Figure 1. a) Schematic diagram of Raman spectra principle. b) Typical structure of biomineralization. c) Multiple structural forms of biomineralization.
The advance of Raman spectroscopic study on biomineralization
Confocal Raman spectroscopy devices typically use an infinite correction objective to focus the pump light and a pinhole module to spatially filter the light to achieve confocal mode (Figure 2a ). The pinhole transmits light from the focal plane to the detector. A diode-pumped solid-state laser is utilized as a monochromatic light source.[85,94,95] A piezoelectric-driven nanopositioner was used to position the specimen to enhance the required accuracy of the scan.[82,85] To detect the Raman mapping signal, a holographic imaging spectrometer with an attached CCD camera was used. In Raman spectroscopy, wavelengths of λ = 512, 785, 830, 980, and 1064 nm are the most common.[96–99]Excitation sources at lower visible wavelengths cause intense photoluminescence (or fluorescence) of organic macromolecules in biomineralized tissue, which can mask Raman peaks to affect the analysis of Raman spectra.[84,100–102] The wavelength of the excitation source is usually chosen to be λ =512 or 785 nm, which will effectively avoid the occurrence of photoluminescence.[103–105] The selection of excitation light power also plays an important role in the analysis of the biomineralized process(Figure 2b ). When the laser power is low, the signal-to-noise ratio of the Raman spectrum will be poor, affecting further analysis results. When the power is high, it may damage the tissue or light quench the signal.
Important for Raman spectroscopy is the acquisition and processing of samples (Figure 2b ). It can make the sample keep the original chemical composition and structure to get better results in Raman spectroscopy analysis. When bone tissue or teeth are obtained, the operation requires avoiding the use of metal instruments to avoid scratching the tissue. The acquired tissue needs to be measured for Raman spectroscopy promptly to prevent tissue denaturation, which can lead to inaccurate results.[106,107] If the timely measurement is not possible, the tissue can be fixed and decalcified by organic matter and placed in cryopreservation or directly in liquid nitrogen for preservation.[108–112] It is noteworthy that during the decalcification process, the hydrochloric acid/ethylenediamine tetraacetic acid (HCl/EDTA) mixture removed most of the minerals. Still, it affected the secondary structure of the collagen.[113] In contrast, the bones were treated with HCl alone, leaving the collagen structure well preserved in the shortest time. And the bone decalcified by CH2O2 had the highest collagen quality parameters. The interference of ethanol used to fix the tissue appears to be relatively less pronounced than that of glycerol.[114,115] The location of dissection, fixation, and embedding has to be considered, which can also impact the results of Raman spectroscopy analysis. During subsequent cryopreservation of the samples, the Raman spectra of the frozen bone tissue showed a significant decrease in the amide I and amide III bands, the proline residues were consistent with fresh tissue, and the mineral crystallinity decreased significantly after only one freezing.[116,117] The mineral carbonate content did not deviate significantly during freeze-thaw.
The direction of incident light and the direction of polarization will also have an impact on the final result. The intensity of the Raman band is not related to the a-axis or b-axis direction of the single hydroxyapatite crystal but only to the c-axis direction.[118] Both minerals and collagen fibers present in biomineralized tissues are directional.[119,120] Significant bands such as ν1 PO43- and amide I, which are used to determine the mineral and organic composition, are very sensitive to the direction of incident light and the direction of polarization,[121] whereas bands like amide III, ν2PO43- and ν4PO43- are less susceptible to the influence of directional objects.[122] Therefore, the incident light direction and polarization direction should be kept consistent among samples in Raman spectroscopy testing. To ensure the confidence of the results, the analysis of relevant Raman spectroscopy should be accurate and avoid errors.