Fluorescence
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Fluorescence. Energy diagram. Energy diagram. Quantum Yield - Q. Rate of depopulation. Red shift. Fluorimeter. Time resolved experiment . Spectrofluorimeter. Frequency domain (phase/modulation) . Excitation/ emmission spectrum :. Fluorophores. Intrinsic fluorophores. Extrinsic probes.

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Fluorescence

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Fluorescence

Fluorescence

Biophysical Tools '04 - Fluorescence


Energy diagram

Energy diagram

Energy diagram

Quantum Yield - Q

Rate of depopulation

Red shift

Biophysical Tools '04 - Fluorescence


Fluorimeter

Fluorimeter

Time resolved experiment

Spectrofluorimeter

Frequency domain (phase/modulation)

Excitation/emmission spectrum:

Biophysical Tools '04 - Fluorescence


Fluorophores

Fluorophores

Intrinsic fluorophores

Extrinsic probes

Solvent Effects

Biophysical Tools '04 - Fluorescence


Anisotropy

I┴

Anisotropy

Time resolved anisotropy

Anisotropy

Motional depolarization

Biophysical Tools '04 - Fluorescence


Phosphorescence

Phosphorescence

Phosphorescence

Øtriplet probes: eosin, erythrosin

S

T

Biophysical Tools '04 - Fluorescence


Anisotropic motion

Anisotropic motion

Hindered rotation

Perrin Plot

A(t)

A∞

Biophysical Tools '04 - Fluorescence


Quenching

Quenching

the rate of quenching = k[Q]

Stern-Volmer plots

2

Fluor (no Q) / fluor (Q=x)

1

Quencher conc.

Biophysical Tools '04 - Fluorescence


Fluorescence

5

donor

4

donor/aceptor

donor (AEDANS)

3

Intensity

2

acceptor

1

acceptor (AF)

0

500

700

400

600

Wavelength (nm)

FRET

Efficiency of transfer:

E = rate of transfer /total rate of relaxation =

E = kFET/(k + kFET ) = 1-tAD/tD = 1- FAD/FD

dipole-dipole interactions

kFET  (R-3)2 ~ 1/R6

Ro distance @ E=0.5 i.e. when kFET = 1/t then:

E = R-6/(Ro-6 + R-6)

Orientation problem: k2

Biophysical Tools '04 - Fluorescence


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