Fve 1200 upright microscope

For intravital imaging, mice were anesthetized with isoflurane, and the spleen was surgically exposed and elevated above the body of the mouse. A glass coverslip was carefully applied to the top of the spleen to create an imaging window. Mice were kept at 37°C using a custom heating platform. Imaging was performed on an Olympus FVE-1200 upright microscope using a 25× 1.04–numerical aperture objective and a Deepsee MaiTai Ti-Sapphire pulsed laser (Spectra Physics) tuned to 870 nm. To maintain temperature and limit infiltrating light, the microscope was fitted with a custom-built incubator chamber heated to 37°C. Z-stack images (512 by 512) were acquired every 60 s with 5-μm steps. For explant imaging, mice were euthanized with CO₂, and spleens were immediately harvested. Spleens were affixed to coverglass using Vetbond (3M) on the medulla. Tiled images were acquired using 320 × 320 Z-stack images with 15-μm steps. Tiled images were stitched using Olympus Fluoview software. Cell tracking, drift correction, and monocyte volume analysis were carried out using Imaris 9.2 (Bitplane).