Off-confocal Raman spectroscopy (OCRS) for subsurface measurements in layered turbid samples

Abstract

We report, for the first time, the development of a depth-sensitive Raman spectroscopy system for investigating subsurface depths in a layered turbid sample using the concept of varying Raman collection zones, while keeping the point of illumination fixed on the surface of the target sample. The system makes use of a conventional confocal Raman configuration and realizes the variation in Raman collection zones employing off-confocal detection. This is effected by moving the tip of the Raman detection fiber (acting as the pinhole aperture) from the focus of the Raman collection objective either by taking the point of detection away from the objective (along its axis) or bringing it closer to the objective (along the same axis), thereby essentially offering two ways of enabling subsurface interrogation at a given time. Another important attraction of the approach is that it can be used for analyzing layered turbid samples at depths beyond the reach of the conventional confocal Raman, though not at the cost of any further modifications in its instrumentation. Furthermore, the illumination point remains fixed on the sample surface and no adjustment is required in the sample arm, which indeed are significant advantages for depth-sensitive measurements in situ from layered turbid samples, particularly those having irregular surfaces (like biological tissues). The ability of the system to recover Raman spectra of the subsurface layer was demonstrated using a layered non-biological phantom and a biological tissue sample.