The tracking microscope was built predominantly using optics and an optical cage system from Thorlabs. The basic optical path consists of a 4x NA 0.1 Olympus PLAN objective (Thorlabs #RMS4X) with a 150 mm tube lens (Thorlabs #AC254-150-A-ML), yielding a magnification of 3.33X. The image is collected on a monochrome Chameleon3 camera (FLIR, #CM3-U3-13Y3M), with a 1/2" sensor format and 1280 × 1021 pixels (4.8 μm/px at sensor plane, 1.44 μm/px at object plane). This yields a field of view of 1.84 × 1.47 mm. We illuminated the sample with a 780 nm, 18 mW IR LED (Thorlabs #LED780E) in a transillumination configuration. The LED was diffused using a ground glass diffuser (Thorlabs #DG10-120), and collimated onto the sample using a f=16 mm aspheric condenser lens (Thorlabs #ACL25416U) placed 16 mm from the diffuser surface.
The microscope path also included a 4.5 mW, 532 nm laser diode module (Thorlabs #CPS532) for optogenetic stimulation. The laser beam (3.5 mm diameter) was combined with the main optical path using a 550 nm dichroic (Thorlabs #DMLP550R), then focused on the back focal point of the objective using a f = 100 mm planoconvex lens (Thorlabs #LA1509-A-ML). This illuminated a circular region roughly 1.6 mm in the sample plane, with a calculated average intensity of 150 μW/mm2.
The microscope path also included a 4.5 mW, 532 nm laser diode module (Thorlabs #CPS532) for optogenetic stimulation. The laser beam (3.5 mm diameter) was combined with the main optical path using a 550 nm dichroic (Thorlabs #DMLP550R), then focused on the back focal point of the objective using a f = 100 mm planoconvex lens (Thorlabs #LA1509-A-ML). This illuminated a circular region roughly 1.6 mm in the sample plane, with a calculated average intensity of 150 μW/mm2.