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BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
Internal course 2014
January 14th
FLUORESCENCE MICROSCOPY
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
FLUORESCENCE
MICROSCOPY
Why do we need it?
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BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
3
Missing specificity
UNSTAINED SPECIMEN
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
Histological stain (Absorption)
like e.g H&E staining
(Hematoxilin and Eosin staining)
Fluorescent dyes:
Sensitivity
DIFFERENT STAINING
STRATEGIES
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
FLUORESCENCE
MICROSCOPY
Basic principles
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BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
ORIGIN OF FLUORESCENCE
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
ABSORPTION
400 450 500 550 600 650 700
0
50
100
150
200
250
10
-3*/ (
M-1c
m-1)
wavelength / nm
CY3
CY5
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
Jablonski Diagram
(very simplified)
FLUORESCENCE
ENERGY DIAGRAM
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
Excitation and emission
spectra are not discrete.
EXCITATION AND EMISSION
SPECTRA
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
EXCITATION AND EMISSION
SPECTRA
Scattered excitation light can be efficiently separated
from fluorescence
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
The profile of the emission spectra are independent
of the excitation wavelength
EXCITATION AND EMISSION
SPECTRA
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
1. Excitation 10-15 s
2. Internal conversion 10-12 s
3. Solvent relaxation 10-11 s
JABLONSKI DIAGRAM SIMPLIFIED
1
2
3
4h ex h emm
5
6
T1
S0
S1
4. Fluorescence 10-9 s
5. Intersystem crossing 10-9 s
6. Phosphorescence 10-3 s
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
BLEACHING
Bleaching is irreversible (=fluorophore is destroyed)
Bleaching is dependent on the excitation power
Bleaching can also cause photodamage
“bleached”
“bleached”
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
FLUORESCENCE
MICROSCOPY
Detection
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BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
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 be removed or attenuated by a factor up to ≈ 1011
Most excitation light
FLUORESCENCE DETECTION
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
Sample
Objective
Excitation Light
EPIFLUORESCENCE
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
Sample
Objective
Fluorescence
Back-scattered
excitation light:
IE/100
EPIFLUORESCENCE
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
Sample
Excitation LightDichroic mirror
(passes green but reflects blue light)
Objective
EPIFLUORESCENCE
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
Sample
Detector
Fluorescence
Back-scattered
excitation light
IE/100
Dichroic mirror(passes green but reflects blue light)
Objective
EPIFLUORESCENCE
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
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
EPIFLUORESCENCEREAL WORLD
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
Sample
Detector
Back-scattered
excitation light
IE/100
Dichroic mirror(passes green but reflects blue light)
Emission filter(passes fluorescence but not
back-scattered excitation light)
Back-scattered
excitation light
IE/10,000
Back-scattered
excitation light
IE/1011
Objective
Fluorescence
EPIFLUORESCENCEREAL WORLD
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
Sample
HBO
Detector
Scattered light
Dichroic mirror
Emission
Filterwheel
Excitation
Filterwheel
Alexa 488
488nm
520nm
Objective
EPIFLUORESCENCE
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
Sample
HBO
Detector
Scattered light
Double dichroic mirror
(l1 = 505nm +l2 = 560nm)
Emission
Filterwheel(Bandpass)
Excitation
Filterwheel(Bandpass)
Alexa 488
488nm
520nm
Objective
DOUBLE FLUORESCENCE
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
Sample
HBO
Detector
Scattered light
Double dichroic mirror
Alexa 555
550nm
590nm
Objective
Emission
Filterwheel(Bandpass, Longpass)
Excitation
Filterwheel(Bandpass)
DOUBLE FLUORESCENCE
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
FLUORESCENCE
MICROSCOPY
Implementation
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BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
IMPLEMENTATION OF
EPIFLUORESCENCE
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
IMPLEMENTATION OF
EPIFLUORESCENCE
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
TYPICAL FILTER PROFILES
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
FILTER CHARACTERISTICS AND
NOMENCLATURE
29
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
ANATOMY OF AN INTERFERENCE
FILTER
30
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
Use longpass filter in the emission!
