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Intravital imaging of osteocytes in mouse calvaria using third harmonic generation microscopy
Tokarz, Danielle1; Cisek, Richard2; Wein, Marc N.3; Turcotte, Raphael1; Haase, Christa1; Yeh, Shu-Chi A.1; Bharadwaj, Srinidhi1; Raphael, Anthony P.1,4; Paudel, Hari1; Alt, Clemens1; Liu, Tzu-Ming5; Kronenberg, Henry M.3; Lin, Charles P.1
2017-10-24
Source PublicationPLOS ONE
ISSN1932-6203
Volume12Issue:10
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.

DOIhttp://doi.org/10.1371/journal.pone.0186846
URLView the original
Indexed BySCI
Language英语
WOS Research AreaScience & Technology - Other Topics
WOS SubjectMultidisciplinary Sciences
WOS IDWOS:000413568900049
PublisherPUBLIC LIBRARY SCIENCE
The Source to ArticleWOS
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Cited Times [WOS]:16   [WOS Record]     [Related Records in WOS]
Document TypeJournal article
CollectionFaculty of Health Sciences
Corresponding AuthorTokarz, Danielle; Lin, Charles P.
Affiliation1.Advanced Microscopy Program, Center for Systems Biology and Wellman Center for Photomedicine, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, United States of America
2.Department of Physical and Chemical Sciences, University of Toronto, Mississauga, Ontario, Canada
3.Endocrine Unit, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, United States of America
4.Dermatology Research Centre, The University of Queensland Diamantina Institute, Translational Research Institute, Brisbane, Queensland, Australia
5.Institute of Biomedical Engineering and Molecular Imaging Center, National Taiwan University, Taipei, Taiwan. Faculty of Health Sciences, University of Macau, Macau SAR, China
Recommended Citation
GB/T 7714
Tokarz, Danielle,Cisek, Richard,Wein, Marc N.,et al. Intravital imaging of osteocytes in mouse calvaria using third harmonic generation microscopy[J]. PLOS ONE,2017,12(10).
APA Tokarz, Danielle.,Cisek, Richard.,Wein, Marc N..,Turcotte, Raphael.,Haase, Christa.,...&Lin, Charles P..(2017).Intravital imaging of osteocytes in mouse calvaria using third harmonic generation microscopy.PLOS ONE,12(10).
MLA Tokarz, Danielle,et al."Intravital imaging of osteocytes in mouse calvaria using third harmonic generation microscopy".PLOS ONE 12.10(2017).
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