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Fluorescence Microscopy

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Employ simple, non plan lenses to minimize. internal elements. ... Use High Quantum Efficiency Detector in Camera. Adapted from E.D.Salmon. Live Cell Considerations ... – PowerPoint PPT presentation

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Title: Fluorescence Microscopy


1
Fluorescence Microscopy
  • Ken Jacobson

2
What we will cover
  • What is fluorescence?
  • Fluorescence microscopy light sources,
  • filters, objectives
  • Special considerations autofluorescence
    photobleaching
  • TIRF
  • Class Exercises

3
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
4
Fluorescence fundamentals
  • Fluorescence prop. to Light Absorbedxquantum
    yield
  • F I ? c x Q
  • Q photons emitted/photons absorbedlt1

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Acridine orange
9
Rhodamine 123-potential based stain
10
NBD ceramide
11
Rh-phalloidin anti-integrin
12
Rh-anti tubulin DAPI
13
GFP Structure fluorphore formed by
cyclization of Ser65, Tyr66 and Gly 67
M. Ormo et al, Sci. 2731392, 1996
14
Patterson, G. et al. J Cell Sci 2001114837-838
15
The microscope as a filter fluorometer
with focusing optics
16
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
19
Arc lamps
CAUTION lamps at hi pressure do not touch glass
envelopes
20
TECHNICAL DATA OF THE LIGHT SOURCES FOR
INCIDENT-LIGHT FLUORESCENCE MICROSCOPY
From C. Zeiss
Note small arcs with high luminous density will
be brightest
21
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.
24
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

25
Maximizing image brightness
(B)excitation efficiency (NA)2

gt B (NA)4collection efficiency
(NA)2
at high NA,
26
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
31
Cut off filters
32
Bandpass Filters
Most bandpass filters are interference type with
multilayer dielectric coatings that pass or
reject certain wavelengths with great selectivity
33
Interference filter definitions
34
Figure 5a shows a band pass emitter filter
35
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.

36
Multiple Band-Pass Filters
From E.D. Salmon
37
Multi-Wavelength Immunofluorescence Microscopy
38
Special issues autofluorescencewhich causes
unwanted background obscuring weak signals
39
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
40
Special issues photobleaching
41
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.

42
Photochemistry often begins from the long-lived
triplet state
43
1D photon 1D 3D
isc
44
  • 1D photon 1D 3D
  • isc
  • (i) 3D O oxidized dye

45
  • 1D photon 1D 3D
  • isc
  • (i) 3D O oxidized dye
  • (ii) 3D 3O2 1O2 1D

46
  • 1D photon 1D 3D
  • isc
  • (i) 3D O oxidized dye
  • (ii) 3D 3O2 1O2 1D
  • 1D photobleached dye
  • 1O2
  • other substrates ox. substrate

47
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.
48
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

49
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

50
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

51
Summary Fluorescence Imaging
52
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
53
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
54
Total internal fluorescence TIRF microscopy
55
EPI
TIRF
56
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
57
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?
58
Evanescent Wave
Z
n2
n1
qc
Intensity in Z direction I(z)Ie-z/d d lt
l30-300nm
qi
59
Early design circa 1980 by Dan Axelrod
60
TIRF excitation using the objective
Iino and Kusumi, J. Fluorescence (2001)
61
Iino and Kusumi, J. Fluorescence (2001)
62
EPI
TIRF
63
FRAP fluorescence recovery after photobleaching

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FRAP of EGFP-FAK in a focal adhesion
67
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
68
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
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