Conception of the oblique light sheet microscope (SCAPE)

The SCAPE microscope was built almost identically to the second version of the SCAPE microscope issued in 2019⁴⁶. We added a few components to: (1) hold the sample and automate its translation; (2) record the tail movement; and (3) build a transmitted light image of the larval brain to be able to insert pipettes or electrodes in the brain to manipulate neural circuits. A 488-nm collimated laser was used to illuminate the sample (Coherent, OBIS 1220123), whose intensity is controlled by a filter wheel built with a range of neutral densities (catalog no. FW212CNEB, Thorlabs). The laser beam is shaped from a Gaussian beam to a homogeneous extended line using a Powell lens (Laser Line LOCP-8.9R30-1.0). The illumination light hits a 1D Galvo system (Gversus011/M 1D Large Beam (10 mm) Diameter Galvo System, Thorlabs) before going through an illumination telescope and at the back focal plane of the objective (1-U2B965 XLUMPlanFL ×20/0.95 W objective lens, Olympus). The galvanometer system scans the imaging plane by rotating the beam in the back focal plane of the objective. The laser beam is decentered from the center of the back focal plane of the objective to create the tilted light sheet. The objective illuminates the sample with a thin titled light sheet and collects fluorescence photons in the orthogonal direction. The emitted light is descanned and magnified by a detection system built of two telescopes, and corrects the light sheet angle using a system of two objectives (catalog no. MRD00205, CFI Plan Apochromat Lambda ×20/0.75 NA/1.0 mm attached to the detection telescope, and catalog no. MRD00105, CFI Plan Apochromat Lambda ×10/0.45 NA/4.0 mm attached to the camera system, both Morrel) mounted in a titled geometry to form a 2D image on a complementary metal–oxide–semiconductor sensor (Zyla sCMOS Zyla-5.5-CL10, Andor) orthogonal to the light beam propagation. A DAQ board controls the communication between electronic devices and the computer (NI USB-6361, National Instruments). To this initial setup, we added a sample holder (catalog no. MLS203P2, Thorlabs), an xyz motorized translation (catalog no. PT3/M-Z8, Thorlabs) controlled by KDC components (catalog no. KDC101, Thorlabs) to hold and move the 3.5-cm glass bottom Petri dish where the zebrafish larvae were placed. We also added components below the sample holder to record tail movement that are further described in the behavior recording and analysis section. The camera was controlled by the Andor Solis software, which was interfaced with a MATLAB program written by the Hillman lab to control the acquisition.