Intravital imaging of osteocytes in mouse calvaria using third harmonic generation microscopy.
Intravital imaging of osteocytes in mouse calvaria using third harmonic generation microscopy.
复制标题
DOI：
10.1371/journal.pone.0186846
复制
发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Lin CP
中科院分区：
文献类型：
--
作者：
Tokarz D;Cisek R;Wein MN;Turcotte R;Haase C;Yeh SA;Bharadwaj S;Raphael AP;Paudel H;Alt C;Liu TM;Kronenberg HM;Lin CP
骨细胞是骨骼中最丰富的细胞，具有多种功能，包括机械传感和骨重塑活动的调节。由于骨细胞嵌入骨基质中，它们的不可接近性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。本研究的目的是研究三次谐波发生 (THG) 显微镜作为一种无创技术对活体小鼠腔隙-小管网络 (LCN) 进行高分辨率成像的用途。通过结合二光子和三光子荧光显微镜进行 THG 成像，我们发现 THG 信号是从腔隙的骨-间质液边界产生的，而间质液-骨细胞边界显示较弱的 THG 信号。泪管也可以通过 THG 成像轻松可视化，与垂直于光轴（平行于图像平面）定向的泪管相比，相对于光轴以小角度定向的泪管表现出更强的信号强度。通过离体测量薄骨样本与厚骨样本中的前向与外延检测到的 THG 信号，我们发现从完整骨的 LCN 中外延收集的 THG 包含向后定向和后向散射的前向 THG 的叠加。作为生物学应用的一个例子，THG 被用作无标记成像技术来研究缺乏组蛋白脱乙酰酶 4 和 5（HDAC4、HDAC5）的活体小鼠 LCN 的结构变化。进行三维分析并揭示了 HDAC4/5 双敲除小鼠和野生型小鼠之间在每体积骨细胞数量和每腔隙表面积的骨小管数量方面存在统计学显着差异。骨细胞密度和树突投射的这些变化发生时腔隙大小没有差异。这项研究表明，活体小鼠 LCN 的 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。
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.
登录
查看更多内容
影响因子：
4.1
作者：
Kalajzic, Ivo;Matthews, Brya G.;Torreggiani, Elena;Harris, Marie A.;Pajevic, Paola Divieti;Harris, Stephen E.
通讯作者：
Harris, Stephen E.
影响因子：
4
作者：
Barad, Y;Eisenberg, H;Silberberg, Y
通讯作者：
Silberberg, Y
DOI：
10.1109/jstqe.2010.2065796
发表时间：
2011-07-01
影响因子：
4.9
作者：
Cisek, Richard;Barzda, Virginijus;Shik, Alexander
通讯作者：
Shik, Alexander
DOI：
10.1007/978-1-61779-415-5_26
发表时间：
2012-01-01
期刊：
BONE RESEARCH PROTOCOLS, SECOND EDITION
影响因子：
--
作者：
Dallas, Sarah L.;Veno, Patricia A.
通讯作者：
Veno, Patricia A.
影响因子：
3.6
作者：
FREUND, I;DEUTSCH, M
通讯作者：
DEUTSCH, M