Lecture 4: How to Measure FRET

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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)
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
1.0
E 
0.8
Energy
Transfer
Donor
Acceptor
E
0.6
1
1  ( R / R0 )
6
0.4
Ro  50 Å
0.2
0.0
0
25
50
75
100
R (Å)
D
A
Time
Dipole-dipole Distant-dependent
Energy transfer
Look at relative amounts
of green & red
R0
D
D
A
A
Time
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 reradiate, often with
polarization.
Good dyes: QY ≈ 1;
Absorption ≈ 100,000 cm-1M-1
( A = ebc)
Energy
Thermal
relaxation
[Picosec]
Absorption
[Femtosec]
Fluorescence (krad )
& Non-radiative (kn.r.)
Thermal relaxation
[Picosec]
k = krad + kn.r.
t = 1/k = trad + tn.r.
QY = krad/(krad + kn.r)
FRET : competition between donor deactivating by
internal processes and by acceptor being nearby
E 
1
1  ( R / R0 )
Energy Transfer = function (kET, knd)
Energy
Transfer
Acceptor
Donor
E.T. = kET/(kET + knd)
E.T. = 1/(1 + knd/kET)
How is kET dependent on R?
Kn.d.
kET
(Surprisingly,) it depends on R-6.
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
6

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