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Bchem 471 > Fluorescense 3 > Flashcards

Fluorescense 3 Flashcards

1
Q

What is nonpolarized light

Polarized light

A

Nonpolarized is when the light is coming from all different directions

Polarized if when the light goes in one direction as a wave

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2
Q

When we see polarized light what do we actually see

A

The vector going just up and down

But we imagine the polarized light as a wave

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3
Q

What is depolarization

A

This is where the chromophore absorbs light only when the electric vector (the polarized light) is parallel to the transition moment of the chromophore

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4
Q

What is an isotopic and inosotropic solution

A

Isotropic is when the molecules are randomly oriented

Anisotropic is when they are uniform in one direction

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5
Q

If fluorophores have been excited by polarized light, when the emit what direction will the light be

Why

A

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

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6
Q

Over time after a fluorophore has absrobed polarized light , how will it emit

A

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

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7
Q

What is an example of fluorescent polarization and depolarization

How does each sample affect the fluorophore

A

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

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8
Q

How is the fluorimeter with polarized light different than the regular fluorimeter

What does this change

A

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

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9
Q

What is fluorescence anisotropy independent of

And normalized by

A

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

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10
Q

Slide 7 diagram

A

Idk

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11
Q

What are the two extremes for anisotropy

A

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

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12
Q

What is the range of values for anisotropy

A

0-1

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13
Q

What is the other word for anisotropy used in old literature

Are the extremes for it the same

A

Polarization

Yes but they are different if under other conditions

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14
Q

If you have a hydrated spherical molecule in a solution, if it’s very viscous what does this do to the rational correlation time

A

The time is higher

Meaning the rotational time is slower, higher anisotropy (since they aren’t moving as much)

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15
Q

How do we do measurements of the anisotropy

A

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

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16
Q

What are the limitations for the measurement method of anisotropy

A

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

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17
Q

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

A

Plot 1/r vs T/n

The y intercept is 1/r0

Slope is R tau f/r0 Vh

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18
Q

When calculating anisotropy what are we assuming

A

The molecule is spherical and hydrated

It is steady state anisotropy

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19
Q

Derivation of equation for finding size

A

In notes

20
Q

What is the first step to doing an anisotropy experiment

What do we have to make sure

A

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)

21
Q

What is the second step of the anisotropy experiment

What are the limitations

A

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

22
Q

How do you get the y intercept of the anisotropy plot (1/r vs temp/viscosity)

A

Extrapolate the plot to a very high viscosity

This makes the x value very small

23
Q

For bigger molecules the correlation time is

A

Higher, so they rotate slower

24
Q

Is the protein in a hydrated solution aslo hydrated?

A

Yes, is it not bare if it’s in an aqueous solution

This changes the rotation corellation

25
Q

What is a reporter molecule

A

Another word for the fluorophore

26
Q

If a reporter molecule is bound to a heavier thing what happens to the anisotropy

A

The rotational correlation is higher so anisotropy is higher

This means the reporter rotates less quickly

27
Q

What does the energy transfer part of a thing losing energy after getting excited include

A

Needs two chromophores with one being the donor (of energy) and one as the acceptor

28
Q

In the donor acceptir pair what has to be fluorophore

A

The donor has to be a fluorophore but the acceptor doesn’t

The acceptor can be either chromophore or fluorophore

29
Q

What are the steps to energy transfer

A

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

30
Q

What does the energy transfer depend on

A

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

31
Q

What does the energy transfer depend on

A

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

32
Q

Fret means

A

Forster resonance energy transfer

Not fluorescence because the acceptor doesn’t need to fluoresce

33
Q

Is the distance is greater than 10nm (100A) in energy transfer what happens

A

No energy transfer

34
Q

If the dipoles are 90 to each other can energy transfer happen

A

No , need to be same orientation

35
Q

How is the distance between the donor and acceptor (R) calculated

A

Through the efficiency of energy transfer equations

36
Q

How is the distance between the donor and acceptor (R) calculated

A

Through the efficiency of energy transfer equations

37
Q

The distance between the donor and acceptor is usually

A

15 to 60 A

Kind of like the thickness of a membrane or diameter of a protein

38
Q

How do we find R0 on a efficiency vs distance diagram

A

Go to the half way point on the plot and down to the corresponding distance value

That’s r0

39
Q

Slide 22 1/2

A

Idk ask her

40
Q

What three things can help us calc efficiency of energy transfer

A

Quantum yeild

Intensity

Lifetime of flurescense

41
Q

What is the experiment of the efficiency of energy transfer

A

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)

42
Q

Slide 24

A

Explain

43
Q

What is the assumption we’re making when we find R0

A

We’re assuming that the orientation of the donor acceptor pair is fixed (not changing)

Or averaged (randomly oriented to get that value)

44
Q

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

A

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

45
Q

Slide 26 and 27

A

Idk

46
Q

Why would we want the Forster distance (R0) equal to the DA pair distance (R)

A

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

Bchem 471 (24 decks)

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