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Fundamentals of Fluorescence
Microscopy
E. D. Salmon
University of North Carolina at Chapel Hill
References: Murphy Book;
http://micro.magnet.fsu.edu/primer/techniques/
Fluorescence; and
www.chroma.com
Basic Concept of Absorption and
Emission
Common Fluorophores Have Complex
Electronic Structures
Excitation and Emission Spectra
Jablonski Diagram
Basic Features of Fluorescence
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Excitation occurs in 10-15 sec
Emission occurs in 10-12 – 10-8 sec
Usually broad excitation spectrum w peak
Usually broad emission spectrum w peak
Stokes shift is separation of Ex. & Em peaks
Iem = Iexeclj
Photobleaching: Rate depends on Iex ,environment
Fluorophore Parameters
• Absorption coefficient at peak absorption
• Quantum efficiency at peak emission
• Photostability (e.g. fluorescein has 10,000
excitations before bleaching event)
• Stokes Shift
• Widths of excitation and emission spectra
• Fluorescence is polarized: absorption and
emission usually for E vector in plane of
conjugated bonds
Quantum Yields
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Compound
Acridine Orange
Benzene
Eosin
Fluorescein
Rhodamine-B
Chlorophyl-A
Solvent
Ethanol
Ethanol
Water
Water
Ethanol
Ethanol
Ex. l (nm) Quantum Yield
366
0.46
248
0.04
366
0.16
366
0.92
535
0.97
644
0.23
Molecular Fluorescent Probes
• Specific Fluorescent Dyes (e.g. DAPI)
• Covalently bind fluorescent dye to purified protein
• Fluorescent Antibodies (e.g immunofluorescent
labeling with primary and fluorescent secondary
antibodies)
• Express in cells Green (C,Y,R) Fluorescent Protein
(G, C,Y, R-FP) fused to protein of interest
There are Different Fluorescent Molecules for Different Jobs
See Molecular Probes Catalog; Sigma Catalog; CloneTech for GFP
Green Fluorescent Protein (GFP)
CloneTech
MultiWavelength
Fluorescence
Imaging
Basic Concept of Epi-Fluorescence
Microscopy
Hg-Arc Lamp
Xenon Arc-Lamp Spectra
Arc Lamps for Epi-Fluorescence
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Lamp Type:
XBO 150W/1 XBO 75W/2 HBO 200W/2 HBO 100W/2 HBO 50W/3
Current:
DC
DC
DC
DC
DC
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Rate Power (watts):
150
75
Luminous Flux (lumens):
3000
950
Light Intensity (Candella):
300
100
Avg. Brightness (cd/cm):
15000
40000
Arc Size (w x h in millimeters):
0.50 x 2.20 0.25 x 0.50
Life (Hours):
1200
400
200
100
50
10000
2200
1300
1000
260
150
40000
170000
90000
0.60 x 2.20
0.25 x 0.25 0.20 x 1.35
400
200
200
Quartz-Halogen (Tungsten Filament)
Lamp Spectra
Current
Lasers Have Line Spectra
Ploem-Type Epi-Illuminator
EpiFluorescence
Microscope
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Arc Lamp Housing
Lamp Alignment
Alignment of Arc and Mirror Images at
Objective Back Focal Plane (Use
Centering-Screen or white Card on Stage
W/O Objective)
Filter Cubes
Filter Cubes Are Not Inter-Changeable
Between Different Manufactures
Basic Design Features
Exciter and Barrier Filters are Designed
to Separate Emission Light from
Excitation Light
Problems in Filter Design: Example
Absorption and Emission Spectra
The Dichromatic Mirror Further Isolates
the Emission Light from the Excitation
Light
Modern Interference-Reflection filter Design Can Give Sharp
Cut-Off with High Transmission Efficiency for the Pass Wavelengths.
See web-sites for “Chroma Technology” and “Omega Optical”
Multi-Wavelength Immunofluorescence
Microscopy
Fluorophores for Triple-Label
Multiple
Band-Pass
Filters
Multiple
Band-Pass
Filters
Choose WideBand Emission
Filters for Single
Fluorophore to
Maximize
Sensitivity
Chroma Technology Corp. is an employee- owned company that
produces the world's finest optical filters and filter sets. The
company specializes in the design and manufacture of optical
filters and coatings for applications which require the greatest
precision in color separation, optical quality and signal purity. For
more about us, see our About Chroma page. Welcome to our new
website! This site is under construction, so if you don't find what
you need please give us a call at (800) 824-7662.
Handbook of Optical Filters
for Fluorescence Microscopy:
Download a copy of our "Handbook of Optical Filters for
Fluorescence Microscopy"
in Adobe Acrobat PDF format.
www.chroma.com
Multi-Wavelength Immunofluorescence
Microscopy
MultiWavelength
Fluorescence
Imaging
Multi-wavelength Fluorescence Imaging
MultiWavelength
Fluorescence
Imaging
Ploem-Type Epi-Illuminator
Parameters for Maximizing Sensitivity
• Use High Objective NA and Lowest Magnification:
Ifl ~ IilNAobj4/Mtot2
• Use high efficiency filters
• Use as few optical components as possible
• Close Field Diaphragm down as far as possible
• Buy the newest objective: select for best efficiency
• Match magnification to camera resolution:
MMax = 3*Pixel Size of Detector/Optical
Resolution
E.g.: 3*7 mm/[0.6 *520nm/1.4] = 91X
• Reduce Photobleaching
• Use High Quantum Efficiency Detector in Camera
Reducing Photobleaching
• For fixed specimens use anti-fade
compounds: These reduce oxygen effects
• 95% glycerol works quite well
• For live specimens, reduce oxygen with:
- Oxyrase
- Catalase + glucose + glucose-oxidase
Reducing Photobleaching: Anti-Fade
Reagents for Fixed Specimens
• p-phenylenediamine: The most effective reagent for FITC. Also
effective for Rhodamine. Should be adjusted to 0.1% pphenylenediamine in glycerol/PBS for use. Reagent blackens when
subjected to light exposure so it should be stored in a dark place. Skin
contact is extremely dangerous.G. D. Johnson & G. M. Araujo (1981)
J. Immunol. Methods, 43: 349-350
• DABCO (1,4-diazabi-cyclo-2,2,2-octane): Highly effective for FITC.
Although its effect is slightly lower than p-phenylenediamine, it is
more resistant to light and features a higher level of safety.G. D.
Johnson et. al., (1982) J. Immunol. Methods, 55: 231-242.
• n-propylgallate: The most effective reagent for Rhodamine, also
effective for FITC. Should be adjusted to 1% propylgallate in
glycerol/PBS for use. H. Giloh & J. W. Sedat (1982), Science, 217:
1252-12552.
• mercapto-ethylamine: Used to observe chromosome and DNA
specimens stained with propidium iodide, acridine orange, or
Chromomysin A3. Should be adjusted to 0.1mM 2mercaptotheylamine in Tris-EDTAS. Fujita & T. Minamikawa (1990),
Experimental Medicine, 8: 75-82
Use High Quantum Efficiency Camera
Detector: e.g. ORCA cooled CCD
Cdc20 Persists
At Kinetochores
Throughout
Mitosis and
Exhibits Fast
Kinetics:
FRAP t1/2 =
[4 sec (attached)
25 sec (unattached]
Green:
GFP-Cdc20
At
Kinetochores
Red:
Phase Contrast
Images of PtK1
Tissue Cells