Figure 2. a) Schematic diagram of the optical path of Raman spectrometer. b) The relevant study conditions of Raman spectroscopy for biomineralization.
Physicochemical study of Raman spectroscopy on biomineralization
It is known that each peak illustrated by Raman spectroscopy corresponds to a specific molecular bond vibration, which includes both the single chemical bond and the vibration of a group composed of several chemical bonds involved in biomineralization. The Raman spectral peaks in biomineralized tissues include those attributed to inorganic and organic substances.[123–125]The main mineral component in teeth and bones is hydroxyapatite, which exhibits ν1PO43-, ν2PO43-, ν3PO43-, and ν4PO43- fundamental frequency modes in the Raman spectrum (Table 1 ). The sharpest and most intense band, ν1 PO43-, is associated with the symmetric stretching of the oxygen atom tetrahedra around the phosphorus atom, with the main peak located at 960 cm-1, representing the characteristic peak of hydroxyapatite.[59,126] Collagen plays a role in the induction of hydroxyapatite nucleation and growth in bone and tooth mineralization.[127–129] The three main amide bands of collagen are amide I (1675 cm-1), amide II (1565 cm-1), and amide Ⅲ (1272 cm-1). Among them, ν1PO43- and amide Ⅲ are susceptible to the direction of incident light and the direction of polarization. This phenomenon can be used to analyze the orientation of the crystal and collagen fibers.[3,33,130,131] In addition to the characteristic peaks of collagen and hydroxyapatite, there are other peaks, including ν1CO32-, ν2CO32-, ν4CO32-, ν(C-C), δ(CH2) and ν(O-H), etc. (Table 1 ).
Pathological mineralization, such as tumor micro mineralization, urinary stones, and atherosclerosis, is distinct from the mineral and organic composition of physiological mineralization.[7,8,132–134] In atherosclerosis, the characteristic marker bands of the protein are located at 1181, 1209, 1272, 1410, 1565, and 1675 cm-1, corresponding to ν(C-O-C), ω(CH2), amide III, β(CH2), amide II and amide I, respectively (Table 1 ). One of the minerals belongs to carbonate apatite, which also has the characteristic peak of ν1 PO43- at 960 cm-1.[135]Microcalcification of tumor tissue is usually due to oversized tumor tissue, resulting in deep tumors not receiving nutrients These tumor cells eventually calcify into minerals after necrosis.[66,136] The calcified composition is mainly that of apatite. However, due to the heterogeneity of the tumor, differences in organic matter composition can exist. The cause of urinary stones is the nucleation of insoluble microcrystals in the urethra and kidney tubules.[64,137] These insoluble microcrystals are various types of calcium oxalate crystals.[138,139] In the urinary environment, the nucleation and dissolution processes of calcium oxalate crystals are in a dynamic equilibrium, while the true stone formation process is still unknown.
Morphology and other characterization of biomineralization
As described above, the specific mineralization information could be readily obtained by Raman spectroscopy. Meanwhile, other characterization strategies have been also utilized to analyze the morphology and crystal morphology of the biomineralization. Scanning electron microscopy (SEM) provides nanoscale resolution of sample morphological features and, together with an energy dispersive spectrometer (EDS), it can provide quantitative elemental analysis of mineralized tissues.[140,141] X-ray diffraction (XRD) is the main method for studying the physical phase and crystal structure of minerals.[9,142,143] The crystallographic characteristics such as composition, crystalline shape, intra-molecular bonding patterns, molecular conformation, and conformation are obtained by the diffraction phenomenon of minerals irradiated by X-rays to different degrees. X-ray computed tomography uses precisely collimated x-ray beams to obtain macroscopic structural images of biomineralized tissues.[144,145]Multiple analysis techniques are needed to obtain comprehensive nano-structural information on the general nature of relevant biomineralization.