50 years of scanning electron microscopy of bone—a comprehensive overview of the important discoveries made and insights gained into bone material properties in health, disease, and taphonomy

Abstract

Bone is an architecturally complex system that constantly undergoes structural and functional optimisation through renewal and repair. The scanning electron microscope (SEM) is among the most frequently used instruments for examining bone. It offers the key advantage of very high spatial resolution coupled with a large depth of field and wide field of view. Interactions between incident electrons and atoms on the sample surface generate backscattered electrons, secondary electrons, and various other signals including X-rays that relay compositional and topographical information. Through selective removal or preservation of specific tissue components (organic, inorganic, cellular, vascular), their individual contribution(s) to the overall functional competence can be elucidated. With few restrictions on sample geometry and a variety of applicable sample-processing routes, a given sample may be conveniently adapted for multiple analytical methods. While a conventional SEM operates at high vacuum conditions that demand clean, dry, and electrically conductive samples, non-conductive materials (e.g., bone) can be imaged without significant modification from the natural state using an environmental scanning electron microscope. This review highlights important insights gained into bone microstructure and pathophysiology, bone response to implanted biomaterials, elemental analysis, SEM in paleoarchaeology, 3D imaging using focused ion beam techniques, correlative microscopy and in situ experiments. The capacity to image seamlessly across multiple length scales within the meso-micro-nano-continuum, the SEM lends itself to many unique and diverse applications, which attest to the versatility and user-friendly nature of this instrument for studying bone. Significant technological developments are anticipated for analysing bone using the SEM.

Peptide-Enriched Silk Fibroin Sponge and Trabecular Titanium Composites to Enhance Bone Ingrowth of Prosthetic Implants in an Ovine Model of Bone Gaps

“…The BSE-SEM analysis was performed to specifically discriminate the mineralized matrix in contact with the implant. This technique is known to specifically detect highly calcified matrix, but it is unable to identify the osteoid due to its low level of calcification (Shah et al, 2019). By contrast, the specific histological dichromic staining used to compute the tissue fractions by optical microscopy enabled to sharply distinguish osteoid matrix and highly mineralized bone (Fabbro et al, 2017;Varoni et al, 2018;Shah et al, 2019;Ragone et al, 2020).…”

“…Backscattered electron microscopy (BSE-SEM) offers a considerable insight into the mineralized tissues at the implantbone interface (Shah et al, 2019). BSE-SEM is particularly useful to discern one material from another, since the yield of the collected backscattered electrons increases monotonically with the specimen's atomic number, thus allowing to distinguish elements with atomic number differences of at least three (Rocchietta et al, 2007).…”

Ultrastructural Pathology of Atherosclerosis, Calcific Aortic Valve Disease, and Bioprosthetic Heart Valve Degeneration: Commonalities and Differences

“…The consecutive grinding of the sample permits layer-by-layer scanning that is principally similar to the sectioning but does not cause any damage to the mineralised tissues. Although backscattered scanning electron microscopy was earlier utilised for the investigation of calcium phosphate transformation in human samples [ 20 , 21 , 22 , 23 ], it has never been applied in cardiovascular pathology.…”

Three-dimensional Structural Interrelations between Cells, Extracellular Matrix and Mineral in Vertebrate Mineralization

“…Such information is difficult to obtain because it requires nanometer resolution and a large field of view in the range of tens of microns, in order to visualize several cells with their processes (21). In this study, focused ion beam-scanning electron microscopy (FIB-SEM) (22) was used to examine simultaneously cells, ECM, and mineral deposits in turkey leg tendon (TLT) as a model system for collagen mineralization. Normally mineralizing tendons are representative of other vertebrate mineralizing tissues and are structurally more advantageous for study and analysis (23,24).…”