Intravital imaging of orthotopic and ectopic bone
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(2020) 40:26
Inflammation and Regeneration
REVIEW
Open Access
Intravital imaging of orthotopic and ectopic bone Kunihiko Hashimoto1,2, Takashi Kaito1 , Junichi Kikuta2 and Masaru Ishii2*
Abstract Bone homeostasis is dynamically regulated by a balance between bone resorption by osteoclasts and bone formation by osteoblasts. Visualizing and evaluating the dynamics of bone cells in vivo remain difficult using conventional technologies, including histomorphometry and imaging analysis. Over the past two decades, multiphoton microscopy, which can penetrate thick specimens, has been utilized in the field of biological imaging. Using this innovative technique, the in vivo dynamic motion of bone metabolism-related cells and their interactions has been revealed. In this review, we summarize previous approaches used for bone imaging and provide an overview of current bone tissue imaging methods using multiphoton excitation microscopy. Keywords: Intravital imaging, Multiphoton microscopy, Regeneration, Bone, Ectopic bone
Background Bone homeostasis is dynamically regulated internally and externally via interactions among osteoblasts, osteoclasts, osteocytes, other organs, and other cell types. Conventional histological evaluation of bone tissue has been performed on histological sections ex vivo; this evaluation is limited to “static” analysis of cell morphology and gene and protein expression. Bone histomorphometric analysis can quantify the rates of bone formation and resorption during a certain period but cannot visualize real-time “dynamic” behaviors, interactions, and functions among osteoblasts, osteoclasts, osteocytes, and other cell types. Because conventional laser beams used for microscopic observation cannot penetrate the thick mineralized cortical bone, numerous approaches including micro-computed tomography imaging [1–4], Raman microspectroscopy imaging [5, 6], and magnetic resonance imaging [7] have been utilized for indirectly visualizing the inside of the bone through time-lapsed in vivo imaging. However, it remains difficult to perform real-time analysis of bone dynamics. * Correspondence: [email protected] 2 Department of Immunology and Cell Biology, Graduate School of Medicine & Frontier Biosciences, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan Full list of author information is available at the end of the article
The advent of multiphoton microscopy has launched a new era in the field of biological imaging of the bone. The longer wavelength light sources used in multiphoton excitation microscopy enable deeper tissue penetration because less scattering occurs, and there is less laser-induced damage to the tissue; the lower levels of photobleaching of the imaged fluorophores by using near-infrared lasers enables longer observation times compared with those of conventional fluorescent microscopy. Using this innovative technique, the dynamic motion of osteoblasts and osteoclasts and their interactions inside skeletal bone have been clarified. We recently established a novel i
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