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Fig. 2 Analysis of zebrafish sleep/wake behaviour at the frame-by-frame resolution with the FramebyFrame R package. (a) 3D model of the 96-square well mesh-bottom plate. Available at github.com/francoiskroll/FramebyFrame (copy archived at Kroll, 2025a). (b) Example of an 8-day (208 hr total) video-tracking experiment. scrambled-injected control larvae were in a mesh-bottom plate placed in a water bath containing paramecia. Larvae were tracked for 63 hr on a 14 hr:10 hr light:dark cycle (white and dark grey backgrounds, respectively), then switched to constant dim light (30 lux) for 145 hr (subjective nights are framed). The larvae were tracked from 4 to 13 dpf and all appeared healthy at the end of the experiment. The trace is the mean ± SEM across larvae of the activity (sum of Δ pixels/10 min). The arrow indicates when the water in the bath was replaced and fresh paramecia were supplemented, causing a spike in activity. (c) The FramebyFrame R package calculates 17 parameters from the Δ pixel timeseries of each larva. The parameters are grouped in three categories: activity parameters, active bout parameters, and sleep parameters. Here, sleep parameters calculated by the FramebyFrame R package are annotated on the first ~9 min of the Δ pixel timeseries for one wild-type larva starting at lights off. These parameters describe the sleep behaviour of each larva during each day or night. Sleep parameters are also calculated for days, except for sleep latency. This plot is connected to (b) for illustrative purposes only; the data was not from the same experiment. (d) Example of an active bout parameter—active bout length—annotated on the sample data from (c). For all the activity and active bout parameters calculated by the FramebyFrame R package, see Figure 2—figure supplement 1.