Protocol to analyze marrow B lineage cell dynamics by in vivo three-photon microscopy in intact mouse tibia

Three-photon microscopy (3PM) allows deep-tissue imaging beyond the capabilities of two-photon microscopy (2PM) owing to infrared excitation. Here, we present a protocol for time-lapse 3D imaging of B lymphocytes in the tibia marrow of fluorescent reporter mice using 3PM at 1,650 nm. We describe steps for verifying microscope performance and tibia imaging. We then detail the cell dynamics analysis, including denoising, cell segmentation, and tracking. This protocol has potential application for immune cell tracking in other optically inaccessible organs in which 2PM fails. For complete details on the use and execution of this protocol, please refer to Rakhymzhan et al.¹.