SINGLE COLOR DETECTION
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
Bandpass emission filters are necessary in multicolor imaging
GFP-detection TRITC-detection
GFP-TRITC DETECTION FILTER
CUBES
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
FLUORESCENCE
MICROSCOPY
Excitation
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BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
FLUORESCENCE MICROSCOPY
• Why do we need fluorescence microscopy?
•Basics about fluorescence
•Fluorescence detection
•Fluorescent excitation
•Fluorescent dyes and staining procedures
•Applications
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
Ideal for excitation of GFP2, CFP and DsRed imaging but
less convenient for EGFP
SPECTRUM OF A MERCURY
ARC LAMP
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
SPECTRUM OF A XENON ARC
LAMP
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
Color Name
Wavelength
(Nanometers
)
Semiconduct
or
Composition
Infrared 880GaAlAs/GaA
s
Ultra Red 660GaAlAs/GaAl
As
Super Red 633 AlGaInP
Super
Orange612 AlGaInP
Orange 605 GaAsP/GaP
Yellow 585 GaAsP/GaP
Incandescen
t
White
4500K (CT) InGaN/SiC
Pale White 6500K (CT) InGaN/SiC
Cool White 8000K (CT) InGaN/SiC
Pure Green 555 GaP/GaP
Super Blue 470 GaN/SiC
Blue Violet 430 GaN/SiC
Ultraviolet 395 InGaN/SiC
Pro:•long lifetime
•Fast switching
•No unwanted excitation
Contra:
•flexibility
•intensity
LIGHT EMITTING DIODES (LED)
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
Halogen 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
LIGHT SOURCES
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
FLUORESCENCE
MICROSCOPY
Staining strategies
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BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
LABELLING CELLULAR
COMPONENTS
Organelle specific dyes/molecules:
Nucleus: DAPI, Hoechst.
Mitochondria: DiOC6 , MitotrackerRed/Green
Endoplasmic Reticulum: DiOC6 , ER tracker
Golgi Aparatus: BODIPY FL
Advantage: easy to use, availability
Disadvantage: limited specificity
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
SMALL MOLECULE STAINING
Mitotracker LysoSensor
DAPI
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
IgG labelled IgG
IgG labelled IgG
ANTIBODIES AS PROBES
Advantages: high-specificity
Disadvantages: availability
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
Major limitation: Targeting in live cells.
ANTIBODY
LABELLING WORKFLOW
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
488 nm
Aequorea victoria
(Jellyfish) Chemistry Nobel price 2008
Osamu
Shimomura
Martin
ChalfieRoger Y.
Tsien
GREEN FLUORESCENT
PROTEIN
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
The fluorescent protein palette: tools for cellular imaging.
Richard N. Day and Michael W. Davidson
Chem. Soc. Rev., 2009, 38, 2887–2921.
GREEN FLUORESCENT
PROTEIN
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
APPLICATIONS OF
FLUORESCENT PROTEINS (FP)
Nathan C. Shaner, Paul A. Steinbach & Roger Y. Tsien
Nat Methods. 2005 Dec;2(12):905-9.
Chudakov DM, Lukyanov S, Lukyanov KA.
Trends Biotechnol. 2005 Dec;23(12):605-13.
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
(A) mOrange2-b-actin-C-7.
(B) mApple-Cx43-N-7.
(C) mTFP1-fibrillarin-C-7.
(D) mWasabi-cytokeratin-N-17.
(E) mRuby-annexin (A4)-C-12.
(F) mEGFP-H2B-N-6.
(G) EBFP2-b-actin-C-7.
(H) mTagRFP-T-mitochondria-N-7.
(I) mCherry-C-Src-N-7.
(J) mCerulean-paxillin-N-22.
(K) mKate-clathrin (light chain)-C-15.
(L) mCitrine-VE-cadherin-N-10.
(M) TagCFPlysosomes-C-20.
(N) TagRFP-zyxin-N-7.
(O) superfolderGFP-lamin B1-C-10.
(P) EGFP-a-v-integrin-N-9.
(Q) tdTomato-Golgi-N-7.
(R) mStrawberry-vimentin-N-7.
(S) TagBFP-Rab-11a-C-7.
(T) mKO2-LC-myosin-N-7.
(U) DsRed2-endoplasmic reticulum-N-5.
(V) ECFP-atubulin-C-6.
(W) tdTurboRFP-farnesyl-C-5.
