FT-IR Spectroscopy of Brain Tissue

The detail method was previously described^{16 (link)}. Briefly, for in situ FT-IR measurements, we used an IRT-7000 IR microscope combined with an FT/IR-6100 spectrometer (Jasco Co., Tokyo, Japan). Spectra were acquired in reflection mode using a 16× Cassegrain lens and collected in the mid-IR range of 700–4000 cm⁻¹ at a resolution of 4 cm⁻¹ over 64 scans from 30 × 30-μm apertures. The reflection spectra were obtained from tissues around brain blood vessels in the cerebral cortex using lattice measurement (x-axis: 7 points, y-axis: 7 points, total of 49 spectra acquired). Sixty-four spectra were acquired from only embedding medium (OCT compound) regions and an average of these spectra was used as a common basal line for analysis of FT-IR spectral data. Smoothing and normalization of the obtained spectra were performed on the region containing the amide bands (1000–2000 cm⁻¹) using Spectra Manager Software Ver. 2 (Jasco International Co., Ltd, Tokyo, Japan). We deconvoluted the spectra for protein secondary structural analysis and calculated ratios of secondary structure contents (α-helix, β-sheet, β-turn, and random coil) from peak intensities of the amide I bands (1600–1700 cm⁻¹). The calculated ratios of secondary structures were visualized using the universal RGB code on the protein mapping analysis software (IR-SSE; JASCO Co., Ltd.)^{21 (link)}.