Deepsee pulsed laser

Two-photon imaging was performed with a Leica TCS SP8 DIVE microscope equipped with a Mai Tai DeepSee pulsed laser (Spectra-Physics). Imaging was performed under an HCX IR APO L25 × /0.95 water immersion objective (Leica). GFP or YFP was excited at 850 nm, FITC or tdTomato was excited at 960 nm, TRITC or PE was excited at 1050 nm, and Alexa Fluor 700 and TRITC were excited at 860 nm. Images of 512 × 512 pixel fields were acquired for data collection and of 1024 × 1024 pixel fields for use as representative images (area of 738 × 738 μm²). Identical imaging conditions were used for paired groups. Images used as landmarks and time-lapse movies were captured under a Leica DFC425 CCD camera.

At the end of physiological recordings brain slices from WT and Cdh2^H150Y littermates, the slice that contained the cells filled with SBFI fluorescent dye (0.5 µM, Thermo Fisher) was placed in an aCSF perfused chamber. Slices were scanned with an Ultima IV two-photon microscope (Bruker) equipped with a Mai Tai DeepSee pulsed laser (Spectra-Physics) and Olympus water-immersion lens ×60. Using two-photon excitation at 740 nm, we focused on the cell and scanned 30–40 slices of dendrites, soma, and axon at 0.5-μm depth intervals. The resulting z-stacks were imported into ImageJ (US National Institutes of Health) for reconstruction and image processing.