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Fluorescence Microscopy Ken Jacobson What we will cover What is fluorescence? Fluorescence microscopy: light sources, filters, objectives Special considerations: autofluorescence & photobleaching TIRF Class Exercises
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Fluorescence Microscopy Ken Jacobson
What we will cover • What is fluorescence? • Fluorescence microscopy: light sources, filters, objectives • Special considerations: autofluorescence & photobleaching • TIRF • Class Exercises
On line resource: Molecular Expressions, a Microscope Primer at: http://www.microscopy.fsu.edu/primer/index.html Important reference on fluorescent probes: The Molecular Probes catalog
Fluorescence fundamentals Fluorescence prop. to [ Light Absorbed]x[quantum yield] F = I [c] x Q Q=[ #photons emitted/#photons absorbed]<1
GFP Structure: fluorphore formed by cyclization of Ser65, Tyr66 and Gly 67 M. Ormo et al, Sci. 273:1392, 1996
The microscope as a filter fluorometer with focusing optics
Basic design of the epi fluorescence microscope Objective acts as condenser; excitation light reflected away from eyes
MICROSCOPE COMPONENTS Identify Major Components And Their Locations And Functions Within Modern Research Light Microscope (See Salmon And Canman, 2000, Current Protocols in Cell Biology, 4.1) Camera Binocular Camera Adapter Eyepiece Epi-Condenser Epi-Lamp Housing Diaphragm Epi-Field Diaphragm Mirror: & Centering Filters Shutter Focus and Beam Switch Centering Magnification Changer Filter Cube Changer Slot for Analyzer Body Tube Focus, Centering Slot for DIC Prism Objective Nosepiece Objective Stage Trans-Lamp Housing Condenser: Diaphragm&Turret Mirror: Centering Focus and Focus Centering Slot for Polarizer Field Diaphragm Upright Microscope Coarse/Fine Filters Lamp: Focus, Centering Stand Specimen Focus and Diffuser
Arc lamps CAUTION: lamps at hi pressure; do not touch glass envelopes
Type Wattage (W) Luminous density (cd/cm2) Arc size h x w (mm) Lifetime (h) High-pressure Mercury lamps HBO 50W/AC HBO 100W/2 50 100 30 000 170 000 1.0 x 0.3 0.25 x 0.25 100 200 High-pressure Xenon lamps XBO 75W/2 75 40 000 0.5 x 0.25 400 Tungsten-Halogen lamps 12V 100W 100 4500 4.2 x 2.3 50 TECHNICAL DATA OF THE LIGHT SOURCES FOR INCIDENT-LIGHT FLUORESCENCE MICROSCOPY From C. Zeiss Note: small arcs with high luminous density will be brightest
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).
Works because the depth of focus of the collector lens on the lamp housing is very long: what’s in focus at the back focal plane is ~ in focus at the specimen plane.
Objectives High transmittance Fluorite lenses: > 350 nm [ok for FURA] Quartz lenses: < 350 nm Employ simple, non plan lenses to minimize internal elements. Neglible autofluorescence or solarization [color change upon prolonged illumination]
1 (NA)4 also B ~ => B ~ , for NA ≤ 1.0 M2 M2 Maximizing image brightness (B)excitation efficiency ~ (NA)2=> B ~ (NA)4collection efficiency ~ (NA)2 at high NA,
MICROSCOPE COMPONENTS Identify Major Components And Their Locations And Functions Within Modern Research Light Microscope (See Salmon And Canman, 2000, Current Protocols in Cell Biology, 4.1) Camera Binocular Camera Adapter Eyepiece Epi-Condenser Epi-Lamp Housing Diaphragm Epi-Field Diaphragm Mirror: & Centering Filters Shutter Focus and Beam Switch Centering Magnification Changer Filter Cube Changer Slot for Analyzer Body Tube Focus, Centering Slot for DIC Prism Objective Nosepiece Objective Stage Trans-Lamp Housing Condenser: Diaphragm&Turret Mirror: Centering Focus and Focus Centering Slot for Polarizer Field Diaphragm Upright Microscope Coarse/Fine Filters Lamp: Focus, Centering Stand Specimen Focus and Diffuser
Filter cube must provide excitation, reflect the excitation onto sample while transmitting emission, and pass the fluorescence
Bandpass Filters Most bandpass filters are interference type with multilayer dielectric coatings that pass or reject certain wavelengths with great selectivity
Filter selection • Broadband filters: more excitation, less contrast [more autofluorescence may be excited]. • Narrowband filters:less signal, more contrast. • Note: eye responds to contrast while detectors respond to signal.
Multiple Band-Pass Filters From E.D. Salmon
Multi-Wavelength Immunofluorescence Microscopy
Special issues: autofluorescencewhich causes unwanted background obscuring weak signals
COMMON SOURCES OF AUTOFLUORESCENCE Autofluorescent SourceTypical 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
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.
Photochemistry often begins from the long-lived triplet state
1D + photon 1D*3D* • isc • (i) 3D*+ [O] oxidized dye
1D + photon 1D*3D* • isc • (i) 3D*+ [O] oxidized dye • (ii) 3D*+3O21O2+1D
1D + photon 1D*3D* • isc • (i) 3D*+ [O] oxidized dye • (ii) 3D*+3O21O2+1D • +1D photobleached dye • 1O2 • + other substrates ox. substrate
Singlet oxygen has a lifetime of ~ 1s and a diffusion coefficient ~ 10 E-5 cm2/s. Therefore, potential photodamage radius from fluor is ~ 50nm.
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
Reducing photobleaching (fixed cells:anti-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
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