1. Fluorescence: get beautiful pictures

2. Lab 1: Bright Field and Fluorescence Optical Microscopy and Sectioning
Ensemble Fluorescence (Location - Loomis Selvin Lab; Instructor(s) - Marco Tjioe and/or another Selvin student)
Part 1: Dye absorption, emission, lifetime, anisotropy
Part 2: Bulk FRET, donor-acceptance donor
Bright Field & Fluorescence Microscopy (Location – IGB; Instructor - Jaya Yodh)
Part 1: Brightfield, Kohler illumination DIC, Phase Contrast, Color, Fluorescence Microscopy
Part 2: Widefield fluorescence 3D stack and deconvolution ✔

3. Basic Set-up, Anisotropy (Polarization), FRET
Physics 598BP
Today: Fluorescence
What is it? Why is it good?
Basic Set-up, Anisotropy (Polarization), FRET
We spent about an hour on signal-to-noise in a dark-field experiment (for example, fluorescence), vs. a light field experiment.
The bottom line is when you have a large background, you canNOT simply subtract it off to see a small signal. Just like you cannot see the stars during the day (where there is a lot of sunlight). That is because, with photons (and many other source), the noise of the background is proportional to the square root of the brightness. So, if you have Nbg photons of background, you will have Nbg1/2 of noise. As long as this is greater than Nsignal you can’t see the signal. Hence, with the stars, the amount of noise in the background is very large because sun is shining during the day, but is absent from the night.
This becomes very clear with fluorescence. Fluorescenceis a dark field experiment, so with background so low, you can see very little signal, which means very low fluorescence. With a high numerical aperture objective (so you can collect all the light), you can even see single molecules! By absorption, which is a light-background experiment (i.e. with no sample you have a bright light, and then you are trying to measure the particles because they cause a decrease in the signal), it is very difficult to see a little amount. So we saw that by diluting a concentrated solution, 10,000 fold, we could still see it by fluorescence although the absorption was so small that we couldn’t see it by absorption.

4. Photobleaching Important: Dye emits 105 107 photons, then dies!
What is fluorescence?
Shine light in, gets absorbed, reemits at longer wavelength
Stokes Shift ( nm)
Excitation
Spectra
Emission
Light In
Light Out
Thermal
relaxation
Fluorescence
& Non-radiative
Absorption
Thermal relaxation
[Femtosec]
[Picosec]
[Nanosec]
Energy
Time (nsec)
Fluorescence
-t/tf
Y = e
Photobleaching Important: Dye emits 105 107 photons, then dies!

5. Why fluorescence? Why so good?
Super-sensitive: see single fluorescent dye!
Why?
Answer: It’s a dark-field technique– shine light and w/o fluorophore being there, (ideally) see nothing. [Recall, that the noise associate with taking a measurement is proportional to how much there is: want to weigh something—it’s hard to do really precisely if object weighs a lot; easier to do if you’re weighing a light object. A bright-field technique has a lot of noise.
2. Lots of different labels for different objects.
Get specificity and see many different objects on same sample.
3. Problem w fluorescence:
must label with a fluorophore
– sometimes, this is difficult/impossible.

6. Fluorescence Microscopy
What is it?
How does it compare in sensitivity to brightfield?
Stokes Shift ( nm)
Excitation
Spectra
Emission
Use dyes which absorb at one wavelength and emit at their own wavelength.
It’s MUCH more sensitive
--in fact, can see down to a single molecule!
Background is potentially ZERO! (It’s a dark-field technique)
But signal is reasonably strong
(ideally, get out one photon for every photon that you put in)
With little background, can see very little, i.e. tremendously sensitive

7. 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)

8. Fluorescence Polarization Dyes have an orientation (will absorb & emit in particular directions) Important for binding assays, FRET assays.
1. Dyes have a transition absorption dipole moment
If light is polarized in direction of dipole, , will absorb light; if polarized perpendicular to light, it won’t absorb it.
Signal proportional to sinQa, Qa is angle between light vector and dipole moment vector.
2. Once molecule is excited, then has probability of emitting, via an emission dipole moment, (which tends to be aligned with the absorption dipole moment).
The probability that it will make it through the analyzer is sinQe, Qe is angle between emission dipole vector and analyzer.
Coordinate system
Excited fluorophores

9. Polarization Can measure the average polarization (easiest),
Or the time-dependent (nanosecond lifetime) polarization (most informative)

10. How to measure FP Anisotropy Polarization (0 to 0.5) (0 to 0.4)
perpendicular ( )
Anisotropy
Polarization
(0 to 0.5)
(0 to 0.4)
parallel ( )
Polarization & Anisotropy: Just slightly different forms
Generally use Anisotropy because of simpler forms when time-dependent.
Also simpler when have multiple components:
A = SciAi where ci is the mole fraction of the ith component.

11. Polarization vs. Anisotropy
Denominators with P, A
Anisotropy
Polarization
The denominator of Anisotropy is simply the total light that would be observed if no polarizers were used. (Come in along x-axis.)
Call Iz = I||
Call Iy = I
Call Ix = I
Ix+ Iy + Iz = 2 I + I||
P defined by analogy with dichroism ratio
Non-polarized light (along x-axis)
Break into Iz, Iy.
Anisotropy is a more useful form for experimental data on complex systems
Generally use Anisotropy because of simpler forms when time-dependent.
Also simpler when have multiple components:
A = SciAi where ci is the mole fraction of the ith component.

12. FP set-up in a microscope
Anisotropy
Perrin equation (Perrin, 1926): A0/A=1+6Dt, including the rotational diffusion coefficient (D), fluorescence lifetime (t) and, more significantly, the fundamental anisotropy (A0) which varies according to wavelength (Lakowicz, 1999; Weber & Shinitzky, 1970).

13. FP applied to binding

14. Competition monitored via FP Homogeneous Assays, no labeling competitors

15. Competition monitored by FP

16. FRET: measuring conformational changes of (single) biomolecules
FRET depends sharly on distance, R-6; 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.

17. FRET is so useful because Ro (2-8 nm) is often ideal
Bigger Ro (>8 nm)
can use FIONA, PAM. STORM -type techniques

18. 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

19. 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

20. Energy Transfer goes like…or ?
Take limit…

21. The End