Charles P. Lin

Histomorphometry and Micro-CT are commonly used to assess bone remodeling and bone microarchitecture. These approaches typically require separate cohorts of animals to analyze 3D morphological changes and involve time-consuming immunohistochemistry preparation. Intravital Microscopy (IVM) in combination with mouse genetics may represent an attractive option to obtain bone architectural measurements while performing longitudinal monitoring of dynamic cellular processesin vivo. In this study we utilized two-photon, multicolor fluorescence IVM together with a lineage tracing reporter mouse model to image skeletal stem cells (SSCs) in their calvarial suture niche and analyze their differentiation fate after stimulation with an agonist of the canonical Wnt pathway (recombinant Wnt3a). Ourin vivohistomorphometry analyses of bone formation, suture volume, and cellular dynamics showed that recombinant Wnt3a induces new bone formation, differentiation and incorporation of SSCs progeny into newly forming bone. IVM technology can therefore provide additional dynamic 3D information to the traditional static 2D histomorphometry.

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.

Photoconversion, an irreversible shift in a fluorophore emission spectrum after light exposure, is a powerful tool for marking cellular and subcellular compartments and tracking their dynamics in vivo. This paper reports on the photoconversion properties of Di‐8‐ANEPPS, a commercially available membrane dye. When illuminated with near‐infrared femtosecond laser pulses, Di‐8‐ANEPPS undergoes multiphoton photoconversion as indicated by the supralinear dependence of the conversion rate ρ_pc on the incident power (), and by the ability to photoconvert a thin optical section in a three‐dimensional matrix. The characteristic emission spectrum changed from red to blue, and ratiometric analysis on single cells in vitro revealed a 65‐fold increase in the blue to red wavelength ratio after photoconversion. The spectral shift is preserved in vivo for hours, making Di‐8‐ANEPPS a useful dye for intravital cell marking and tracking applications. magnified image

The bone marrow is an important site where all blood cells are formed from hematopoietic stem cells and where hematologic malignancies such as leukemia emerge. It is also a frequent site for metastasis of solid tumors such as breast cancer and prostate cancer. Intravital microscopy is a powerful tool for studying the bone marrow with single cell and sub-cellular resolution. To improve optical access to this rich biological environment, plasma-mediated laser ablation with sub-microjoule femtosecond pulses was used to thin cortical bone. By locally removing a superficial layer of bone (local laser osteotomy), significant improvements in multiphoton imaging were observed in individual bone marrow compartments in vivo. This work demonstrates the utility of scanning laser ablation of hard tissue with sub-microjoule pulses as a preparatory step to imaging.

Adult zebrafish are being increasingly used as a model in cancer and stem cell research. Here we describe an integrated optical system that combines a laser scanning confocal microscope (LSCM) and an in vivo flow cytometer (IVFC) for simultaneous visualization and cell quantification. The system is set up specifically for non‐invasive tracking of both stationary and circulating cells in adult zebrafish (casper) that have been engineered to be optically transparent. Confocal imaging in this instrument serves the dual purpose of visualizing fish tissue microstructure and an imaging‐based guide to locate a suitable vessel for quantitative analysis of circulating cells by IVFC. We demonstrate initial testing of this novel instrument by imaging the transparent adult zebrafish casper vasculature and tracking circulating cells in CD41‐GFP/Gata1‐DsRed transgenic fish whose thrombocytes/erythrocytes express the green and red fluorescent proteins. In vivo measurements allow cells to be tracked under physiological conditions in the same fish over time, without drawing blood samples or sacrificing animals. We also discuss the potential applications of this instrument in biomedical research. © 2011 International Society for Advancement of Cytometry

We provide an overview of the methods used to label circulating cells for fluorescence detection by in vivo flow cytometry. These methods are useful for cell tracking in small animals without the need to draw blood samples and are particularly useful for the detection of circulating cancer cells and quantification of circulating immune cells. © 2011 International Society for Advancement of Cytometry.

Detection and quantification of rare circulating cells in biological tissues is an important problem and has many applications in biomedical research. Current methods normally involve extraction of blood samples and counting of cells ex vivo, or the use of microscopy-based fluorescence in vivo flow cytometry. The goal of this work is to develop an instrument for non-invasively enumerating very rare circulating cells in small animals with diffuse light with several orders of magnitude sensitivity improvement versus current approaches. In this work, we describe the design of our system and show that single, fluorescent microspheres can be detected in limb-mimicking optical flow phantoms with varying optical properties chosen to simulate in vivo conditions. Further, we demonstrate single cell counting capabilities using fluorescently (Vybrant-DiD) labeled Jurkat and Multiple Myeloma cells. Ongoing work includes in vivo testing and characterization of our system in mice.