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Fluorescence and filters. Phenomenon of fluorescence. Jablonski diagram: Absorption of photon elevates fluorophore to excited singlet state S 1 ’ Nonradiative decay to lowest energy singlet excited state S 1 3. Decay to ground state by emission of a photon. Probes.invitrogen.com.

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phenomenon of fluorescence
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

everyday examples of fluorescence
Everyday examples of fluorescence
  • Whiteners in fabrics
  • “Blue hair”

"Hey grandma, I dig your blue hair!"

ideal fluorophore characteristics
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
a few dyes
A few dyes

Probes.invitrogen.com

fluorescent proteins data table
Fluorescent proteins data table
  • See http://home.earthlink.net/~pubspectra/
other useful characteristics of fluorophores environmental sensitivity fura 2
Other useful characteristics of fluorophores – Environmental sensitivity – Fura-2
how do we observe fluorescence
How do we observe fluorescence
  • Black light
    • Not enough sensitivity
  • Filters
    • Bleed-through
  • Darkfield fluorescence microscopy
dichroic filters
Dichroic Filters
  • Fabry-Perot Interferometers:
    • Constructive interference leads to transmission

Molecular Expressions Web Site: Interference filters

multiple wavelength interference filters
Multiple-wavelength interference filters

Molecular Expressions Web Site: Interference filters

excitation filters
Excitation filters
  • Note that Dichromatic mirror has bandpass at short wavelengths

Molecular Expressions Web Site: Interference filters

emission filters
Emission filters
  • May be either broadband (colored glass) or bandpass
    • Broadband gives more light
    • Bandpass filters out more autofluorescence, other fluorophores
light souces arc lamps
Light souces: arc lamps

www.microscopyu.com

Advantages of mercury: intense illumination at specific wavelengths

http://www.olympusmicro.com/primer/anatomy/sources.html

xenon arcs
Xenon arcs
  • More uniform power across visible spectrum

http://www.olympusmicro.com/primer/anatomy/sources.html

dangers of arc lamps
Dangers of arc lamps
  • Warm-up period of a new arc lamp
  • Explosion
    • Toxic Hg vapors
  • Solarization of filters
  • Bleaching of filters
  • Retinal phototoxicity!!!!
specimen preparation for fluorescence
Specimen preparation for fluorescence
  • Fixation
    • Formaldehyde
    • Glutaraldehyde (red autofluorescence)
    • Coagulation by ice cold methanol plunge
  • Permeabilization
chemically specific staining
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
amanita phalloides
Amanita phalloides

Image of Amanita phalloides from Abbé Giacomo Bresadola (1927 - 1960) Iconographia mycologica

Death cap mushroom

indirect immunofluorescence
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

our plan
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