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

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.

Hormonal Regulation of Osteocyte Perilacunar and Canalicular Remodeling in the Hyp Mouse Model of X-Linked Hypophosphatemia

“…Calvarial osteocytes were imaged with an in-house three-photon excitation microscope. (23) Images were obtained of four areas of the calvaria bordering the sagittal and coronal sutures in the frontal and parietal bones. Briefly, a 5-MHz repetition rate, 370-fs duration pulses, and 1550-nm wavelength laser (FLCPA-01CCNL41; Calmar, Palo Alto, CA) was coupled to a polarization maintaining large mode area single-mode photonic crystal fiber (LMA40; NKT Photonics, Birkerod, Denmark) for pulse compression.…”

3‐dimensional visualization of implant‐tissue interface with the polyethylene glycol associated solvent system tissue clearing method

“…44,45 A multi-photon microscope is required for its detection. 46,47 resolution, which is far better than any current µCT equipment. In addition, SHG image has no halation surrounding metal implant, which is a common artefact for µCT analysis.…”

Image matters: Development of a novel staining method, the RGB-trichrome, as a reliable tool for the study of the musculoskeletal system

“…Indeed, a recent study raised the intriguing possibility that osteocytes participate in the regulation of fat body mass through sensing changes in bone strain 33 . In this context, osteocytes seem to play central roles in bone physiology and pathophysiology 32, 34–37 ; osteocyte dendritic network being the morphological counterpart of the functional coupling of osteocytes among them and with other bone cell types 26, 29, 30 . Although ground bone tissue sections and classical staining methods, such as silver impregnation 17, 38 , allow visualization of the osteocyte lacuna-canalicular network, the novel high-resolution and 3D imaging technologies have provided during the last decade detailed views of the osteocyte syncytia 26–29, 39, 40 .…”