Deepsee maitai ti sapphire pulsed laser

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).

Mice were anesthetized using isoflurane and secured on a warming plate. The tibial bone was surgically exposed and thinned as previously described^{49 (link)}. All imaging was performed on an Olympus FVE-1200 upright microscope, using 25×1.04 NA objective, and Deepsee MaiTai Ti-Sapphire pulsed laser (Spectra-Physics) tuned to 920 nm. Mice were imaged in a custom-built 37 °C-heated incubator chamber to maintain body temperature. Time-lapse movies were conducted every 1 min for at least 1 hour with 3 μm z spacing. Qtracker 705 (Invitrogen) was used to highlight vascular region and injected at the start of imaging.
All image analysis was conducted using Imaris 9.3 (Bitplane) to track cells and correct drift. For drift correction, autofluorescent sessile macrophages were tracked using semi-automated or manual methods to correct XYZ registrations over time. Ratio channels were used to isolate shCtrl-tdTomato and shTMIGD2-GFP AML cells and subtracted channels were created to isolate vessel signal. Maximal intensity time projection was used to highlight the AML traces over time for the overlap with vascular region. All imaging and conditions were independently conducted at least twice.