Fundamentals of Fluorescence Microscopy

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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.e
  du/primer/techniques/
• Fluorescence and
• www.chroma.com

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Basic Concept of Absorption and Emission
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Common Fluorophores Have Complex Electronic
Structures
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Excitation and Emission Spectra
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Jablonski Diagram
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Basic Features of Fluorescence

• 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

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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

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Quantum Yields

• Compound Solvent Ex. l (nm) Quantum Yield
• Acridine Orange Ethanol 366 0.46
• Benzene Ethanol 248 0.04
• Eosin Water 366 0.16
• Fluorescein Water 366 0.92
• Rhodamine-B Ethanol 535 0.97
• Chlorophyl-A Ethanol 644 0.23

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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

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There are Different Fluorescent Molecules for
Different Jobs
See Molecular Probes Catalog Sigma Catalog
CloneTech for GFP
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Green Fluorescent Protein (GFP)
CloneTech
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Multi-Wavelength Fluorescence Imaging
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Basic Concept of Epi-Fluorescence Microscopy
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Hg-Arc Lamp
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Xenon Arc-Lamp Spectra
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Arc Lamps for Epi-Fluorescence

• Lamp Type
• XBO 150W/1 XBO 75W/2 HBO 200W/2 HBO 100W/2
  HBO 50W/3
• Current
• DC DC
  DC DC DC
• Rate Power (watts)
• 150 75
  200 100 50
• Luminous Flux (lumens)
• 3000 950
  10000 2200 1300
• Light Intensity (Candella)
• 300 100 1000
  260 150
• Avg. Brightness (cd/cm)
• 15000 40000 40000
  170000 90000
• Arc Size (w x h in millimeters)
• 0.50 x 2.20 0.25 x 0.50 0.60 x 2.20
  0.25 x 0.25 0.20 x 1.35
• Life (Hours)
• 1200 400 400
  200 200

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Quartz-Halogen (Tungsten Filament) Lamp Spectra
Current
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Lasers Have Line Spectra
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Ploem-Type Epi-Illuminator
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Epi-Fluorescence Microscope
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Arc Lamp Housing
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Lamp Alignment
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Alignment of Arc and Mirror Images at Objective
Back Focal Plane (Use Centering-Screen or white
Card on Stage W/O Objective)
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Filter Cubes
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Filter Cubes Are Not Inter-Changeable Between
Different Manufactures
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Basic Design Features
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Exciter and Barrier Filters are Designed to
Separate Emission Light from Excitation Light
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Problems in Filter Design Example Absorption and
Emission Spectra
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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
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Multi-Wavelength Immunofluorescence Microscopy
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Fluorophores for Triple-Label
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Multiple Band-Pass Filters
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Multiple Band-Pass Filters
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Choose Wide-Band Emission Filters for Single
Fluorophore to Maximize Sensitivity
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Filters for Fluorescence MicroscopyDownload a
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Fluorescence Microscopy"in Adobe Acrobat PDF
format.www.chroma.com
37
Multi-Wavelength Immunofluorescence Microscopy
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Multi-Wavelength Fluorescence Imaging
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Multi-wavelength Fluorescence Imaging
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Multi-Wavelength Fluorescence Imaging
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Ploem-Type Epi-Illuminator
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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 3Pixel Size of Detector/Optical
  Resolution
• E.g. 37 mm/0.6 520nm/1.4 91X
• Reduce Photobleaching
• Use High Quantum Efficiency Detector in Camera

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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

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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 p-phenylenediamine 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 2-mercaptotheylamine in
  Tris-EDTAS. Fujita T. Minamikawa (1990),
  Experimental Medicine, 8 75-82

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Use High Quantum Efficiency Camera Detector e.g.
ORCA cooled CCD
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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