FIGURE 1.

Emission from eGFP-M₂ upon photobleaching at 514 nm. A, cell coexpressing eGFP-M₂ and eYFP-M₂ was subjected to 12 rounds of photobleaching and spectral analysis to obtain values of k_DA(j) and k_AD(j) for the cell before bleaching (j = 0) and after each successive round (j = 1–12) (●). The stepwise decrease in k_AD(j) was accompanied by an increase in k_DA(j) that arose from donor-dequenching. Each value of k_DA(j) also was adjusted according to Equation 4 to correct for photobleaching of the donor (○). The lines were fitted to the uncorrected (●) and corrected data (○) by linear regression, and the corresponding x-intercepts represent k_D^e and k_D^ec, respectively, as described in the text. B, cell expressing eGFP-M₂ alone was subjected to successive rounds of photobleaching. The emission spectrum was acquired after each step (j), and the intensity at the maximum (k_D(j)) is shown relative to that from the unbleached cell (k_D(0)). The line represents the best fit of Equation 3 to the data, and the fitted value of f_r is 0.989 ± 0.001. Inset, the emission spectrum was recorded before photobleaching (■) and after each successive step j (▴, ▾, ♦, ●, □, ▵, ▿). A.U., arbitrary units.