活体双光子显微镜能够在活体动物中以高时空分辨率进行深层组织成像。然而，骨内骨室和下面的骨髓对光学成像提出了独特的挑战，因为光被厚的矿化骨基质和富含脂肪的骨髓吸收、散射和分散。早期的骨活体成像方法利用颅缝中的间隙来绕过穿透皮质骨的需要。最近，研究人员开发了侵入性方法来使皮质骨变薄或植入成像窗口以对承重长骨中的细胞动力学进行成像。在这里，我们提供了准备动物的分步程序，用于对小鼠胫骨进行微创、无损、纵向活体成像。该方法涉及使用混合骨髓辐射嵌合体来明确地双标记破骨细胞和骨形态。通过简单的皮肤切口和使用导热 T 型腻子构建的成像室暴露胫骨。长达 12 小时的成像过程可以在多个时间点重复，以提供进入骨内膜和骨髓龛的纵向时间窗口。该方法可用于研究骨重塑、癌细胞生命周期和造血作用以及长寿命体液和细胞免疫中的细胞动力学。该程序需要一个小时才能完成，适合具有小动物手术经验的用户。© 2023。Springer Nature Limited。

Intravital two-photon microscopy enables deep-tissue imaging at high temporospatial resolution in live animals. However, the endosteal bone compartment and underlying bone marrow pose unique challenges to optical imaging as light is absorbed, scattered and dispersed by thick mineralized bone matrix and the adipose-rich bone marrow. Early bone intravital imaging methods exploited gaps in the cranial sutures to bypass the need to penetrate through cortical bone. More recently, investigators have developed invasive methods to thin the cortical bone or implant imaging windows to image cellular dynamics in weight-bearing long bones. Here, we provide a step-by-step procedure for the preparation of animals for minimally invasive, nondestructive, longitudinal intravital imaging of the murine tibia. This method involves the use of mixed bone marrow radiation chimeras to unambiguously double-label osteoclasts and osteomorphs. The tibia is exposed by a simple skin incision and an imaging chamber constructed using thermoconductive T-putty. Imaging sessions up to 12 h long can be repeated over multiple timepoints to provide a longitudinal time window into the endosteal and marrow niches. The approach can be used to investigate cellular dynamics in bone remodeling, cancer cell life cycle and hematopoiesis, as well as long-lived humoral and cellular immunity. The procedure requires an hour to complete and is suitable for users with minimal prior expertise in small animal surgery.© 2023. Springer Nature Limited.