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Fundamentals of Fluorescence Microscopy E. D. Salmon University of North Carolina at Chapel Hill References: Murphy Book;

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Presentation on theme: "Fundamentals of Fluorescence Microscopy E. D. Salmon University of North Carolina at Chapel Hill References: Murphy Book;"— Presentation transcript:

1 Fundamentals of Fluorescence Microscopy E. D. Salmon University of North Carolina at Chapel Hill References: Murphy Book; Fluorescence; and

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 sec Emission occurs in – sec Usually broad excitation spectrum w peak Usually broad emission spectrum w peak Stokes shift is separation of Ex. & Em peaks I em = I ex  cl  Photobleaching: Rate depends on I ex,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.  (nm) Quantum Yield Acridine Orange Ethanol Benzene Ethanol Eosin Water Fluorescein Water Rhodamine-B Ethanol Chlorophyl-A Ethanol

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): Luminous Flux (lumens): Light Intensity (Candella): Avg. Brightness (cd/cm): Arc Size (w x h in millimeters): 0.50 x x x x x 1.35 Life (Hours):

17 Quartz-Halogen (Tungsten Filament) Lamp Spectra Current

18 Lasers Have Line Spectra

19 Ploem-Type Epi-Illuminator

20 Epi- Fluorescence Microscope

21

22 Arc Lamp Housing

23 Lamp Alignment

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

25 Filter Cubes

26 Filter Cubes Are Not Inter-Changeable Between Different Manufactures

27 Basic Design Features

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

29 Problems in Filter Design: Example Absorption and Emission Spectra

30 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”

31 Multi-Wavelength Immunofluorescence Microscopy

32 Fluorophores for Triple-Label

33 Multiple Band-Pass Filters

34

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

36 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) Handbook of Optical Filters for Fluorescence Microscopy: Download a copy of our "Handbook of Optical Filters for Fluorescence Microscopy" in Adobe Acrobat PDF format. Chroma "Handbook of Optical Filters for Fluorescence Microscopy"

37 Multi-Wavelength Immunofluorescence Microscopy

38 Multi- Wavelength Fluorescence Imaging

39 Multi-wavelength Fluorescence Imaging

40 Multi- Wavelength Fluorescence Imaging

41 Ploem-Type Epi-Illuminator

42 Parameters for Maximizing Sensitivity Use High Objective NA and Lowest Magnification: I fl ~ I il NA obj 4 /M tot 2 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: M Max = 3*Pixel Size of Detector/Optical Resolution E.g.: 3*7  m/[0.6 *520nm/1.4] = 91X Reduce Photobleaching Use High Quantum Efficiency Detector in Camera

43 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

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: 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: 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: 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

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

46 Green: GFP-Cdc20 At Kinetochores Red: Phase Contrast Images of PtK1 Tissue Cells Cdc20 Persists At Kinetochores Throughout Mitosis and Exhibits Fast Kinetics: FRAP t1/2 = [4 sec (attached) 25 sec (unattached]


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