Fiedler Alexander F, Leben Ruth, Stürmer Herbert, Günther Robert, Matthys Romano, Nützi Reto, Hauser Anja E, Niesner Raluca A
Biophysical Analytics, German Rheumatism Research Center - a Leibniz Institute, Berlin, Germany.
Dynamic and Functional in vivo Imaging, Freie Universität Berlin, Berlin, Germany.
Biomed Opt Express. 2025 Mar 31;16(4):1711-1731. doi: 10.1364/BOE.549311. eCollection 2025 Apr 1.
Intravital imaging of bone marrow provides a unique opportunity to study cellular dynamics and their interaction with the tissue microenvironment, which governs cell functions and metabolic profiles. To optically access the deep marrow of long bones, we previously developed a microendoscopy system for longitudinal two-photon fluorescence imaging of the murine femur. However, this does not provide information on cell functions or metabolism, for which quantification fluorescence lifetime imaging (FLIM) has proven to be a versatile tool. We present and characterize FLIMB, an adapted GRIN-based microendoscopic system capable of performing reliable, co-registered TCSPC-based two-photon excited FLIM and fluorescence imaging in the femur of fluorescent reporter mice, at sub-cellular resolution. Using FLIMB, we demonstrate metabolic imaging via NAD(P)H-FLIM and intracellular Ca signaling via FRET-FLIM in immune cell subsets, in the femoral marrow. This method retains the power to study molecular mechanisms underlying various cell functions in tissue context thus providing new insights into bone biology.
骨髓的活体成像为研究细胞动力学及其与组织微环境的相互作用提供了独特的机会，而组织微环境决定着细胞功能和代谢特征。为了通过光学方式观察长骨的深部骨髓，我们之前开发了一种用于小鼠股骨纵向双光子荧光成像的显微内窥镜系统。然而，该系统无法提供有关细胞功能或代谢的信息，而定量荧光寿命成像（FLIM）已被证明是一种多功能工具。我们展示并表征了FLIMB，这是一种基于梯度折射率（GRIN）的适配显微内窥镜系统，能够在荧光报告基因小鼠的股骨中以亚细胞分辨率进行基于时间相关单光子计数（TCSPC）的可靠、共配准双光子激发FLIM和荧光成像。使用FLIMB，我们在股骨骨髓的免疫细胞亚群中通过NAD(P)H-FLIM进行代谢成像，并通过FRET-FLIM进行细胞内钙信号传导成像。这种方法保留了在组织环境中研究各种细胞功能潜在分子机制的能力，从而为骨生物学提供了新的见解。
