help!! the advantage of using fluorescence anisotropy

Frank Fürst ffrank at rz.uni-potsdam.de
Wed Oct 6 03:20:33 EST 1999


Deepblue wrote:
> 
> Hi everyone!! I find a journal which use "fluorescence anisotropy". But i don't understand the meanings and > advantages of useing it. please let me know it. Thanks your attentions.

You can detect binding of the fluorescent dye to a macromolecule even
if the fluorescence intensity doesn't change.

You excite the fluorophors with linear polarized light (vertically
relative to a horizontal optical bench). If the vector of the exciting
light is vertical, the emitted light is vertical too, but the
direction of maximal intensity is, of course, vertical to the
propagation direction of the exciting beam. 

Then if the fluorophors would not move at all before emission, you
would only get vertically polarized light (you also put a polarizer
before the emission detector).

But the molecules do move, and thus also emit differently polarized
light. The faster they move relative to the average lifetime of the
excited state, the more will the distribution approach randomness.

The point is that small molecules move fast and bigger ones move
slower. Therefore a small fluorophor will display nearly random
distribution of polarization direction, and you will find that the
intensity with vertical emission polarizer is the same as that with
horizontal emission polarizer. 
Now if the small fluorophor binds to a big macromolecule, it now only
moves together with that: It becomes slow. Thus the ratio of vertical
to horizontal emission intensity increases, and you can use this
phenomenon to measure binding constants.

The anisotropy itself is defined as (I= - Ip)/(I= + 2 * Ip) where I=
means parallel intensity = intensity with excitation and emission
polarizer vertical and Ip means perpendicular intensity = intensity
with excitation polarizer vertical and emission polarizer horizontal.
A high anisotropy means a slow movement relative to the fluorescence
lifetime, a low one a fast movement.

Often one can also see the quantity polarization, it is defined as (I=
- Ip)/(I= + Ip) and can be converted to anisotropy.

hope this helps, 
Frank
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