Faculty Collaboration Database

Intravital imaging of osteocytes in mouse calvaria using third harmonic generation microscopy. PLoS One 2017;12(10):e0186846

Date

10/25/2017

Pubmed ID

29065178

Pubmed Central ID

PMC5655444

DOI

10.1371/journal.pone.0186846

Scopus ID

2-s2.0-85032034898 (requires institutional sign-in at Scopus site)   32 Citations

Abstract

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.

Author List

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

Author

Melissa Wein MD Assistant Professor in the Radiology department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Animals
Histone Deacetylases
Intravital Microscopy
Mice
Mice, Knockout
Osteocytes
Skull