I have a question regarding the extraction of the diffusion coefficient from FRAP datas.
To put it back into the context, we performed FRAP experiment (on a spinning-disk microscope with a FRAP module attached to it) on a midbody between two cells, and we are interested in the diffusion of a protein present in the midbody and the cytoplasm of the cells.
As we are particularly interested in the diffusion inside the midbody, we perform the FRAP on an area larger than the beam on the midbody (sometimes the neighborhood cytoplasm is also bleached) and we designed an area of measurement only on the midbody.
My question is : as the formula of the diffusion coefficient is
D = w²/4t
where w is the radius of the circular beam and t the half-time recovery, can I use, as w, not the diameter of the area used to FRAP, but rather the diameter of the area where we make our measurement ?
I can’t really control the diameter of the area to FRAP, but i can easily determine an area to measure the recovery, and keep the same for all.
Thank you very much for your help