Design for Assembly of a Confocal System Applied to Depth Profiling in Biological Tissue Using Raman Spectroscopy

This work presents the development of a Z-depth system for Confocal Raman Spectroscopy (CRS), which allows for the acquisition of Raman spectra both at the surface and at depth profile in heterogeneous samples. The proposed CRS system consists of the coupling of a commercial 785 nm Raman Probe Bifurcated (RPB) with a 20x/0.40 infinity plan achromatic polarizing microscope objective, a Long Working Distance (LWD) of 1.2 cm, and a 50 μm core-multimode optical fiber used as a pinhole filter. With this implementation, it is possible to achieve both a high spatial resolution of approximately 16.2 μm and a spectral resolution of ∼14 cm−1, which is determined by the FWHM of the thin 1004 cm−1 Raman profile band. The system is configured to operate within 400–1800 cm−1 spectral windows. The implementation of a system of this nature offers a favorable cost–benefit ratio, as commercial CRS is typically found in high-cost environments such as cosmetics, pharmaceutical, and biological laboratories. The proposed system is low-cost and employs a minimal set of optical components to achieve functionality comparable to that of a confocal Raman microscope. High signal-to-noise ratio (SNR) Raman spectra (∼660.05 at 1447 cm−1) can be obtained with short integration times (∼25 s) and low laser power (30–35 mW) when analyzing biological samples such as in vivo human fingernails and fingertips. This power level is significantly lower than the exposure limits established by the American National Standards Institute (ANSI) for human laser experiments. Raman spectra were recorded from the surface of both the nails and fingertips of three volunteers, in order to characterize their biological samples at different depths. The measurements were performed in 50 μm steps to obtain molecular structural information from both surface and subsurface tissue layers. The proposed CRS enables the identification of differences between two closely spaced, centered, and narrow Raman bands. Additionally, broad Raman bands observed at the skin surface can be deconvolved into at least three sub-bands, which can be quantitatively characterized in terms of intensity, peak position, and bandwidth, as the confocal plane advances in depth. Moreover, the CRS system enables the detection of subtle, low-intensity features that appear at the surface but disappear beyond specific depth layers.