FIGURE 5.

3DFSR provides improved 3D resolution while accurately estimating E_D, E_A, and R_M for uniformly distributed bound and free molecules. The concentrations of [D], [A], and E[DA] were reconstructed by 3DFSR from widefield images of live cells expressing uniform free CFP and YFP (A) or a linked CFP-YFP molecule (B). 3D stacks of I_D, I_A, and I_F from cells expressing free CFP and YFP were used for direct calculation of FRET stoichiometry (top row, E_D and R_M, E_A images were very similar to E_D). Thirty iterations of 3DFSR (including photobleaching correction) produced improved estimates of [D] and [A], whereas the reconstructed estimates for E[DA] and E_D indicated no complex. Furthermore, R_M was precisely maintained. Cells expressing a linked construct demonstrated a uniform E_D of ∼0.37 and R_M ∼1.0. 3DFSR (including photobleaching correction) generated improved estimates for [D], [A], and E[DA] and accurately returned ratio images with values of 0.37 and 1.0 in all planes. The effects of FRET-enhanced photobleaching and its correction can be observed by comparing the raw E_D and reconstructed E_D in (B). Scale bar is 5 μm.