Confocal Raman Micro-Spectroscopy for Discrimination of Glycerol Diffusivity in Ex Vivo Porcine Dura Mater

Abstract

Dura mater (DM) is a connective tissue with dense collagen, which is a protective membrane surrounding the human brain. The optical clearing (OC) method was used to make DM more transparent, thereby allowing to increase in-depth investigation by confocal Raman micro-spectroscopy and estimate the diffusivity of 50% glycerol and water migration. Glycerol concentration was obtained, and the diffusion coefficient was calculated, which ranged from 9.6 × 10^-6 to 3.0 × 10^-5 cm²/s. Collagen-related Raman band intensities were significantly increased for all depths from 50 to 200 µm after treatment. In addition, the changes in water content during OC showed that 50% glycerol induces tissue dehydration. Weakly and strongly bound water types were found to be most concentrated, playing a major role in the glycerol-induced water flux and OC. Results show that OC is an efficient method for controlling the DM optical properties, thereby enhancing the in-depth probing for laser therapy and diagnostics of the brain. DM is a comparable to various collagen-containing tissues and organs, such as sclera of eyes and skin dermis.

Keywords: collagen type I; dehydration; diffusion coefficients; glycerol; high wavenumber; hydrogen bound water; penetration; topical application.

Figure 1

Schematic illustration of the measurement of DM samples with CRM.

Figure 2

FP Raman spectra of control porcine DM at 50 µm depth (A), 50% glycerol (B), calibration curve of Raman band intensity at 485 cm⁻¹, and concentration of glycerol solution in 0–50% water (C). The red line (in C) is a result of fitting with correlation coefficient (R²) of 0.9941 and the average Raman spectra before and after 60 s treatment at 50 and 200 µm depths (D).

Figure 3

The time dependencies of DM collagen-related Raman bands at 938 (black squares), 1003 (red circles), 1247 (blue upward triangles), 1270 (green downward triangles), and 1665 cm⁻¹ (purple diamonds) after impregnation by 50% aqueous-glycerol solution from 0 to 390 s at depths 50 (A), 100 (B), 150 (C), and 200 µm (D).

Figure 4

Gaussian lines function-based deconvolution of the HWN Raman spectrum of untreated porcine DM at 50 μm (A), 50% glycerol water solution (B), and the average of Raman spectra before and after 90 s treatment at 50 and 200 μm depths (C and D), respectively.

Figure 5

The time-dependent kinetics of 3005 cm⁻¹ (tightly bound water, black), 3270 cm⁻¹ (strongly bound water, red), 3458 cm⁻¹ (weakly bound water, blue), and 3605 cm⁻¹ (unbound water, green) deconvoluted Gaussian lines at depths of 50 (A), 100 (B), 150 (C), and 200 µm (D).

Figure 6

The kinetics of total water content changes in 50% glycerol-treated porcine DM at depths: 50 (black), 100 (red), 150 (blue), and 200 µm (green) depending on treatment time.