Fluorescence Microscopy - EPFL · PT-BIOP course, Fluorescence Microscopy, EPFL 2010 BioImaging...
Transcript of Fluorescence Microscopy - EPFL · PT-BIOP course, Fluorescence Microscopy, EPFL 2010 BioImaging...
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
Fluorescence Microscopy
Dr. Arne SeitzSwiss Institute of Technology (EPFL)
Faculty of Life Sciences Head of BIOIMAGING AND OPTICS – BIOP
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
Fluorescence Microscopy
• Why do we need fluorescence microscopy•Basics about fluorescence•Fluorescent dyes and staining procedures•Fluorescent microscopy•Advanced applications
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
Purpose of fluorescence microscopy
Cells are usually “transparent” and therefore study of dynamic processes is not always easily possible. Thus a staining procedure is needed.
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
Histological stain (Absorption) like e.g H&E staining(Hematoxilin and Eosin staining)
Fluorescent dyes:Sensitivity (single molecule detection is possible)
Different staining strategies
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
The term 'fluorescence' was coined Gabriel Stokes in his 1852 paper[1]; the name was suggested "to denote the general appearance of a solution of sulphate of quinine and similar media". (Phil. Trans. R. Soc. Lond. 1853 143, 385-396 [quote from page 387). The name itself was derived from the mineral fluorite (calcium difluoride), some examples of which contain traces of divalent europium, which serves as the fluorescent activator to provide a blue fluorescent emission. The fluorite which provoked the observation originally, and which remains one of the most outstanding examples of the phenomenon, originated from the Weardale region, of northern England.
(from Wikipedia)
What is fluorescence?
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
12
high energy low energy
The Atomic View
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
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Jablonski Diagram (very simplified)
Fluorescence energy diagram
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
Absorbtion and Emission Spectra of Fluorophores
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
Stokes Shifted
=> Scattered excitation light can be efficiently separated from fluorescence
Excitation and emission spectra of fluorescent dyes
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
Excitation and emission spectra are not discrete.
Excitation and Emission Spectra
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
The profile of the emission spectra are independentof the excitation wavelength
Excitation and Emission Spectra
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
1. Excitation 10-15 s2. Internal conversion 10-12 s3. Solvent relaxation 10-11 s4. Fluorescence 10-9 s
Saturation of excited state possible
5
6
T1
5. Intersystem crossing 10-9 s6. Phosphorescence 10-3 s
Jablonski Diagram (simplified)
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
BleachingBleaching is irreversible (=fluorophore is destroyed)Bleaching is dependent on the excitation powerBleaching can also cause photodamage
“bleached”
“bleached”
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
• High Absoption
• High quantum yield
• High stability, little photobleaching
• Compatibility with biological systems (labeling efficiency)
Some Features of a Useful Fluorophore
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
Specificity (molecules can be specifically labelled)
Sensitivity (single molecule detection is possible)
Fluorescence can report on the environmentof the labelled molecule
Fluorescence Microscopy
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
Organelle Specific Fluorescent Stains
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
DAPI
DAPI binds DNA at AT-rich streches in the minor groove
Fluorescent Stains
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
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Mitotracker LysoSensor
Fluorescent Stains
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
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Fluorophore Labeled Proteins/Antibodies
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
Fluorescein isothiocyanate(FITC)
Fluorescein
Molecules can be specifically labelled
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
IgG labelled IgG
IgG labelled IgG
Molecules can be specifically labelled
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
(e.g. Immunofluorescence)Molecules can be specifically labelled
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
Protein of interest
Production of a specific antibody
Fluorescent labbeleing of the antibody
Staining of cells, tissue etc.
Alternative: Detection via a fluorescently labelled
secondary antibody
Major limitation: Targeting in live cells.
