[Nescafe]

From my understanding is that we shine vertical polarized light on our sample, then measure light intensity both parallel to the polarized light (vertical) and perpendicular (horizontal). If the fluorophore is immobile light remains polarized and the intensity of the vertical polarized light would be high and that of horizontal low. Now if it is mobile the intensity of horizontal light will be high and that of parallel low. I guess my question is, why do we only measure horizontal emitted light and not any depolarized light between the two planes (Vertical and Horizontal). I am confusing myself as I am writing this. I just do not get why we look at two planes and that is it, why not anywhere in between?


Nescafe

[Arkcon]

I've moved this question from the Biochemistry sub-forum, because the question doesn't mention any particular biomolecule, just the fluorescent dye fluorescein, but let us know if I'm missing something, and we can move it back.

As a guess to your application, are you measuring absorbance, or reflectance, or fluorescence?  Because separating the light source, from the fluorescence, even though there is a wavelength difference, is still important for fluorescence instruments.

[Babcock_Hall]

Nescafe, The book Biological Spectroscopy by I. Campbell and R. Dwek has a chapter (5) on fluorescence that is pretty readable.  Molecular motion should reduce polarization in a way that depends on the rotational correlation time of the molecule (the Perrin equation).  As to why one only measures only parallel and perpendicular, I am not sure about the answer to your question, but I might hazard a guess.  Any in-between fluorescence might be resolvable into parallel and perpendicular components.  That is really a question that a chemical physicist or spectroscopist should answer, however.

[Yggdrasil]

Fluorescence polarization assays are fairly common biochemical assays to measure biomolecular interactions, so it probably would make sense to move this topic back to the biochemistry board.

You have the right idea for how the assay works.  Large molecules (which rotate very slowly in solution) when excited with plane polarized light, will emit light with mostly the same polarization as the excitation light.  This occurs because the transition dipole of the fluorophore does not have enough time to rotate substantially between absorption and emission.  Smaller molecules, however, will rotate quickly enough that, in the time between absorption and emission, the transition dipole of the fluorophore will essentially adopt a random orientation.  Thus the emitted light has a random polarization.

Now a little note about plane polarized light.  You can measure the polarization of any orientation of light by measuring the intensity of light along two perpendicular axes.  Vertically polarized light would give 100% of its signal in the vertical polarization and 0% horizontal polarization; horizontally polarized light would give 0% vertical, 100% horizontal; 45º light would give 50% vertical, 50% horizontal; 30º light would give 75% vertical, 25% horizontal; etc.  So this is why you need to only measure the intensities parallel and perpendicular to the excitation light.  

[Nescafe]

Thank you all very much for the response.

Yggdrasil. Your note on the plane polarized light helped me understand it. I will keep reading and ask for your help if I get stuck again!

Thank you sir,

Nescafe.

[Nescafe]

Hi,

I am back again

I read one of the main differences between anisotropy and polarization is in the equation of the two:

Polarization = Iparallel - Iperpendicular/ Iparallel + Iperpendicular

Anisotropy =  Iparallel - Iperpendicular/ ITotal = Iparallel - Iperpendicular/parallel + 2 * Iperpendicular

So I asked the question why is Itotal = 2 * Iperpendicular and found the following answer which I do not understand.... at.... ALL =(

For the reason of symmetry the component polarized in the X direction and Y direction have the same intensity Iperpendicular, but in the given geometry we detect only the one polarized in the x direction.

What?

Help me :S

Nescafe.

[Yggdrasil]

To make it more clear, polarization (p) and anisotropy (r) are defined in terms of the intensities of light parallel I_par and perpendicular I_per to the excitation light's polarization:

[tex]p = \frac{I_{par} - I_{per}}{I_{par}+ I_{per}}[/tex]
[tex]r = \frac{I_{par} - I_{per}}{I_{par}+2I_{per}}[/tex]

These two values are related by:
[tex]r = \frac{2p}{3-p}[/tex]

The polarization value should be fairly straightforward to understand: as the sample goes from completely polarized (I_per = 0) to completely depolarized (I_per = I_par), p goes from 1 to 0.

Now, consider we have a mixture of two populations of molecules, 1 and 2.  If the proportion of molecules are f₁ and f₂ and these molecules have polarizations p₁ and p₂, the overall polarization of the solution, p, is given by:

[tex]\left(\frac{1}{p} - \frac{1}{3}\right)^{-1} = f_1 \left(\frac{1}{p_1} - \frac{1}{3}\right)^{-1} + f_2 \left(\frac{1}{p_2} - \frac{1}{3}\right)^{-1}[/tex]

(for a derviation see Weber 1952 Biochem J. 51:145)

This equation is obviously not so easy to work with.  However, if we use the anisotropy measurement as defined above, the overall anisotropy of the mixture, r, is easy to calculate:

[tex]r = f_1r_1 + f_2r_2[/tex]

This is the main reason for why we have that strange definition for anisotropy.

In case you find it useful, here is a good practical guide for analyzing FP data: http://labs.fhcrc.org/hahn/Methods/biochem_meth/beacon_fluorescence_guide.pdf

[Nescafe]

Thank you so much,

Nescafe.