ROI size in FRAP microscopy (GAP-FRAP) - (Oct/04/2012 )

I´m analyzing gap junctional communication by FRAP using carboxyfluorescein. Once inside the cell this dye can pass the membrane only via gap junctions.
We are using a progenitor cell line that can differentiate into neural phenotypes and we compare communication between non-differentiated and differentiated cells. Due to the alterations in cell morphology it is quiet complicated to do FRAP with the same ROI size.
Has anyone an idea how to solve this problem? Maybe normalization to the ROI area?

thanks

OMG This is EXACTLY what my problem is. An ROI after FRAP that changes shape and size. If I use the same ROI than there is tons of extra area after FRAP but if I used a smaller more defined FRAP than the mean value is artificially increased.

What was your solution for the problem???