FluorescenceMicroscopy Inpharmacology

Fluorescence Microscopy
Wolfgang Graier (wolfgang.graier@kfunigraz.ac.at)
Factin
NFkB(activationbyH2O2)
0
92
23
47
117
147
Pictures:W.FGraier,MBC&MB,Graz,Austria
NOTE:
ThisPowerpointpresentationalsoincludessofarnot
publishedpicturesandresults.Ithasbeenreleasedonly
forteachingtheprinciplesandpossibilitiesofhigh
resolutionmicrsocopytograduateandpostgraduate
students.Thankyouverymuchforyourfairness.
Ifanyotheruseisplanedpleasecontact:
Prof.WolfgangF.Graier
DepartmentofMedicalBiochemistryandMedicalMolecularBiology
KarlFranzensUniversityofGraz
Harrachgasse21/III
A8010Graz
Tel.+433163807560
Fax.+433163809615
Email:wolfgang.graier@kfunigraz.ac.at
BasicsandIntroduction
Fluorescence/Transmissionmicroscopy
Advantage/Drawbackoflightmicroscopy
FluorescenceDyes
GFPs
InstrumentalDevices
Confocallaserscanmicroscopy(CLSM)
Imaginginlivingcells
Deconvolutionmicroscopy
Comparisonoftechniquesavailable
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

blue
limit of
resolution
5 m
Picture:S.Kohlwein,B&FB,Graz,Austria
Fluorescence
microscopy
eyepiece
1st barrier filter
(ex)
2nd barrier filter

(em)
beam-splitting
mirror
light source
objective lens
object
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 !
limits of resolution (wavelength of light)
sample preparation !
3d reconstruction - multi-dimensional
(3d + time, multiple wavelengths, reaction kinetics..)
viability, temperature, oxygen, phototoxicity,

bleaching
dynamics of structures (loss of resolution)
Immunofluorescence Microscopy
protein localization
limits of resolution (wave length of light)
3d reconstruction
resolution > life cells (no dynamics)
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
(FM4-64)
(Nile Red)
vacuoles
(FM4-64, CDCFDA)
nucleus
(DAPI, SYTO)
membranes
Cholesterol: filipin
potential-sensitive dyes: bis-oxonol
endoplasmic
reticulum (DiOC6, Mito-Traker ER)
Cholesteroldistributionin3T3cells(fillipin)
DiOC6
deconvoluted
deconvoluted
Y
10 m
10 m
Y
10 m
10 m
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
FluorescenceDyes
Conjugates
Conjugates
Substrates
Substrates
Agonists
Agonists
Chelators
Chelators
Immunfluorescence
Conjugates
Conjugates
Principles:
primaryantibody
secondaryantibody
(dyecoupled)
Samples:
Alexa,CyX
Pictures:MolecularProbes
Substrates
Substrates
120
4,5Diaminofluorescein
(DAF)
110
+ L-N
100 Bradykinin
L-Arginine
0120
240
360
480
600
720
840
960
Time
(mi
Agonists
Agonists
A
BODIPYRyanodine
Chelators
Chelators
Fura2
EM510
Ca2+
Na+
H+
K+
Cl
Ca2+
F.I.
..
340
..
360
Wavelenghts(nm)
380
EX
..
Targetingofchelatorsbyspecificgroups(e.g.fattyacids)
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
2 Photon
1 Photon
ex
em
ex
em
Time-resolved fluorescence microscopy
fluorescence
dye 1 (e.g. background)

time window
dye 2
time (nsec)
FRET (Cameleon)
FRAP
emBFP
exBFP
BFP
Calmodulin/M13
GFP
Ca++
emBFP
emGFP
P
BF
GF
P
exBFP
du
lin
lm
ca
Ca++
OrganellspecificexpressionofanCa2+sensitiveproteine
Cameleons(developedbyR.Y.Tsien)
ERtaggedCameleons
MitaggedCameleons
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
resolution:
Dichroic
mirror
>100 nm (x/y)
>300 nm (z)
Objective lens
Focal plane
Specimen
in-focus rays
out of focus rays
picture element (pixel; e.g. 60x60 nm)
Single optical section
multiple optical sections

z
3d reconstruction

depth
cover slide
focal spot
Yeast Light Microscopy
100 x
Microfluorometry
Microfluorometry
Filt errad
Monitor
Xe-Lampe
Blende
Zelle
Verst rker
Pipette
>600 nm Filter
Camera
Zelle
Lampe
Microskop
Blende
Videorecorder
Optischer
Prozessor
510 nm Filter
A/ D-Wandler
Computer
PM
Microfluorometry:
SimultaneouslyrecordingsofCa2+andioncurrents
st
2000
1 spike
0.9
1500
0.8
0.7
Current: I (pA)
1000
0.6
2+
bulk
0.5
: Ratio (F
500
0.4
0.3
0
Histamine
30
[Ca
32
360
/F
380
0.2
34
36
38
40
42
44
Elapsed Time (s)
46
48
50
FluorescenceImaging
Deconvolutionmicroscopy
Pointspreadfunction
Focus
Outoffocusfluorescence
2Dreconstruction
Deconvolutionmicroscopyallowstimeresolved
twodimensionalfluorescencerecordingsinhighxy
resolutionandapp.200to300mthickslices(pixel)
3Dreconstruction
pixel(xyplane).......voxel(xyzplane)
Confocal vs Deconvolution
pinhole
Out-of-focus light
low (10 p/px)
Signal to noise ratio
Serial lines (time scan)
n.a.
slow
Image acquisition
fast
== f(object) >
Imaging quality
== f(object)
100 m
Thick samples
<< 100 m
# laser lines
Excitation
Spectral lamp
PSF & comput.
Costs
high (10
p/px)

FluorescenceMicroscopy Inpharmacology

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Fluorescence Microscopy

Wolfgang Graier (wolfgang.graier@kfunigraz.ac.at)

Vital dyes, GFP and derivatives

Limitation of light microscopy

2nd barrier filter

Organelle-specific, pH, membrane potential, ion

GFP, BFP, RFP, YFP; Aequorin; GFP and FRET

Life Cell Microscopy

limits of resolution (wavelength of light)

viability, temperature, oxygen, phototoxicity,

limits of resolution (wave length of light)

sample preparation, preparation artifacts (fixation,

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

Microscopic analysis of yeast organelles in vivo

Blue/Green/Yellow/Red fluorescent proteins

Green Fluorescent Protein Cloning Strategies

N, C-terminal fusions <> targeting signals !

GFP C-terminal chromosomal fusion

dye 1 (e.g. background)

Electronic Light Microscopy

100 x 100 x 300 nm

cell viability, structure dynamics

The Confocal Principle

The Confocal Principle

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

Single optical section

multiple optical sections

The Confocal Principle

Yeast Light Microscopy

low (10 p/px)

Signal to noise ratio

Serial lines (time scan)

PSF & comput.

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