1. Today’s Topic (02/02/15) How did 1st week of labs go?
Marco covered absorption and emission of organic fluorophores last week, and this week will be doing ensemble FRET and polarization experiment.
Jaya: Brightfield & Fluorescence microscopy.
Today: FRET (Lab 2)

2. How do you measure how far apart donor is from acceptor?
Example of FRET
Fluorescein
Rhodamine
Fluorescein: Donor
Rhodamine Acceptor
How do you measure how far apart donor is from acceptor?

3. Fluorescence Resonance Energy Transfer (FRET)
Spectroscopic Ruler for measuring nm-scale distances, binding
R (Å) E
Ro  50 Å
Energy
Transfer
Donor
Acceptor
Dipole-dipole Distant-dependent
Energy transfer
Time
Time
Look at relative amounts
of green & red

4. How to measure by donor quenching
Energy Transfer = 1 – IDA/ID
Energy Transfer = 1 – tDA/tD
Latter is concentration independent, which is important if you’re measuring in a sample where it’s not easy to determine concentration, e.g. inside of a cell.
Why does lifetime of donor get shorter in the presence of an acceptor?

5. Fluorophores & Quantum Yield q.y. = # photons out/photons in.
Have ≥ 1 electron that is free to move. Excitation light moves e’s around, i.e. a dipole, and it can re-radiate, often with polarization.
Good dyes: QY ≈ 1;
Absorption ≈ 100,000 cm-1M-1
( A = ebc)
Thermal
relaxation
Fluorescence (krad )
& Non-radiative (kn.r.)
Absorption
Thermal relaxation
[Femtosec]
[Picosec]
Energy
k = krad + kn.r.
t = 1/k = trad + tn.r.
QY = krad/(krad + kn.r)

6. Energy Transfer = function (kET, knd)
FRET : competition between donor deactivating by internal processes and by acceptor being nearby
Energy
Transfer
Donor
Acceptor
Energy Transfer = function (kET, knd)
E.T. = kET/(kET + knd)
E.T. = 1/(1 + knd/kET)
How is kET dependent on R?
Kn.d.
(Surprisingly,) it depends on R-6.
kET
E.T. = 1/(1 + knd/kET) = 1/(1 + knd/aR-6) = 1/(1 + R6 knd/a)
E.T. = 1/(1 + (R6/Ro6)) = 1/(1 + (R/Ro)6) where Ro6 = E.T.-independent constants