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.