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Fundamentals of Fluorescence Microscopy

Fundamentals of Fluorescence Microscopy. E. D. Salmon University of North Carolina at Chapel Hill References: Murphy Book; http://micro.magnet.fsu.edu/primer/techniques/ Fluorescence; and www.chroma.com. Basic Concept of Absorption and Emission.

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Fundamentals of Fluorescence Microscopy

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  1. Fundamentals of Fluorescence Microscopy E. D. Salmon University of North Carolina at Chapel Hill References: Murphy Book; http://micro.magnet.fsu.edu/primer/techniques/ Fluorescence; and www.chroma.com

  2. Basic Concept of Absorption and Emission

  3. Common Fluorophores Have Complex Electronic Structures

  4. Excitation and Emission Spectra

  5. Jablonski Diagram

  6. 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

  7. 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

  8. 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

  9. 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

  10. There are Different Fluorescent Molecules for Different Jobs See Molecular Probes Catalog; Sigma Catalog; CloneTech for GFP

  11. Green Fluorescent Protein (GFP) CloneTech

  12. Multi-Wavelength Fluorescence Imaging

  13. Basic Concept of Epi-Fluorescence Microscopy

  14. Hg-Arc Lamp

  15. Xenon Arc-Lamp Spectra

  16. 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

  17. Quartz-Halogen (Tungsten Filament) Lamp Spectra Current

  18. Lasers Have Line Spectra

  19. Ploem-Type Epi-Illuminator

  20. Epi-Fluorescence Microscope

  21. Arc Lamp Housing

  22. Lamp Alignment

  23. Alignment of Arc and Mirror Images at Objective Back Focal Plane (Use Centering-Screen or white Card on Stage W/O Objective)

  24. Filter Cubes

  25. Filter Cubes Are Not Inter-Changeable Between Different Manufactures

  26. Basic Design Features

  27. Exciter and Barrier Filters are Designed to Separate Emission Light from Excitation Light

  28. Problems in Filter Design: Example Absorption and Emission Spectra

  29. 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”

  30. Multi-Wavelength Immunofluorescence Microscopy

  31. Fluorophores for Triple-Label

  32. Multiple Band-Pass Filters

  33. Multiple Band-Pass Filters

  34. Choose Wide-Band Emission Filters for Single Fluorophore to Maximize Sensitivity

  35. Chroma Technology Corp. is an employee- owned company that produces the world's finest optical filters and filter sets. The company specializes in the design and manufacture of optical filters and coatings for applications which require the greatest precision in color separation, optical quality and signal purity. For more about us, see our About Chroma page.Welcome to our new website! This site is under construction, so if you don't find what you need please give us a call at (800) 824-7662.Handbook of Optical Filters for Fluorescence Microscopy:Download a copy of our "Handbook of Optical Filters for Fluorescence Microscopy"in Adobe Acrobat PDF format.www.chroma.com

  36. Multi-Wavelength Immunofluorescence Microscopy

  37. Multi-Wavelength Fluorescence Imaging

  38. Multi-wavelength Fluorescence Imaging

  39. Multi-Wavelength Fluorescence Imaging

  40. Ploem-Type Epi-Illuminator

  41. 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 = 3*Pixel Size of Detector/Optical Resolution E.g.: 3*7 mm/[0.6 *520nm/1.4] = 91X • Reduce Photobleaching • Use High Quantum Efficiency Detector in Camera

  42. 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

  43. 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

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

  45. 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

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