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Fluorescence Microscopy

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  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.fsu.edu/primer/index.html Important reference on fluorescent probes: The Molecular Probes catalog

  4. Fluorescence fundamentals Fluorescence prop. to [ Light Absorbed]x[quantum yield] F = I  [c] x Q Q=[ #photons emitted/#photons absorbed]<1

  5. Acridine orange

  6. Rhodamine 123-potential based stain

  7. NBD ceramide:

  8. Rh-phalloidin & anti-integrin

  9. Rh-anti tubulin & DAPI

  10. GFP Structure: fluorphore formed by cyclization of Ser65, Tyr66 and Gly 67 M. Ormo et al, Sci. 273:1392, 1996

  11. Patterson, G. et al. J Cell Sci 2001;114:837-838

  12. The microscope as a filter fluorometer with focusing optics

  13. Basic design of the epi fluorescence microscope Objective acts as condenser; excitation light reflected away from eyes

  14. 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

  15. Common non-laser light sources

  16. Arc lamps CAUTION: lamps at hi pressure; do not touch glass envelopes

  17. 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

  18. 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).

  19. 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.

  20. 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]

  21. 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,

  22. Filters: the key to successfulfluorescence microscopy

  23. 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

  24. Filter cube must provide excitation, reflect the excitation onto sample while transmitting emission, and pass the fluorescence

  25. Cut off filters

  26. Bandpass Filters Most bandpass filters are interference type with multilayer dielectric coatings that pass or reject certain wavelengths with great selectivity

  27. Interference filter definitions

  28. Figure 5a shows a band pass emitter filter

  29. 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.

  30. Multiple Band-Pass Filters From E.D. Salmon

  31. Multi-Wavelength Immunofluorescence Microscopy

  32. Special issues: autofluorescencewhich causes unwanted background obscuring weak signals

  33. 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

  34. Special issues: photobleaching

  35. 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.

  36. Photochemistry often begins from the long-lived triplet state

  37. 1D + photon 1D*3D* isc

  38. 1D + photon 1D*3D* • isc • (i) 3D*+ [O] oxidized dye

  39. 1D + photon 1D*3D* • isc • (i) 3D*+ [O] oxidized dye • (ii) 3D*+3O21O2+1D

  40. 1D + photon 1D*3D* • isc • (i) 3D*+ [O] oxidized dye • (ii) 3D*+3O21O2+1D • +1D photobleached dye • 1O2 • + other substrates ox. substrate

  41. 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.

  42. 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

  43. 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

  44. 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