通过体内三光子显微镜分析完整小鼠胫骨中骨髓B淋巴细胞动态变化的实验方案。
Protocol to analyze marrow B lineage cell dynamics by in vivo three-photon microscopy in intact mouse tibia.
作者信息
Bias Anne, Fiedler Alexander F, Günther Robert, Leben Ruth, Beckers Ingeborg, Hauser Anja E, Rakhymzhan Asylkhan, Niesner Raluca A
机构信息
Dynamic and Functional in vivo Imaging, Freie Universität Berlin, 14163 Berlin, Germany; Biophysical Analytics, German Rheumatology Research Center - a Leibniz Institute, 10117 Berlin, Germany; Medical Physics/Physical Engineering, Berlin University of Applied Science and Technologies, Berlin, Germany.
Dynamic and Functional in vivo Imaging, Freie Universität Berlin, 14163 Berlin, Germany; Biophysical Analytics, German Rheumatology Research Center - a Leibniz Institute, 10117 Berlin, Germany.
出版信息
STAR Protoc. 2025 May 14;6(2):103824. doi: 10.1016/j.xpro.2025.103824.
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..
三光子显微镜（3PM）由于采用红外激发，能够实现双光子显微镜（2PM）所无法企及的深层组织成像。在此，我们展示了一种使用1650纳米的三光子显微镜对荧光报告基因小鼠胫骨骨髓中的B淋巴细胞进行延时三维成像的方案。我们描述了验证显微镜性能和胫骨成像的步骤。然后，我们详细介绍了细胞动力学分析，包括去噪、细胞分割和跟踪。该方案在双光子显微镜无法适用的其他光学上难以触及的器官中的免疫细胞跟踪方面具有潜在应用价值。有关本方案使用和执行的完整详细信息，请参考拉赫姆扎恩等人的研究。
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