Mapping Bone Surface Composition Using Real-Time Surface Tracked Micro-Raman Spectroscopy

Abstract

The surface of bone tells a story – one that is worth a thousand words – of how it is built and how it is repaired. Chemical (i.e., composition) and physical (i.e., morphology) characteristics of the bone surface are analogous to a historical record of osteogenesis and provide key insights into bone quality. Analysis of bone chemistry is of particular relevance to the advancement of human health, cell biology, anthropology/archaeology, and biomedical engineering. Although scanning electron microscopy remains a popular and versatile technique to image bone across multiple length scales, limited chemical information can be obtained. Micro-Raman spectroscopy is a valuable tool for nondestructive chemical/compositional analysis of bone. However, signal integrity losses occur frequently during wide-field mapping of non-planar surfaces. Samples for conventional Raman imaging are, therefore, rendered planar through polishing or sectioning to ensure uniform signal quality. Here, we demonstrate ν₁ PO₄^3- and ν₁ CO₃^2- peak intensity losses where the sample surface and the plane of focus are offset by over 1–2 μm when underfocused and 2–3 μm when overfocused at 0.5–1 s integration time (15 mW, 633 nm laser). A technique is described for mapping the composition of the inherently irregular/non-planar surface of bone. The challenge posed by the native topology characteristic of this unique biological system is circumvented via real-time focus-tracking based on laser focus optimization by continuous closed-loop feedback. At the surface of deproteinized and decellularized/defatted sheep tibial cortical bone, regions of interest up to 1 mm² were scanned at micrometer and submicrometer resolution. Despite surface height deviations exceeding 100 μm, it is possible to seamlessly probe local gradients in organic and inorganic constituents of the extracellular matrix as markers of bone metabolism and bone turnover, blood vessels and osteocyte lacunae, and the rope-like mineralized bundles that comprise the mineral phase at the bone surface.