Figure 2. System schematics that integrated tracking, whole brain functional imaging, and real time behavioral analysis.

Larval zebrafish swam in a customized chamber with an optically transparent ceiling and floor. The water-filled chamber was mounted on a high-speed three-axis stage (PI M686 and PI P725KHDS). Customized LED rings generated dark field illumination of the zebrafish. The scattered light was collected by four cameras: two cameras below the chamber were used for x-y plane tracking and low magnification real-time (RT) analysis, respectively; two cameras above the chamber and after the imaging objective were used for Z autofocus and high magnification RT analysis. The positional information of the larval zebrafish, acquired from the tracking and autofocus system, was converted to feedback voltage signals to drive the three-axis stage and to compensate for fish movement. The functional imaging system, described in Figure 1, shared the same imaging objective placed above the swimming chamber. The 3D tracking, RT behavioral analysis, and functional imaging system were synchronized for accurate correlation between neural activity and behavioral output.

Figure 2—figure supplement 1. Characterization of the autofocus system.

(a) Autofocus camera behind a one-dimensional lenslet array captured triplet images of the fish head (up). Its autocorrelation function was computed (bottom). (b) Central line profile of the autocorrelation function was extracted and inter-fish distance was computed as local maximums in the autocorrelation function. (c) Axial shift of the fish head, calibrated by moving the piezo at a constant interval, changed linearly (red line) with inter-fish distance.