Label-free imaging of bone multiscale porosity and interfaces using third-harmonic generation microscopy

Abstract

Interfaces provide the structural basis of essential bone functions. In the hierarchical structure of bone tissue, heterogeneities such as porosity or boundaries are found at scales ranging from nanometers to millimeters, all of which contributing to macroscopic properties. To date, however, the complexity or limitations of currently used imaging methods restrict our understanding of this functional integration. Here we address this issue using label-free third-harmonic generation (THG) microscopy. We find that the porous lacuno-canalicular network (LCN), revealing the geometry of osteocytes in the bone matrix, can be directly visualized in 3D with submicron precision over millimetric fields of view compatible with histology. THG also reveals interfaces delineating volumes formed at successive remodeling stages. Finally, we show that the structure of the LCN can be analyzed in relation with that of the extracellular matrix and larger-scale structures by simultaneously recording THG and second-harmonic generation (SHG) signals relating to the collagen organization.

Intravital Multiphoton Imaging of the Bone and Bone Marrow Environment

“…Parallel collagen bundles in compact and trabecular bone elicit strong SHG signals. The signal intensities have been shown to directly correlate with the packing density and degree of fiber alignment . Areas of low collagen content, such as bone marrow cavities and osteocyte lacunae, remain dark (Fig.…”

“…Label‐free second and third harmonic generation (SHG/THG) microscopy has emerged as a powerful imaging approach to visualize the calcified and densely packed network of collagen fibers in the bone providing detailed information on the bone microarchitecture, endosteal surfaces, and the bone porosity . SHG signals result from the near‐simultaneous arrival of two photons at dense, noncentrosymmetric structures, such as collagen fibers.…”

“…The THG signal is elicited by three incoming photons that are converted into one emitted photon at interfaces and heterogeneities in tissues, such as water–lipid interfaces or the endosteal surface in bone . As nonlinear optical processes, both SHG and THG signals decrease sharply away from the imaging focus allowing optical sectioning and three‐dimensional tissue reconstruction without the need for a pinhole . In general, the THG signal is less probable than the SHG signal; however, the signal intensity of both harmonic generations strongly depends on the spatial orientation and the scattering properties of the tissue and the imaging depth.…”

2D and 3D Segmentation of Uncertain Local Collagen Fiber Orientations in SHG Microscopy

“…Collagen structures in SHG images have been analyzed in several publications [2,25,5,1,22,15,11]. They were analyzed in tissue [2] and bones [5,25]. Rao et al [15] presented how Fourier analysis can be used to investigate the orientation of collagen fibers.…”

Three‐dimensional intravital imaging in bone research

“…Most recently, third‐harmonic generation (THG) has also been used for label‐free imaging in bone. In particular, THG can even image the star‐shaped osteocytes together with the porous lacuno‐canalicular network (LCN), which is a cellular level structure that reveals the geometry of osteocytes within bone matrices , as shown in Figure B . Tokarz et al showed that THG signal is mainly produced from the bone‐interstitial fluid boundary of the lacuna, while it is weaker from the interstitial fluid‐osteocyte boundary.…”