Spontaneous or Stimulated? Investigating Raman’s Detection Limits in Aqueous Environments

Raman spectroscopy is a powerful method for analyzing chemical compositions across diverse samples. Spontaneous Raman scattering (spRS) provides complete Raman spectra but typically yields low signal levels, requiring long signal integration times. In contrast, stimulat-ed Raman scattering (SRS) produces much stronger signals, allowing for rapid spectral acquisition, and has been widely used to accelerate chemical imaging. Despite advances in both techniques, it remains unclear which method offers superior limits of detection (LODs) for molecules in biological samples. While theoretical comparisons under ideal conditions suggest that performance depends on photon flux and integration time, no experimental studies have directly compared the LOD of spRS and SRS spectroscopy. In this study, we systemati-cally compare the LOD of frequency-domain spRS and SRS for water-soluble analytes in aqueous solutions, a common environment for biological specimens. We first introduce a simplified LOD estimation methodology by taking only three measurements to derive the dilu-tion maximum (DM). This methodology achieved LOD estimates closely aligned with those obtained through serial dilution. Using this approach, we assessed the impact of various parameters, including Stokes laser power, the presence of beam scanning, number of pixels, and pixel dwell time, on the LOD of SRS spectroscopy and determined optimal conditions for SRS. Our results demonstrate that at short inte-gration times, SRS exhibits shot-noise-limited performance, with LOD inversely proportional to the square root of the acquisition time. At longer integration times, photothermal effects can cause deviations from ideal LOD scenarios. Under optimized experimental conditions, spectral-domain SRS confidently detects ~700 μM dimethyl sulfoxide in water and 1 g/L glucose and bovine serum albumin in aqueous solutions without spectral averaging. This performance can surpass that of spRS spectroscopy, even with equal or longer integration times.