Fig. 5.

Adaptive prospective optical gating minimises cardiac photo-injury and photobleaching. a Acquisition of multiple fluorescent z-stacks in a zebrafish heart (transgene myl7:GFP; 3 dpf) using a retrospective optical gating protocol (blue; mean (line) with standard deviation (band)) causes an increase, followed by a fall, in heart rate over a 1.6 h imaging period ($n=6$ fish). Meanwhile 1.6 h of continuous image acquisition using our adaptive prospective optically-gated protocol (orange; mean (line) with standard deviation (band)), with stacks acquired every 5 min, causes no change in heart rate ($n=5$ fish). Plots for individual fish can be seen in Supplementary Fig. 4. b, d The high energy per stack required for retrospective gating (R) is significantly reduced with adaptive prospective (AP) gating. c When exposed to light levels required for retrospective gating (using typical parameters reported in the literature, and again acquiring $z$-stacks at 5 min intervals), the fluorescence signal from a bleach-susceptible fluorophore (myl7:mKate-CAAX) is halved after only 3 $z$-stacks (blue). In contrast, adaptive prospective optical gating achieves 150 $z$-stacks before showing an equivalent fall in fluorescence intensity (orange). Time course shown in Supplementary Video 9. Data show one representative fish for each condition