Fluorescence and filters
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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 gfp
Fluorescent proteins: GFP

Tsien Lab (UCSD)


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


Ph sensitivity bcecf
pH Sensitivity: BCECF Environmental sensitivity – Fura-2


Ca sensitive fluorescent proteins cameleons
Ca Environmental sensitivity – Fura-2++-sensitive fluorescent proteins: Cameleons

Probes.invitrogen.com


How do we observe fluorescence
How do we observe fluorescence Environmental sensitivity – Fura-2

  • Black light

    • Not enough sensitivity

  • Filters

    • Bleed-through

  • Darkfield fluorescence microscopy


Epifluorescence microscopy
Epifluorescence microscopy Environmental sensitivity – Fura-2

Nikon MicroscopyU


Essence of epifluorescence microscope dichroic mirror
Essence of epifluorescence microscope: Dichroic mirror Environmental sensitivity – Fura-2

www.microscopyu.com


Dichroic filters
Dichroic Filters Environmental sensitivity – Fura-2

  • Fabry-Perot Interferometers:

    • Constructive interference leads to transmission

Molecular Expressions Web Site: Interference filters


Multiple wavelength interference filters
Multiple-wavelength interference filters Environmental sensitivity – Fura-2

Molecular Expressions Web Site: Interference filters


Excitation filters
Excitation filters Environmental sensitivity – Fura-2

  • Note that Dichromatic mirror has bandpass at short wavelengths

Molecular Expressions Web Site: Interference filters


Emission filters
Emission filters Environmental sensitivity – Fura-2

  • 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 Environmental sensitivity – Fura-2

www.microscopyu.com

Advantages of mercury: intense illumination at specific wavelengths

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


Xenon arcs
Xenon arcs Environmental sensitivity – Fura-2

  • More uniform power across visible spectrum

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


Dangers of arc lamps
Dangers of arc lamps Environmental sensitivity – Fura-2

  • 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 Environmental sensitivity – Fura-2

  • Fixation

    • Formaldehyde

    • Glutaraldehyde (red autofluorescence)

    • Coagulation by ice cold methanol plunge

  • Permeabilization


Chemically specific staining
Chemically specific staining Environmental sensitivity – Fura-2

  • 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 Environmental sensitivity – Fura-2

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

Death cap mushroom


Indirect immunofluorescence
Indirect immunofluorescence Environmental sensitivity – Fura-2

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 Environmental sensitivity – Fura-2

  • 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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