FIG. 12.

Whole-brain functional imaging at single-cell resolution in behaving 5-dpf transgenic larval zebrafish expressing nuclear-localized calcium indicator elavl3:H2B-GCaMP6s. (a) Maximum-intensity projections of calcium activity are color-coded in time over the 30-min recording window. Active neurons that exhibit fluorescence change during the recording appear as colored dots. Volume of 400 × 800 × 250 (x–y–z) μm³ was sampled by 52 z-planes (4.8 µm z-steps) at 0.5 Hz and 11× magnification. Figure 13 (Multimedia view) shows a 3D rendered movie of the same specimen. (b) Magnified volume renderings of neuron 1 (left) and neuron 2 (right) outlined in the x–y projection in (a), demonstrating cellular resolution in whole-brain 2P-SPIM imaging. The standard deviation projection along the temporal axis was used to generate the renderings, and a false-color (hot) lookup table was used to aid visualization. (c) Representative single-neuron activity traces extracted from the whole-brain recordings, calculated as ∆F/F from the manually selected neurons in (a). (d) Plot shows the total sum intensity of the entire imaged volume as a function of time, normalized to the average intensity of the first 5 min of acquisition (gray line at y = 1). Total intensity exhibits less than a 0.5% decrease in magnitude after the 30-min recording window, indicating that photobleaching is negligible. (e) Analysis of tail flick behavior as a function of time. Tail flicks (movements) obtained by taking the absolute difference in the pixel intensities from the tail region of the wide-field view of the sample, as shown in Fig. 5(b), normalized by the average image intensity corresponding to when there is no tail movement (gray lines). A threshold was applied to classify tail flicks and subsequently confirmed by manual inspection. Tail flicks (red arrowheads), indicating intended swimming behavior, occur throughout the recorded time window (average of ∼9 flicks ±2.2 every 5 min), thus suggesting minimal stress from the imaging conditions used. Scale bars: (a) 100 µm and (b) 5 µm.