BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
FLUORESCENCE MICROSCOPY
PT BIOP COURSE 2012
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
FLUORESCENCE MICROSCOPY
• Why do we need fluorescence microscopy
•Basics about fluorescence
•Fluorescent dyes and staining procedures
•Fluorescent microscopy
•Advanced applications
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
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.
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
Histological stain (Absorption)
like e.g H&E staining
(Hematoxilin and Eosin staining)
Fluorescent dyes:
Sensitivity / Selectivity
DIFFERENT STAINING STRATEGIES
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
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?
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
ABSORBTION/EMISSION SPECTRA
OF FLUOROPHORES
Scattered excitation light can be efficiently separated
from fluorescence
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
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
4. Fluorescence 10-9 s Saturation of excited
state possible
5
6
T1
5. Intersystem crossing 10-9 s
6. Phosphorescence 10-3 s
JABLONSKI DIAGRAM (SIMPLIFIED)
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
FLUORESCENT MICROSCOPY
• Why do we need fluorescence microscopy
•Basics about fluorescence
•Fluorescent dyes and staining procedures
•Fluorescent microscopy
•Advanced applications
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 Light Dichroic 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
EPIFLUORESCENCE (REAL 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
EPIFLUORESCENCE (REAL WORLD)
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
Sample
HBO
Detector
Scattered light
Dichroic mirror
Emission
Filterwheel
Excitation
Filterwheel
Alexa 488
488nm
520nm
Objective
TYPICAL SET-UP FOR 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
SET-UP 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)
SET-UP FOR DOUBLE FLUORESCENCE
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
IMPLEMENTATION OF
EPIFLUORESCENCE
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
Longpass Bandpass Shortpass
TYPICAL FILTER PROFILES
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
DAPI GFP TexasRed
TYPICAL TRIPLE BANDPASS
FILTER
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
SINGLE COLOR DETECTION (E.G. GFP)
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
Use longpass filter in the emission!
SINGLE COLOR DETECTION
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
DOUBLE 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
TRIPLE FILTER CUBE
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
Color glass filters (cheap, limited in wavelengths)
Interference filters (high flexibility in wavelengths)
TYPES OF FILTERS TYPICALLY
USED
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
Must fit the fluorescent dyes
Must fit the Detectors
LIGHT SOURCES
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
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 880 GaAlAs/GaA
s
Ultra Red 660 GaAlAs/GaAl
As
Super Red 633 AlGaInP
Super
Orange 612 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
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.
SUMMARY
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
Specificity (molecules can be specifically labelled)
Sensitivity (single molecule detection is possible)
Fluorescence can report on the environment
of the labelled molecule
FLUORESCENCE MICROSCOPY
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
DAPI
DAPI binds DNA at
AT-rich stretches in
the minor groove
FLUORESCENT STAINS
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
Mitotracker LysoSensor
FLUORESCENT STAINS
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
IgG labelled IgG
IgG labelled IgG
MOLECULES CAN BE SPECIFICALLY
LABELLED
Fluorescein isothiocyanate
(FITC)
Fluorescein
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
(e.g. Immunofluorescence)
MOLECULES CAN BE
SPECIFICALLY LABELLED
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
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
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
488 nm
Aequorea victoria
(Jellyfish) Chemistry Nobel price 2008
Osamu
Shimomura
Martin
Chalfie Roger Y.
Tsien
GREEN FLUORESCENT PROTEIN
(GFP)
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
Two Most common applications of GFP variants
From Chudakov et al, Trends Biotech., 2005
APPLICATIONS OF FLUORESCENT
PROTEINS (FP)
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
FLUORESCENT PROTEINS
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
10mm
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
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
• High Absoption
• High quantum yield
• High stability, little photobleaching
• Compatibility with biological systems
(labeling efficiency)
FEATURES OF A USEFUL FLUOROPHORE
BIOIMAGING AND
OPTICS PLATFORM
EPFL–SV–PTBIOP
FLUOROPHORES BRIGHTNESS 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
Dye Class Examples Brightnes
s
Environme
nt
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
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
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