Mice were anesthetized using isoflurane and secured on a warming plate. The tibial bone was surgically exposed and thinned as previously described49 (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.
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.