
(lem)
beam-splitting
mirror
light source
objective lens
object
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
....
• http://www.probes.com (Molecular Probes)
Microscopic analysis of yeast organelles in vivo
mitochondria
(DASPMI , Mito-Traker Mi)
endocyt. vesicles
lipid particles
(Nile Red)
(FM4-64)
vacuoles
(FM4-64, CDCFDA)
nucleus
(DAPI, SYTO)
membranes
Cholesterol: filipin
potential-sensitive dyes: bis-oxonol
endoplasmic
reticulum (DiOC6, Mito-Traker ER)
Cholesterol distribution in 3T3 cells (fillipin)
Pictures: W.F Graier, MBC & MB, Graz, Austria
DiOC6
deconvoluted
deconvoluted
Pictures: W.F Graier, MBC & MB, Graz, Austria
Pictures: W.F Graier, MBC & MB, Graz, Austria
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 C-terminal chromosomal fusion
YFG
pUG plasmid template
GFP
kanMX
PCR
transformation
G418 selection
YFG
GFP
kanMX
Fluorescence Dyes
Conjugates
Substrates
Agonists
Chelators
Immunfluorescence
Conjugates
Principles:
primary antibody
secondary antibody
(dye coupled)
Samples:
Alexa, Cy-X
Pictures: Molecular Probes
Substrates
4,5-Diaminofluorescein
(DAF)
Agonists
A
D
BODIPY- Ryanodine
B
C
E
F
Pictures: W.F Graier, MBC & MB, Graz, Austria
Chelators
Fura-2
Ca2+
Na+
H+
K+
Cl..
..
..
Targeting of chelators by specific groups (e.g. fatty acids)
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
2 Photon
1 Photon
lex
lem
lex
lem
Fluorescence Microscopy
Time-resolved fluorescence microscopy
dye 1 (e.g. background)
time window
dye 2
time (nsec)
Fluorescence Microscopy
FRET (Cameleon)
FRAP
l
emBFP
l
exBFP
BFP
Calmodulin/M13
GFP
Ca++
l
emBFP
l
exBFP
o
Ca++
l emGFP
Organell-specific expression of an Ca2+-sensitive proteine
Cameleons (developed by R.Y. Tsien)
ER-tagged-Cameleons
Mi-tagged-Cameleons
Pictures: W.F Graier, MBC & MB, Graz, Austria
Electronic Light Microscopy
(local concentration !)
sensitivity
resolution
rec. speed
100 x 100 x 300 nm
msec – sec
cell viability, structure dynamics
The Confocal Principle
Photomultiplier
Confocal detector
aperture
optical
Illuminating aperture
Point source
Dichroic
mirror
resolution:
>100 nm (x/y)
>300 nm (z)
Objective lens
Focal plane
Specimen
in-focus rays
out of focus rays
The Confocal Principle
picture element (pixel; e.g. 60x60 nm)
z
x
Single optical section
y
multiple optical sections
z
3d reconstruction
The Confocal Principle
depth
cover slide
‘‘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 Ca2+ and ion currents
B
1st spike
2000
1
0.9
0.8
0.7
1000
500
/F
36 0
0.4
: Ratio (F
0.5
bulk
0.6
[Ca 2+]
Current: I (pA)
1500
)
38 0
0.3
0
Histamine
30
32
0.2
34
36
38
40
42
44
Elapsed T ime (s)
46
48
50
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
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)
3D reconstruction
pixel (x-y plane) .......voxel (x-y-z plane)
Confocal vs Deconvolution
pinhole
Out-of-focus light
PSF & comput.
low (10 p/px)
Signal to noise ratio
high (104 p/px)
√
Serial lines (time scan)
n.a.
slow
Image acquisition
fast
== f(object) >
Imaging quality
== f(object)
100 µm
Thick samples
<< 100 µm
# laser lines
Excitation l
Spectral lamp
Costs