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Fluorescence and filters

Fluorescence and filters

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Fluorescence and filters

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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