(X) mEmerald-EB3-N-7.
(Y) mPlum-CENP-B-N-22.
FLUORESCENT PROTEINS
The fluorescent protein palette: tools for cellular imaging.
Richard N. Day and Michael W. Davidson, Chem. Soc. Rev., 2009, 38, 2887–2921.
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
Re
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FlAsH;ReAsH Fluoresceine, Oxazine
TMP-tag Fluorescein, Atto655
SNAP-tag Tetramethylrhodamine (TMR), Atto633
HaloTag TMR
Coumarine ligase Coumarin
TAG TECHNOLOGY
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
LABELLING STRATEGIES
SUMMARY
Fixed specimens
• Small, organelle specific molecules
• Pro: availability, stability
• Con: low specificity
• Antibody labeled with fluorophore
• Pro: flexibility, specificity
• Con: complex production, stability
Living specimens
• Small, organelle specific molecules
• Pro: ease of use,
• Con: Toxicity, permeability, specificity
• Fluorescent Proteins
• Pro: intrinsic labeling, known stoichiometry, flexibility
• Con: slow, genetic tools
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
FLUOROPHORESBRIGHTNESS DEFINITION
•ε : molar decadic
extinction coefficient
E= ε(l)*c*d
E=-lg T
•QE: Quantum efficiency
Brightness: B=QE* ε(l)
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
FLUOROPHORESBRIGHTNESS DEFINITION
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
Dye Class Examples Brightness Environment
Sensitivity
Photo-
stability
Bio-
compability
Coumarin AlexaFluor350 + ++++ + +++
Fluorescein FITC +++ ++++ + +++
BOPIDY BOPIDY FL +++ +++ ++ ++++
AlexaFluor AlexaFluor488 ++++ ++ ++++ ++++
Quantum
Dots
QDot605 +++++ + +++++ +
Cyanines CY3 +++ ++ ++++ +++
Styryl Dyes FM 1-43 ++ +++++ ++ ++
Rhodamines TRITC +++ +++ ++++ ++
Fluorescent
Proteins
EGFP ++ ++ ++ +++++
Table modified from: J. B Pawley, Handbook of Confocal Microscopy, 3rd edition, p. 355
FLOUROPHORES
COMPARISON
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
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
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
FLUORESCENCE
MICROSCOPY
Resolution
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BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
RESOLUTION
Lateral resolution
Axial resolution
2NA2
nR
z
l
NA61.0
l R
l=540 nm, NA=1.4, n=1.52: 235 nm - lateral, 838 nm - axial
Shortest distance between two points on a
specimen that can still be distinguished by the
observer or camera as separate entities.
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
POINT SPREAD FUNCTION
1E-6
1E-5
1E-4
1E-3
0.01
0.1
1
1E-6
1E-5
1E-4
1E-3
0.01
0.1
1
NA=0.8 NA=1.4
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
LOW PASS FILTERING
Object
Imag
e
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
1 nm
10-9m
1 µm
10-6m
1 mm
10-3m
1 cm
10-2m
1 m1 Å
10-10m
Cells Worm HouseflyOrganelles
light microscopy
resolution limit
Homo sapiens
Light
MicroscopyProteins
RNA
Atom
Molecule
LIGHT MICROSCOPY IN LIFE
SCIENCE
Lateral resolution
obj
AiryNA
r 061.0l
Lord Rayleigh
sin2
0min
nd
l
Ernst Abbe
sinnNA58
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
• Epifluorescence microscopy set-up is very sensitive.
• Ideal excitation light sources should fit the dyes in use.
• Specificity (molecules can be specifically labelled)
• Sensitivity (single molecule detection is possible)
• Fluorescence can report on the environment
of the labelled molecule
SUMMARY
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
1. Lecture
Biomicroscopy I + II, Prof. Theo Lasser, EPFL
2. Books
a) Principle of fluorescence spectroscopy,
Joseph R. Lackowicz, Springer 2nd edition (1999)
3. Internet
a) http://micro.magnet.fsu.edu
b) b) Web sites of microscope manufactures
Leica
Nikon
Olympus
Zeiss
4. BIOp
EPFL, SV-AI 0241, SV-AI 0140
http://biop.epfl.ch/
MORE ABOUT FLUORESCENCE
MICROSCOPY