Intravital imaging of osteocytes in mouse calvaria using third harmonic generation microscopy.

Danielle Tokarz,Marc N Wein,Srinidhi Bharadwaj,Henry M Kronenberg,Anthony P Raphael,Raphaël Turcotte,Shu-Chi A Yeh,Clemens Alt,Christa Haase,Tzu-Ming Liu,Charles P Lin,Hari Paudel,Richard Cisek,Paul Mcneil

doi:10.1371/journal.pone.0186846

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.

Highlights

Osteocytes are the most abundant cell in bone, forming a large surface area network referred to as the osteocytic lacunar-canalicular network (LCN)
Due to the fact that osteocytes and their processes are surrounded by interstitial fluid, it is likely that third harmonic generation (THG) microscopy is visualizing two distinct heterogeneities: one between the bone and the interstitial fluid, and another between the interstitial fluid and the cell
THG microscopy with laser excitation wavelengths of 1550 nm to 1700 nm can be used for quantitative analysis of osteocytes in mouse calvarium in vivo

Summary

Introduction

Osteocytes are the most abundant cell in bone, forming a large surface area network referred to as the osteocytic lacunar-canalicular network (LCN). Existing high resolution imaging techniques to visualize the osteocyte LCN ex vivo include light microscopy, scanning electron microscopy, transmission electron microscopy, confocal microscopy, X-ray computed tomography, transmission X-ray microscopy, and ptychographic computed tomography [1]. These techniques require ex vivo sample preparation such as thin sectioning of embedded or cryopreserved tissues or plastic-embedded thick tissue sectioning and serial milling [2]. Three-dimensional imaging can be more achieved with plastic-embedded thick tissue sections; harsh embedding conditions and tissue sectioning artefacts often limit the representation of the images to the in vivo condition [2]

Objectives

Methods

Results

Conclusion