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Bios 532
Fluorescence
Vocabulary
Fluorophore - molecule that emits fluorescence.
Excitation - absorption of a photon.
Emission - release of a photon.
Luminescence - the emission of
light from electronically excited
states of a molecule:
Fluorescence
Phosphorescence
Efficiency of fluorescence is measured in
terms of the quantum yield.
 = # of photons emitted
# of photons absorbed
S2
internal conversion
S1
hABS hABS
S0
hFLUOR
Quenching
Energy gets transferred to the quencher,
usually through collisions with a
nearby residue or molecule. This reduces
photon emissions and decreases
fluorescence intensity.
3 amino acids emit UV fluorescence:
Tryptophan
Tyrosine
Phenylalanine
Fluorescence Measurements
1. Increased sensitivity, 10-5 M abs vs. 10-8 M flour.
2. Rapid process for measuring kinetics, 10-9 sec.
3. Measure at 90º to incident beam.
4. Dirt, lint, air bubbles, all scatter light significantly.
5. Photobleaching of sample can be a problem.
6. Light passes through a prism or diffraction grating
to set excitation wavelength. The setting is chosen
according to Bragg’s Law, 2dhkl sin =n . The
problem is n - you get , 2, 3, etc., so filters and
are sometimes used.
Filters
1. Longpass - block shorter , pass longer 
2. Bandpass - allows only one region of spectrum
through, blocks both longer and shorter 
3. Interference - similar to bandpass, but with a more
narrow  range and better cutoffs on either side
4. Solution filters - used mostly in photochemistry
Lactose Repressor Protein
The E. coli lac Operon and lac Repressor
I
lacR regulatory
P
O
control
elements
Z
Y
structural genes
lac operon
A
Calculations and Write-up
1. Determination of Kd - we have a function in IGOR
2. When your results differ from published results, postulate
reasons this may be true.