Proteins can be specifically labelled
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
Quantum Dots
ener
gy
band gap
h+
e-
valence band
conduction band
hνWannierexcitonen
ergy
band gap
h+
e-
valence band
conduction band
hνWannierexciton
Band gap increasesParticle Size decreases
Picture from: Chan WCW et al. Current Opinion in Biotechnology 2002, 13: 40-46
Size quantization effect
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
Quantum dotsAdvantages
Quantum yield – Similar, slightly lower as organic dyes
Absorption – Lager cross-section– Reduced photo-bleaching-rate
ZnS-capped CdSe QDs compared with Rhodamine6G– 20 time brighter– 100-200 times more stable
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
Sensitivity (Single Fluorophores)
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
Autofluorescent Proteins
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
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488 nm
Aequorea victoria(Jellyfish) Chemistry Nobel price 2008
Osamu Shimomura
Martin Chalfie
Roger Y. Tsien
Green Fluorescent Protein (GFP)
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
Two Most common applications of GFP variants
From Chudakov et al, Trends Biotech., 2005
Applications of fluorescent proteins (FP)
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
10µm
nucleus nucleolus nuclear envelope cytoplasm
mitochondria peroxisomes microtubules
focal adhesions endoplasmic reticulum Golgi plasma membrane
nucleus + cytoplasm
http://gfp-cdna.embl.de/index.html
Protein Localization
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
Organelles and molecules can be labeled by:• Organelle and protein specific fluorescent stains (e.g. Dapi).• Labeling of antibodies/proteins with fluorophores.• Autofluorescent proteins (e.g. GFPs).
Live cell imaging:• FP (Fluorescent proteins, e.g. GFP) are the method of
choice to label proteins or organelles.• Injection of labeled antibodies is possible.• Organelle specific stains like e.g. DAPI can be toxic for the
cell.
Summary
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
Fluorescent Microscopy
• Why do we need fluorescence microscopy•Basics about fluorescence•Fluorescent dyes and staining procedures•Fluorescent microscopy•Advanced applications
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
Sample
Excitation light (IE)
Fluorescent light (IFL)
IE/IFL = 104 for strong fluorescence
IE/IFL = 1010 for weak fluorescence (e.g. in situ hybrid.)
In order to detect the fluorescence at 10% background the excitation light must beremoved or attenuated by a factor up to ≈ 1011
Most excitation light
Fluorescence detection
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
Sample
Objective
Excitation Light
Epifluorescence
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
Sample
Objective
Fluorescence
Back-scattered excitation light:
IE/100
Epifluorescence
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
Sample
Excitation LightDichroic mirror
(passes green but reflects blue light)
Objective
Epifluorescence
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
Sample
Detector
Fluorescence
Back-scattered excitation light
IE/100
Dichroic mirror(passes green but reflects blue light)
Objective
Epifluorescence
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
Sample
Detector
Back-scattered excitation light
IE/100
Dichroic mirror(passes green but reflects blue light)
Back-scattered excitation light
IE/10,000
Objective
Fluorescence
Epifluorescence (real world)
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
Sample
Detector
Back-scattered excitation light
IE/100
Dichroic mirror(passes green but reflects blue light)
Emission filter(passes fluorescence but notback-scattered excitation light)
Back-scattered excitation light
IE/10,000
Back-scattered excitation light
IE/1011
Objective
Fluorescence
Epifluorescence (real world)
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
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Sample
HBO
Detector
Scattered light
Dichroic mirror
Emission Filterwheel
Excitation Filterwheel
Alexa 488
488nm
520nm
Objective
Typical Set-Up for Epifluorescence
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
Sample
HBO
Detector
Scattered light
Double dichroic mirror(λ1 = 505nm +λ2 = 560nm)
Emission Filterwheel(Bandpass)
Excitation Filterwheel(Bandpass)
Alexa 488
488nm
520nm
Objective
Set-Up for Green-Red Double Fluorescence
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
Sample
HBO
Detector
Scattered light
Double dichroic mirror
Alexa 555
550nm
590nm
Objective
Emission Filterwheel
(Bandpass, Longpass)
Excitation Filterwheel(Bandpass)
Set-Up for Green-Red Double Fluorescence
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
Implementation of Epifluorescence
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
Implementation of Epifluorescence
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
Implementation of Epifluorescence
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
Transmission• Focus on the specimen• Close field diaphragm• Focus condenser until field diaphragm is seen sharp• Center field diaphragm• Close field diaphragm up to 80 – 90 %• Remove eyepiece, look down to the aperture diaphragm• Center (if possible) aperture diaphragm• Open/Close aperture diaphragm up to 80 – 90 %
Fluorescence• Focus on the specimen• Swing in focusing aid (if available) • Focus image of arc sharply• Swing out focusing aid• Close field diaphragm• Center field diaphragm
Köhler illumination in Epifluorescence
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
Typical filter profiles
Longpass Bandpass Shortpass
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
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Typical Triple Bandpass Filter
DAPI GFP TexasRed
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
Single Color Detection (e.g. GFP)
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
Single Color Detection (e.g. GFP)
Use longpass filter in the emission!
