Fluorescence Microscopy

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Fluorescence Microscopy

• Ken Jacobson

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What we will cover

• What is fluorescence?
• Fluorescence microscopy light sources,
• filters, objectives
• Special considerations autofluorescence
  photobleaching
• TIRF
• Class Exercises

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On line resource Molecular Expressions, a
Microscope Primer at http//www.microscopy.fs
u.edu/primer/index.html
Important reference on fluorescent probes The
Molecular Probes catalog
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Fluorescence fundamentals

• Fluorescence prop. to Light Absorbedxquantum
  yield
• F I ? c x Q
• Q photons emitted/photons absorbedlt1

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Acridine orange
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Rhodamine 123-potential based stain
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NBD ceramide
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Rh-phalloidin anti-integrin
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Rh-anti tubulin DAPI
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GFP Structure fluorphore formed by
cyclization of Ser65, Tyr66 and Gly 67
M. Ormo et al, Sci. 2731392, 1996
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Patterson, G. et al. J Cell Sci 2001114837-838
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The microscope as a filter fluorometer
with focusing optics
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Basic design of the epi fluorescence microscope
Objective acts as condenser excitation light
reflected away from eyes
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Common non-laser light sources
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Arc lamps
CAUTION lamps at hi pressure do not touch glass
envelopes
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TECHNICAL DATA OF THE LIGHT SOURCES FOR
INCIDENT-LIGHT FLUORESCENCE MICROSCOPY
From C. Zeiss
Note small arcs with high luminous density will
be brightest
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Aligning the light source
The epi fluorescence microscope is a reflected
light microscope with the arc of the lamp imaged
at the back focal plane of the objective, ideally
just filling the back aperature (Koehler
illumination).
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Works because the depth of focus of the collector
lens on the lamp housing is very long whats in
focus at the back focal plane is in focus at
the specimen plane.
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Objectives

• High transmittance
• Fluorite lenses ? gt 350 nm ok for FURA
• Quartz lenses ? lt 350 nm
• Employ simple, non plan lenses to minimize
• internal elements.
• Neglible autofluorescence or solarization color
• change upon prolonged illumination

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Maximizing image brightness
(B)excitation efficiency (NA)2

gt B (NA)4collection efficiency
(NA)2
at high NA,
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Filters the key to successfulfluorescence
microscopy
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Filter cube must provide excitation, reflect the
excitation onto sample while transmitting
emission, and pass the fluorescence
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Cut off filters
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Bandpass Filters
Most bandpass filters are interference type with
multilayer dielectric coatings that pass or
reject certain wavelengths with great selectivity
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Interference filter definitions
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Figure 5a shows a band pass emitter filter
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Filter selection

• Broadband filters more excitation, less contrast
  more autofluorescence may be excited.
• Narrowband filtersless signal, more contrast.
• Note eye responds to contrast while detectors
  respond to signal.

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Multiple Band-Pass Filters
From E.D. Salmon
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Multi-Wavelength Immunofluorescence Microscopy
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Special issues autofluorescencewhich causes
unwanted background obscuring weak signals
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COMMON SOURCES OF AUTOFLUORESCENCE Autofluoresce
nt Source Typical Emission Wavelength
(nm) Typical Excitation Wavelength (nm)
Flavins
520 to 560
380 to 490 NADH
and NADPH 440 to 470

360 to 390 Lipofuscins
430 to 670
360 to 490 Elastin
and collagen 470 to 520

440 to 480 Lignin
530
488
Chlorophyll 685 (740)

488 From Biophotonics International
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Special issues photobleaching
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Photobleaching

• Photochemical lifetime fluorescein will
• undergo 30-40,000 emissions before bleaching.
  (QYbleaching 3x10-5)
• At low excitation intensities, pb occurs but at
  lower rate.
• Bleaching is often photodynamic--involves light
  and oxygen.

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Photochemistry often begins from the long-lived
triplet state
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1D photon 1D 3D
isc
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• 1D photon 1D 3D
• isc
• (i) 3D O oxidized dye

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• 1D photon 1D 3D
• isc
• (i) 3D O oxidized dye
• (ii) 3D 3O2 1O2 1D

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• 1D photon 1D 3D
• isc
• (i) 3D O oxidized dye
• (ii) 3D 3O2 1O2 1D
• 1D photobleached dye
• 1O2
• other substrates ox. substrate

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Singlet oxygen has a lifetime of 1?s and a
diffusion coefficient 10 E-5 cm2/s. Therefore,
potential photodamage radius from fluor is 50nm.
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Reducing Photobleaching (live cells)

• Deoxygenate Oxyrase (Ashland, OH)--bacterial
• membrane fragments that reduce oxygen to water
• if glucose present
• OR
• Catalase glucose glucose-oxidase to use mol
• oxygen

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Reducing photobleaching (fixed cellsanti-fades)

• Increase viscosity of medium (e.g. 95 glycerol)
• Add singlet oxygen quenchers and free radical
  traps (e.g. histidine, water soluble
• caratenoids)
• Exotic build triplet state quenchers into flour

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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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Summary Fluorescence Imaging
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Parameters for Maximizing Sensitivity

• Use High Objective NA and Lowest Magnification
• Ifl IilNAobj4/Mtot2
• -Buy the newest objective select for best
  efficiency
• Close Field Diaphragm down as far as possible
• Use high efficiency filters
• Use as few optical components as possible
• Reduce Photobleaching
• Use High Quantum Efficiency Detector in Camera

Adapted from E.D.Salmon
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Live Cell Considerations

• Minimize photobleaching and photodamage
  (shutters)
• Use heat reflection filters for live cell imaging
• Image quality Maximize sensitivity and signal to
  noise (high transmission efficiency optics and
  high quantum efficiency detector)
• Phase Contrast is Convenient to Use with
  Epi-Fluorescence
• Use shutters to switch between fluorescence and
  phase
• Phase ring absorbs 15 of emission and slightly
  reduces resolution by enlarging the PSF

Adapted from E.D. Salmon
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Total internal fluorescence TIRF microscopy
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EPI
TIRF
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Refraction
Snells Law n1sin(?1)n2sin(?2)
n2
q2
n1
q1
when n1 gt n2 (dense to less dense), light is bent
away from the normal upon entering the less dense
medium i.e. q2 gt q1
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Total internal reflection and the critical angle
n1 sin(qc) n2 sin(90?)
n2
qr 90?
n1
qc
The critical angle for the glass-water interface
? 67.5?
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Evanescent Wave
Z
n2
n1
qc
Intensity in Z direction I(z)Ie-z/d d lt
l30-300nm
qi
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Early design circa 1980 by Dan Axelrod
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TIRF excitation using the objective
Iino and Kusumi, J. Fluorescence (2001)
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Iino and Kusumi, J. Fluorescence (2001)
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EPI
TIRF
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FRAP fluorescence recovery after photobleaching

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FRAP of EGFP-FAK in a focal adhesion
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FLIP--fluorescence loss in photobleaching
Repeated photobleaching of a GFP-ER membrane
protein causes Loss of fluorescence from entire
ER--gtmembrane continuity
Lippincott-Schwartz et al, Nat. Cell Biol. 2001
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Set magnification so that the PSF corresponds to
2-3 pixels on camera
Example MMax 3Pixel Size of Detector/Optical
Res. pixel size 7 mm NA 1.4 ? 520
nm measure of PSF dimension 0.61 ?/NA
MMax 37 mm/0.6 520nm/1.4 91X