Analysis of Trabecular Bone Architecture using Two Photon Fluorescence Microscopy

Abstract

Biomechanical competence of trabecular bone is dependent on the makeup of its architecture. Alterations in the trabecular architecture can lead to fractures in metabolic bone diseases like osteoporosis. Therefore, it is paramount to understand the signaling mechanisms that dictate these changes in bone growth and fracture repair. Two photon fluorescence microscopy revolutionized the imaging of biological specimens utilizing its unique capabilities. The three-dimensional (3D) imaging based on nonlinear excitation of the fluorophores brings multiple advantages for imaging skeletal tissue. However, noise generated by the subsurface signal and auto-fluorescence of the local tissue make imaging of trabecular bone problematic. Imaging of calcified tissue presents a unique challenge to address the aberrations produced through the noise generated. Also a general practice of immunolabeling of the plasticized bone for antigen stability are to be optimized. We demonstrate here for the first time using two-photon fluorescence imaging of trabecular bone and its architecture identifying the structural differences and cell populations lining the trabecular cavity and also the cells embedded in it. Furthermore, we developed a shortened method of immunohistochemistry for plastic embedded bone tissue providing antigen stability for antibody labeling. Two photon fluorescence imaging greatly reduces photo damage and helps image of specimens of uneven planes to submicrometer resolution making this an ideal source for imaging in vivo signaling of trabecular bone. We demonstrate here labelling of multi colored fluorophores measuring Smad and ERK activity in trabecular bone growth in mice that are systemically injected with Bone Morphogenetic Protein 2 (BMP2). We optimized the conditions for in vivo imaging of bone tissue that is calcified and plasticized. We demonstrate here two photon fluorescence microscopy of the trabecular bone can be used for understanding the molecular mechanisms which control bone growth and development in vivo.