Protocol to analyze marrow B lineage cell dynamics by in vivo three-photon microscopy in intact mouse tibia
Bias A. Fiedler A.F. Günther R. Leben R. Beckers I. Hauser A.E. Rakhymzhan A. Niesner R.A.
20 June 2025Cell Press
STAR Protocols
2025#6Issue 2
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
Biophysics , Immunology , Microscopy
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Dynamic and Functional in vivo Imaging, Freie Universität Berlin, Berlin, 14163, Germany
Biophysical Analytics, German Rheumatology Research Center – a Leibniz Institute, Berlin, 10117, Germany
Medical Physics/Physical Engineering, Berlin University of Applied Science and Technologies, Berlin, Germany
Rheumatology and Clinical Immunology, Charité – Universitätsmedizin, Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Berlin, 10117, Germany
Immune Dynamics, German Rheumatology Research Center – a Leibniz Institute, Berlin, 10117, Germany
Department of Fundamental Medicine, Higher School of Medicine, Al-Farabi Kazakh National University, Almaty, 050040, Kazakhstan
Dynamic and Functional in vivo Imaging
Biophysical Analytics
Medical Physics/Physical Engineering
Rheumatology and Clinical Immunology
Immune Dynamics
Department of Fundamental Medicine
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