Tokarz Danielle, Cisek Richard, Wein Marc N, Turcotte Raphaël, Haase Christa, Yeh Shu-Chi A, Bharadwaj Srinidhi, Raphael Anthony P, Paudel Hari, Alt Clemens, Liu Tzu-Ming, Kronenberg Henry M, Lin Charles P
Advanced Microscopy Program, Center for Systems Biology and Wellman Center for Photomedicine, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, United States of America.
Department of Physical and Chemical Sciences, University of Toronto, Mississauga, Ontario, Canada.
PLoS One. 2017 Oct 24;12(10):e0186846. doi: 10.1371/journal.pone.0186846. eCollection 2017.
Osteocytes are the most abundant cell in the bone, and have multiple functions including mechanosensing and regulation of bone remodeling activities. Since osteocytes are embedded in the bone matrix, their inaccessibility makes in vivo studies problematic. Therefore, a non-invasive technique with high spatial resolution is desired. The purpose of this study is to investigate the use of third harmonic generation (THG) microscopy as a noninvasive technique for high-resolution imaging of the lacunar-canalicular network (LCN) in live mice. By performing THG imaging in combination with two- and three-photon fluorescence microscopy, we show that THG signal is produced from the bone-interstitial fluid boundary of the lacuna, while the interstitial fluid-osteocyte cell boundary shows a weaker THG signal. Canaliculi are also readily visualized by THG imaging, with canaliculi oriented at small angles relative to the optical axis exhibiting stronger signal intensity compared to those oriented perpendicular to the optical axis (parallel to the image plane). By measuring forward- versus epi-detected THG signals in thinned versus thick bone samples ex vivo, we found that the epi-collected THG from the LCN of intact bone contains a superposition of backward-directed and backscattered forward-THG. As an example of a biological application, THG was used as a label-free imaging technique to study structural variations in the LCN of live mice deficient in both histone deacetylase 4 and 5 (HDAC4, HDAC5). Three-dimensional analyses were performed and revealed statistically significant differences between the HDAC4/5 double knockout and wild type mice in the number of osteocytes per volume and the number of canaliculi per lacunar surface area. These changes in osteocyte density and dendritic projections occurred without differences in lacunar size. This study demonstrates that THG microscopy imaging of the LCN in live mice enables quantitative analysis of osteocytes in animal models without the use of dyes or physical sectioning.
骨细胞是骨骼中最丰富的细胞，具有多种功能，包括机械传感和调节骨重塑活动。由于骨细胞嵌入骨基质中，难以获取使得体内研究存在问题。因此，需要一种具有高空间分辨率的非侵入性技术。本研究的目的是探讨使用三次谐波产生（THG）显微镜作为一种非侵入性技术，用于对活小鼠的骨陷窝-骨小管网络（LCN）进行高分辨率成像。通过将THG成像与双光子和三光子荧光显微镜相结合，我们发现THG信号由骨陷窝的骨间质液边界产生，而间质液-骨细胞边界的THG信号较弱。骨小管也可通过THG成像清晰地可视化，相对于光轴呈小角度取向的骨小管比垂直于光轴（平行于图像平面）取向的骨小管表现出更强的信号强度。通过在体外对薄骨和厚骨样本中前向与背向检测的THG信号进行测量，我们发现从完整骨的LCN背向收集的THG包含向后定向和向前背向散射的THG叠加。作为一个生物学应用的例子，THG被用作一种无标记成像技术，以研究组蛋白脱乙酰酶4和5（HDAC4、HDAC5）均缺乏的活小鼠LCN中的结构变化。进行了三维分析，结果显示HDAC4/5双敲除小鼠和野生型小鼠在每体积骨细胞数量和每个骨陷窝表面积的骨小管数量上存在统计学显著差异。骨细胞密度和树突状突起的这些变化在骨陷窝大小上没有差异。本研究表明，对活小鼠的LCN进行THG显微镜成像能够在不使用染料或物理切片的情况下对动物模型中的骨细胞进行定量分析。
