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Understanding Fluorescence and Filters in Microscopy

Learn the phenomenon of fluorescence, Jablonski diagram, and fluorescence probes for cell imaging. Discover ideal fluorophore characteristics, fluorescent proteins, and how to observe fluorescence using filters, microscopy techniques, and specimen preparation. Explore multiple-wavelength interference filters and light sources in microscopy.

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Understanding Fluorescence and Filters in Microscopy

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  1. Fluorescence and filters

  2. Phenomenon of fluorescence • Jablonski diagram: • Absorption of photon elevates fluorophore to excited singlet state S1’ • Nonradiative decay to lowest energy singlet excited state S1 • 3. Decay to ground state by emission of a photon Probes.invitrogen.com

  3. Everyday examples of fluorescence • Whiteners in fabrics • “Blue hair” "Hey grandma, I dig your blue hair!"

  4. Non-radiative decay to triplet state leads to photbleaching Molecular Expressions website

  5. Ideal fluorophore characteristics • High quantum efficiency • Slow photobleaching • For live cells: excitation wavelengths non-phototoxic to cessl • Little overlap with autofluorescence • Mammalian cells: Flavoprotein, pigment • Plant cells: chlorophyll

  6. A few dyes Probes.invitrogen.com

  7. Fluorescent proteins: GFP Tsien Lab (UCSD)

  8. Fluorescent proteins data table • See http://home.earthlink.net/~pubspectra/

  9. Other useful characteristics of fluorophores – Environmental sensitivity – Fura-2

  10. pH Sensitivity: BCECF

  11. Ca++-sensitive fluorescent proteins: Cameleons Probes.invitrogen.com

  12. How do we observe fluorescence • Black light • Not enough sensitivity • Filters • Bleed-through • Darkfield fluorescence microscopy

  13. Epifluorescence microscopy Nikon MicroscopyU

  14. Essence of epifluorescence microscope: Dichroic mirror www.microscopyu.com

  15. Dichroic Filters • Fabry-Perot Interferometers: • Constructive interference leads to transmission Molecular Expressions Web Site: Interference filters

  16. Multiple-wavelength interference filters Molecular Expressions Web Site: Interference filters

  17. Excitation filters • Note that Dichromatic mirror has bandpass at short wavelengths Molecular Expressions Web Site: Interference filters

  18. Emission filters • May be either broadband (colored glass) or bandpass • Broadband gives more light • Bandpass filters out more autofluorescence, other fluorophores

  19. Light souces: arc lamps www.microscopyu.com Advantages of mercury: intense illumination at specific wavelengths http://www.olympusmicro.com/primer/anatomy/sources.html

  20. Xenon arcs • More uniform power across visible spectrum http://www.olympusmicro.com/primer/anatomy/sources.html

  21. Dangers of arc lamps • Warm-up period of a new arc lamp • Explosion • Toxic Hg vapors • Solarization of filters • Bleaching of filters • Retinal phototoxicity!!!!

  22. Specimen preparation for fluorescence • Fixation • Formaldehyde • Glutaraldehyde (red autofluorescence) • Coagulation by ice cold methanol plunge • Permeabilization

  23. Chemically specific staining • Histochemistry – stain is a fluorophore • H&E – not specific, not fluorescent • Nissl stain – Nucleic acid spefic, but not fluorescent • DAPI • Chemically labelled toxins • Fluorescent α bungarotoxin • Rhodamine phalloidin

  24. Amanita phalloides Image of Amanita phalloides from Abbé Giacomo Bresadola (1927 - 1960) Iconographia mycologica Death cap mushroom

  25. Indirect immunofluorescence Bradwell A R, Hughes R G, Harden E L (2003)Atlas of HEp-2 patterns, 2nd editionPrinters KNP Group Ltd, Redditch, UKISBN: 0704424371

  26. Our plan • DAPI to detect chromatin • Rhodamine phalloidin to detect f-actin • Monoclonal anti-tubulin (DM1a) to bind to microtubules • Wash • FITC goat-anti-mouse to detect DM1a • Widefield epifluorescence microscopy

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