W 1.0 na objective

All experiments were performed on a LSM MP710 2-photon imaging system (Zeiss). Cells were identified for whole-cell patch clamp and imaging using either widefield infrared illumination captured with a DAGE IR-1000 camera (DAGE-MTI). This was preferable to patching GFP-labelled cells due to the sparse labeling and ease in identifying healthy neurons with transmitted illumination. GFP and/or Rhod-2 imaging was achieved by 2-photon excitation with a Ti:Sapphire Chameleon Ultra II 2-photon laser (Coherent) tuned to 850 nm. Images were acquired with a Zeiss 20X-W/1.0 NA objective at a pixel resolution of either 512 × 512 or 256 × 256 for fast Rhod-2 Ca²⁺-imaging. Emission light was split with a 575 nm longpass filter, and green and red emissions were filtered with 535/50 nm and 630/75 nm bandpass filters, respectively (all from Chroma Tech). Emission light was collected with LSM BiG GaAsP detectors from Zeiss, and data were acquired using Zen software (Zeiss) and analyzed in Fiji.

Acute slices from CX3CR1^+/EGFP C57BL/6 mice were imaged immediately after recovery using a Coherent Chameleon Ultra II laser (mode‐locked pulse train at 80 MHz at 920 nm) with a Zeiss LSM 7 MP microscope and Zeiss 20x‐W/1.0 NA objective. Green fluorescence was detected by a 520/60 nm filter (Chroma tech) and GaAsP photo‐multiplier tube (PMT; Zeiss LSM BiG). Images were acquired as a z‐stack (zoom factor 2.8; 151.82 × 151.82 μm xy scale, 8‐line averaging) 18 μm thick, centered approximately 150 μm below the slice surface (2 μm slice interval) in the stratum radiatum region of the CA1 hippocampus. Following a 10‐min baseline imaging period, a lesion was created by focusing the laser to the region of interest and scanning at 800 nm at 100% power for approximately 30 s. Microglial response to this lesion was then imaged for an additional 15 min using the same imaging parameters as baseline.
For motility analysis, baseline movies were maximum projected and loaded into a custom MATLAB program. This program quantifies the number of new pixels (additions) and number of removed pixels (retractions) across time as the Motility Index. To quantify the microglial response to lesion, a circular region of interest with a diameter of 30 μm was centered on the lesion response region, and the mean intensity was measured at each frame.