FIG. 5.
SPIM imaging of intracellular calcium for capturing neuronal activity. (a) Schematic of apparatus for imaging of neural activity during various behaviors in the larval zebrafish. Sheets of laser light are synthesized by quickly scanning the pulsed illumination beam (red) with galvo mirrors (G). 2P light-sheets are delivered to the agarose-embedded head of the animal with excitation objectives (EO) from the side and front arms. The side masks cover each eye on the sides of a horizontally oriented zebrafish, while the front mask covers both eyes, enabling access to neurons between the eyes. 2P-excited calcium fluorescence signal is collected through an upright detection objective (DO) and onto a scientific CMOS camera. A triggerable wide-field camera is positioned below the sample chamber (SC) to provide a wide-field, low-resolution view of the sample, as shown in (b). During a typical neural imaging experiment, the zebrafish larva is mounted in a caddy, which in turn is mounted to the dive bar (DB) underneath the DO. Within the caddy, the zebrafish’s head is immobilized in agarose, while the tail is free, permitting the monitoring of zebrafish behavior through tail movement. SL—scan lens, TL—tube lens, SC—sample chamber, and L—camera lens. The third illumination arm, emission filter, detection TL, scientific camera, light-emitting diode, and filter for behavior channel are not shown. Insets in (b) highlight that the calcium fluorescence channel (green) is recorded from the zebrafish brain, while the behavioral channel (dark red) monitors the tail movement of the animal. Scale bar: (b) 400 |