Supplementary Figure 3: Rapid photobleaching occurs when performing simultaneous scanning of two distinct lasers in an intact, living sample.

With a commercial confocal microscope, we imaged individual nuclei within embryonic zebrafish at 1 day post fertilization (dpf) expressing nuclear-targeted Cerulean FP (see Online Methods). a, Nuclei were scanned continuously for 30 frames using a 458 nm laser alone (left column), a 561 nm laser alone (center column), or a simultaneous combination of 458 and 561 nm lasers (right column). Images were taken of the nuclei before (top row) and after (bottom row) the continuous time-lapse to monitor decreases in fluorescence intensity. No significant photobleaching was seen in the time-lapses with the 561 nm laser alone (< 10% decrease in fluorescence intensity, N = 5 nuclei). b, We quantified the rate of photobleaching within the imaged nuclei by fitting the time-lapse data from the left and right columns with a bi-exponential function (see Online Methods). The rate of Cerulean photobleaching increased nonlinearly when the 458 and 561 nm lasers illuminated the sample simultaneously (R²~1, N = 5 nuclei). Scale bar, 5 µm.