Release flux and FRET ratio in a cell with spatially heterogeneous biosensor expression. (A) Biosensor concentration (invariant image) calculated as described in Eq. A6 of Sztretye et al. (2011). (B) FRET ratio R. (C) Histogram of pixel values of R. The distribution is narrow, which is consistent with a lack of effect on R of the variation in [biosensor]. (D) R(t) in line scans along dashed line in A, averaged over regions a (high [biosensor]) and b (low) indicated by line segments at the bottom of A. The average concentration of biosensor was 7.54 µM and 1.54 µM, respectively, in a and b. Note slightly greater R in region b. (E and F) $R•net(t)$ and its integral Rel(t) (Eq. 4) in regions a and b. No difference is visible in spite of the large change in biosensor concentration. ID: 072309a image 29.