Lsm big

Fixed hippocampal slices were imaged with a two-photon Coherent Chameleon Ultra II laser with a Zeiss LSM 7 MP microscope. Using a Zeiss 20×-W/1.0 NA objective, GFP was excited at 920 nm, and emission was detected by a photomultiplier tube (Zeiss LSM BiG) after passing through a 535 ± 25 nm filter. Images were taken in the stratum radiatum of CA1 hippocampus at a depth of 150 µm ± 25 µm. Stacks were imaged at 16 bit, with 1024 × 1024 pixels, 16-line averaging, a zoom of 2.8, and z-step distance of 1 µm. After acquisition, background signal was removed from all images using Fiji’s rolling ball (radius = 25 pixels) background subtraction.

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.