Characterization of human trabecular bone across multiple length scales: A correlative approach combining X-ray tomography with LaserFIB and plasma FIB-SEM

Three-dimensional correlative multimodal and multiscale imaging is an emerging method for investigating the complex hierarchical structure of biological materials such as bone. This approach synthesizes images acquired across multiple length scales, for the same region of interest, to provide a comprehensive view of the material structure of a sample. Here, we develop a workflow for the structural analysis of human trabecular bone using a femtosecond laser to produce a precise grid to facilitate correlation between imaging modalities and identification of structures of interest, in this case, a single trabecula within a volume of trabecular bone. Through such image registration, high resolution X-ray microscopy imaging revealed fine architectural details, including the cement line and bone cell lacunae of the selected bone unit. The selected bone volume was exposed with a combination of manual polishing and site-specific femtosecond laser ablation and then examined with plasma focused ion beam-scanning electron microscopy. This reliable and versatile correlation approach has the potential to be applied to a variety of biological tissues and traditional engineered materials. The proposed workflow has the enhanced capability for generating highly resolved structural data for a better understanding of the architectural features of a material spanning its macroscopic to nanoscopic levels.