Plan apochromat 10x 0

Histological images were obtained with a Keyence BZ-X fluorescence microscope (for regular 2D histology), a Zeiss LSM 880 confocal microscope (with Airyscan, when applied), or a custom-built two-photon microscope. The Zeiss 880 confocal microscope was used for all optical-fiber Z stack/volume imaging with a 10X air objective (Plan-Apochromat 10X/0.45). Zoom was adjusted up to 2 and images were taken at 5 or 10 μm steps. For Figure 6A; 25X immersion lens was used (Plan-Apochromat 25X/0.5 Imm Korr DIC). Whenever comparative quantification was used, laser intensities and filters were matched. Two-photon Z stack images were taken at 512 lines/frame, 125 μs dwell time, with laser wavelengths of 940 nm (10–50 mW) for GCaMP and 1100 nm for 647–nm labeled antibody (10–20 mW). The field of view determined the imaging dimensions, for example, 450X450 and 550X550 μm for mouse #2 and #3, respectively (Figure 4). For two-photon cell registration with IHC, we found that to observe stained cells imaging with a longer wavelength was more efficient from the exposed tissue side. In addition, two-photon imaging with two different wavelengths shows chromatic aberrations. Therefore, autofluorescence markers were identified in the images at both wavelengths, and translation in XYZ was applied if needed (up to 25 μm). Images were analyzed in ImageJ, MATLAB, and/or Imaris (Bitplane).