Intravital imaging of osteocytes in mouse calvaria using third harmonic generation microscopy.
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 hi...
| Main Authors: | , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2017-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC5655444?pdf=render |
| _version_ | 1818476177651662848 |
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| author | Danielle Tokarz Richard Cisek Marc N Wein Raphaël Turcotte Christa Haase Shu-Chi A Yeh Srinidhi Bharadwaj Anthony P Raphael Hari Paudel Clemens Alt Tzu-Ming Liu Henry M Kronenberg Charles P Lin |
| author_facet | Danielle Tokarz Richard Cisek Marc N Wein Raphaël Turcotte Christa Haase Shu-Chi A Yeh Srinidhi Bharadwaj Anthony P Raphael Hari Paudel Clemens Alt Tzu-Ming Liu Henry M Kronenberg Charles P Lin |
| author_sort | Danielle Tokarz |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-10T09:22:04Z |
| format | Article |
| id | doaj.art-34f4d0b94dfe4ddb8a5f191b352a9eed |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-10T09:22:04Z |
| publishDate | 2017-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-34f4d0b94dfe4ddb8a5f191b352a9eed2022-12-22T01:54:40ZengPublic Library of Science (PLoS)PLoS ONE1932-62032017-01-011210e018684610.1371/journal.pone.0186846Intravital imaging of osteocytes in mouse calvaria using third harmonic generation microscopy.Danielle TokarzRichard CisekMarc N WeinRaphaël TurcotteChrista HaaseShu-Chi A YehSrinidhi BharadwajAnthony P RaphaelHari PaudelClemens AltTzu-Ming LiuHenry M KronenbergCharles P LinOsteocytes 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.http://europepmc.org/articles/PMC5655444?pdf=render |
| spellingShingle | Danielle Tokarz Richard Cisek Marc N Wein Raphaël Turcotte Christa Haase Shu-Chi A Yeh Srinidhi Bharadwaj Anthony P Raphael Hari Paudel Clemens Alt Tzu-Ming Liu Henry M Kronenberg Charles P Lin Intravital imaging of osteocytes in mouse calvaria using third harmonic generation microscopy. PLoS ONE |
| title | Intravital imaging of osteocytes in mouse calvaria using third harmonic generation microscopy. |
| title_full | Intravital imaging of osteocytes in mouse calvaria using third harmonic generation microscopy. |
| title_fullStr | Intravital imaging of osteocytes in mouse calvaria using third harmonic generation microscopy. |
| title_full_unstemmed | Intravital imaging of osteocytes in mouse calvaria using third harmonic generation microscopy. |
| title_short | Intravital imaging of osteocytes in mouse calvaria using third harmonic generation microscopy. |
| title_sort | intravital imaging of osteocytes in mouse calvaria using third harmonic generation microscopy |
| url | http://europepmc.org/articles/PMC5655444?pdf=render |
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