Miniature Fluorescence Microscopy for Calcium Imaging in Behaving Mice

For time-lapse imaging in behaving mice we used an integrated miniature fluorescence microscope (nVista 3.0, Inscopix) as previously described^{26 (link)}. At least two weeks after the glass guide tube implantation, we inserted a microendoscope consisting of a metal guide cannula (~3.1 mm length, 1.8 mm outer diameter) and a single gradient refractive index (GRIN) lens (4.0 mm length, 1.0 mm diameter) into the implanted glass tube and examined Ca²⁺ indicator expression in the operated mice (Inscopix data acquisition software, Inscopix). We then affixed the microendoscope within the glass guide tube using ultraviolet-curing adhesive (Flow-It A3, Pentron). Next, we attached the miniature microscope’s magnetic base plate to the dental acrylic surface with the ultraviolet-curing adhesive. At least a day later, the mice were habituated wearing the miniature microscope in their home cage for 30 minutes for 4–5 days. To record mouse behavior in the home cage, we used an overhead monochrome camera (GigE Vision, Basler AG), which we synchronized with the miniature microscope. Ca²⁺ imaging was performed at 10 Hz. Imaging sessions consisted of 18-min-long trials.