1. Today’s take-home lessons: FRET (i. e
Today’s take-home lessons: FRET (i.e. what you should be able to answer at end of lecture)
✓How to measure: Donor Intensity, Lifetime
[More Today] Acceptor Intensity (very good way but more difficult)
✓Why is E.T. a to (R/Ro)-6
[it depends on the near-field component of the electric field, which goes like R-3 with some normalization constant Ro-3
[Today:] Why it depends on orientation of donors and acceptors?

2. Why does E.T. go like (R/Ro)-6

3. FRET: measuring conformational changes of biomolecules
FRET useful for 20-80Å
Distance dependent interactions between green and red light bulbs can be used to deduce the shape of the scissors during the function.

4. 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

5. Example of FRET (on DNA)
E.T. by decreases in donor emission.
Need to compare two samples,
d-only, and D-A.
Where are the donors intensity, and excited state lifetime in the presence of acceptor;
without the acceptor.
Donor
quenching
Acceptor
“sensitized”
emission
The more Energy Transfer (donor and acceptor closer together)…
more donor decreases, more the acceptor increases.

6. Example of FRET (on DNA) Measured w Acceptor’s Sensitized Emission
E.T. by increase in acceptor fluorescence and compare it to residual donor emission.
Need to compare one sample at two l and also measure their quantum yields.
Ideal case, where direct acceptor’s emission = 0
(Next slide, subtract it off)
Donor
quenching
Acceptor
“sensitized”
emission
Energy Transfer (E) is an indication of excitations, not emission. Must divide by quantum yields.
How do you measure quantum yields?
Compare to known lifetime (e.g. SR101 = 1)

7. Example of FRET (on DNA) Measured w Acceptor’s Sensitized Emission
Donor
quenching
Acceptor
“sensitized”
emission
Real case, where direct acceptor’s emission ≠ 0.
How to subtract it off?
Take three samples, donor-only, acceptor only, donor-acceptor.
How to determine E.T.?
Problems with this?
Looking at Intensity?
You have to have right concentrations

8. Example of FRET (on DNA) Measured w Acceptor’s Sensitized Emission
Real case, where direct acceptor’s emission ≠ 0.
How to subtract it off?
Donor
quenching
Acceptor
“sensitized”
emission
Use two lasers, one to induce FRET, the other where only the acceptor excites.
(e.g. 488 nm for Fl: 532 nm for TMR: see next slide)
Then take an acceptor-only sample, excite at the two wavelengths and multiply by the ratio of the acceptors absorption at these two wavelengths.
(You do not need a particular concentration here. You just need to know the relative absorptions at 2 wavelengths)