Fluorescense 3 Flashcards
What is nonpolarized light
Polarized light
Nonpolarized is when the light is coming from all different directions
Polarized if when the light goes in one direction as a wave
When we see polarized light what do we actually see
The vector going just up and down
But we imagine the polarized light as a wave
What is depolarization
This is where the chromophore absorbs light only when the electric vector (the polarized light) is parallel to the transition moment of the chromophore
What is an isotopic and inosotropic solution
Isotropic is when the molecules are randomly oriented
Anisotropic is when they are uniform in one direction
If fluorophores have been excited by polarized light, when the emit what direction will the light be
Why
The emmited light will also be polarized and in the same plane as the polarized excitation light
This is because during the fluorescence the is no time to change the direction of the light
Over time after a fluorophore has absrobed polarized light , how will it emit
The molecules now have time to adopt diff orientations (now isotropic)
meaning now when they emit its isn’t polarized light that the emits
The light is emitted in multiple directions
What is an example of fluorescent polarization and depolarization
How does each sample affect the fluorophore
So you have two solutions
One with antibody analyte and fluorophore
One with just antibody and fluorophore
Polarized light goes into both solutions
In the sample with analyte, the analyte binds more to the antibody than the fluorophore so now there are more free fluorophores.
In the sample without analyte, more of the fluorophores are bound to the antibody
Because the more of the fluorophores are bound to the antibody, they rotate slowly in the solution (anisotropic) and their light remains polarized
Because they are more free in the other solution , they are able to rotate freely and the light emmited is depolarized
How is the fluorimeter with polarized light different than the regular fluorimeter
What does this change
there is the light polarizer in the path of the light
So now the light reaching the sample is vertically polarized
The light coming off the sample will be vertically and horizontally polarized because the molecules have had time to rotate
Then the polarizer at the detector to sample the polarized light before it reaches the detector
What is fluorescence anisotropy independent of
And normalized by
It’s independent of total fluorescence intensity (because it only measure the parallel and perpendicular intensity)
Also independent of the concentration of the fluorophore (if there’s no inner filter effect)
so it’s normalized by the total intensity (the denominator of the equation)
IDK
Slide 7 diagram
Idk
What are the two extremes for anisotropy
The sample is rigid or there’s no time to rotate (then in the equation there is no perp emmision)
This means that r= para/para = 1
There is enough time for random orientation (so amount in para = perp)
This means that the numerator is zero
R=0
What is the range of values for anisotropy
0-1
What is the other word for anisotropy used in old literature
Are the extremes for it the same
Polarization
Yes but they are different if under other conditions
If you have a hydrated spherical molecule in a solution, if it’s very viscous what does this do to the rational correlation time
The time is higher
Meaning the rotational time is slower, higher anisotropy (since they aren’t moving as much)
How do we do measurements of the anisotropy
We have a pulse of light that shines on the molecules
The pulse of light stops
And afterward we measure the decay (the decrease in anisotropy) overtime
What are the limitations for the measurement method of anisotropy
The pulses of light given to the sample have to be shorter than the decay time
The detection system (of the decay) has to measure very fast, on the ns timescale
If you use the final result of the Perrin equation (an equation in the form of y=mx +b)
What can you do to find the size of the molecule in the solution
Plot 1/r vs T/n
The y intercept is 1/r0
Slope is R tau f/r0 Vh
When calculating anisotropy what are we assuming
The molecule is spherical and hydrated
It is steady state anisotropy
Derivation of equation for finding size
In notes
What is the first step to doing an anisotropy experiment
What do we have to make sure
We covalently label protein with the fluorophore
Make sure that the lifetime of the flurofore it is the same as the rotational correlation time (time for fluor to occur is same as time it takes for molecule to rotate)
Make sure the fluorophore is tightly bound to the protein (so that the fluorophore isn’t dangling on the protien)
What is the second step of the anisotropy experiment
What are the limitations
We measure the anisotropy over a range T/N (temp over viscosity)
There’s a limited range of temp for the proteins:
If you go to a higher temp, the protein denatures
If the temp is too low it’s freezing
How do you get the y intercept of the anisotropy plot (1/r vs temp/viscosity)
Extrapolate the plot to a very high viscosity
This makes the x value very small
For bigger molecules the correlation time is
Higher, so they rotate slower
Is the protein in a hydrated solution aslo hydrated?
Yes, is it not bare if it’s in an aqueous solution
This changes the rotation corellation
What is a reporter molecule
Another word for the fluorophore
If a reporter molecule is bound to a heavier thing what happens to the anisotropy
The rotational correlation is higher so anisotropy is higher
This means the reporter rotates less quickly
What does the energy transfer part of a thing losing energy after getting excited include
Needs two chromophores with one being the donor (of energy) and one as the acceptor
In the donor acceptir pair what has to be fluorophore
The donor has to be a fluorophore but the acceptor doesn’t
The acceptor can be either chromophore or fluorophore
What are the steps to energy transfer
The donor gets excited by a photon
There is dipole dipole interaction between D* and A
This leads to FRET from D* to A
But photons are not emmited in this process
What does the energy transfer depend on
There needs to be overlap between the emmision spectrum of the donor and the excitation of the acceptor
The distance between the donor and acceptor , the energy transfer is proportional to 1/R^6
So if higher distances, less energy transfer
Depends on the orientation (angle) of the dipoles and the D and A
What does the energy transfer depend on
There needs to be overlap between the emmision spectrum of the donor and the excitation of the acceptor
The distance between the donor and acceptor , the energy transfer is proportional to 1/R^6
So if higher distances, less energy transfer
Depends on the orientation (angle) of the dipoles and the D and A
Fret means
Forster resonance energy transfer
Not fluorescence because the acceptor doesn’t need to fluoresce
Is the distance is greater than 10nm (100A) in energy transfer what happens
No energy transfer
If the dipoles are 90 to each other can energy transfer happen
No , need to be same orientation
How is the distance between the donor and acceptor (R) calculated
Through the efficiency of energy transfer equations
How is the distance between the donor and acceptor (R) calculated
Through the efficiency of energy transfer equations
The distance between the donor and acceptor is usually
15 to 60 A
Kind of like the thickness of a membrane or diameter of a protein
How do we find R0 on a efficiency vs distance diagram
Go to the half way point on the plot and down to the corresponding distance value
That’s r0
Slide 22 1/2
Idk ask her
What three things can help us calc efficiency of energy transfer
Quantum yeild
Intensity
Lifetime of flurescense
What is the experiment of the efficiency of energy transfer
They labeled two ends of the protein trans helices ,one with donor other with acceptor
They made the helices different lengths and measured the efficiency of energy transfer for each D and A
From this they found R0 by making a plot of the energy transfer at each length (distance)
Slide 24
Explain
What is the assumption we’re making when we find R0
We’re assuming that the orientation of the donor acceptor pair is fixed (not changing)
Or averaged (randomly oriented to get that value)
In proteins, tyr transfers its energy to trp what would have to be true for this to happen?
But trp doesnt transfer to tyr why?
IDK
It would have to be close enough and in the right orientation
because we need an overlap in the emmision of trp and excitation of tyr
When the protein have trp and tyr , and denatures, the tyr fluorescence will be observed
Slide 26 and 27
Idk
Why would we want the Forster distance (R0) equal to the DA pair distance (R)
Because at the point we are close to 100% max efficiency
and any slight changes in the distance could change this Et since the slope of the curve is so high
