Question

1.      Define anisotropy and list the equation that describes fluorescence anisotropy.

2.      How do you think increasing temperature will affect the measured fluorescence anisotropy in this experiment (i.e. will it increase or decrease)? Why?

Answer 1

Ans 1:

These minerals do affect polarization of light passing through them, so a component of the light is able to pass through the upper polar. These minerals will appear dark or extinct every 90 degrees of rotation of the microscope stage. Any grains which become extinct will become light again under a rotating stage

Anisotropic Minerals are one or two directions, through the mineral, along which light behaves as though the mineral were isotropic. This direction or these directions are referred to as the optic axis.

This technique relies on the fluorescent dyes to visualize molecular motion. A fluorescent labeled body such as an oligonucleotide is allowed to bind to the target molecule. Then the solution is excited by vertically polarized light, which excites only the fluorescent tags that are oriented properly relative to the incoming light. The vertical and horizontal emissions from the fluorescent tags are then measured and are used to calculate the anisotropy and is called as the r value.

r = I _II – I _┴ / I _II + 2I _┴

I _II = is the vertical emission and I _┴ is the horizontal emission.

Ans 2: The major effect of temperature is on the rotational mobility of the fluorophore, where a higher temperature leads to a shorter rotational correlation time.

The fluorescence anisotropies are measured over a range of T/ח values. At higher temperature the macromolecules may denature and at low temperature the solvent may freez or the macromolecule may not be soluble. Fluorophores bound to protein result in segmental motion which is independent of rational diffusion.