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
Double Color Detection (e.g. GFP and TRITC)
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
GFP-TRITC Detection Filter Cubes
Bandpass emission filters are necessary in multicolor imaging
GFP-detection TRITC-detection
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
Triple Filter Cube
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
Color glass filters (cheap, limited in wavelengths)
Interference filters(high flexibility in wavelengths)
Types of filters typically used
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
Light Sources
Must fit the fluorescent dyes
Must fit the Detectors
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
Light sourcesHalogen lamp
• Continuous spectrum: depends on temperature• For 3400K maximum at 900 nm• Lower intensity at shorter wavelengths• Very strong in IR
Mercury Lamp (HBO)• Most of intensity in near UV• Spectrum has a line structure • Lines at 313, 334, 365, 406, 435, 546, and 578 nm
Xenon lamp (XBO)• Even intensity across the visible spectrum• Has relatively low intensity in UV• Strong in IR
Metal halide lamp (Hg, I, Br)• Stronger intensity between lines• Stable output over short period of time• Lifetime up to 5 times longer
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
Spectrum of a mercury arc lamp
⇒Ideal for excitation of GFP2, CFP and DsRed imaging butless convenient for EGFP
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
Spectrum of a Xenon arc lamp
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
Summary
Epifluorescence microscopy set-up is very sensitive.
Bandpass detection filters are necessary for multicolor detection.
Ideal excitation light sources should fit the dyes in use.
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
What is special aboutfluorescence microscopy?
Specificity (molecules can be specifically labelled)
Sensitivity (single molecule detection is possible)
Fluorescence can report on the environmentof the labelled molecule
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
1 nm10-9m
1 µm10-6m
1 mm10-3m
1 cm10-2m
1 m1 Å10-10m
Cells Worm HouseflyOrganelles
LMlimit
Human
FRAPFCSFRETPALM,STORM
Molecular dynamics,molecular interactions
FRAP: Fluorescence recovery after photobleaching
FCS: Fluorescence correlation spectroscopy
FRET: Fluorescence resonance energy transfer
Light microscopyElectron microscopy
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
Quantum Yield ”Q” = number of emitted versus absorbed photons
Q =kf
kf+ knr
τ =1
kf+ knr
Lifetime “ τ ” = average time moleculespends in the excited state
k= events/sec
Q =τ
τ 0knr = ∑ ki
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
Nonfluorescent relaxation can be due to:
Sen
sitiz
edE
mis
sion
Tk
FRET
Donor Acceptor
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
Fluorescence Resonance Energy Transfer (FRET)
Exc
itatio
n
Fluo
resc
ence
1−= DD kτDonor Fluorescence
Exc
itatio
n
Fluo
resc
ence
Sen
sitiz
edE
mis
sion
( ) 1−+= TDD kkτ
Tk
QuenchedDonor Fluorescence
SensitizedEmission
Excitation
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
Summary
Fluorescence is dependant on the environment of the molecule.Parameters which can change due to the environment are:• Intensity• Fluorescence lifetime•Frequency (=spectral shift)
Fluorescence can be used as a reporter of the environment.
Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010
BioImaging &Optics Platform
More about fluorescence microscopy1. Lecture
Biomicroscopy I + II, Prof. Theo Lasser, EPFL
2. Booksa) Principle of fluorescence spectroscopy,
Joseph R. Lackowicz, Springer 2nd edition (1999)
3. Interneta) http://micro.magnet.fsu.edub) b) Web sites of microscope manufactures
LeicaNikonOlympusZeiss
4. BIOpEPFL, SV-AI 0241, SV-AI 0140http://biop.epfl.ch/
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