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)

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?

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

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?

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)

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