Photobleaching-assisted extraction of Raman spectral features from fluorescence-enhanced background variation

We present a photobleaching-assisted method for extracting Raman spectra from fluorescence-dominated signals without hardware modifications. This method involves sequential spectral acquisitions during the photobleaching process and applies a least-squares-based decomposition algorithm to isolate the photostable Raman components from the decaying fluorescence background. This procedure reduces the instrument-derived spectral variations that arise with increasing background intensity, thereby improving the detectability of weak and overlapping Raman bands. The prerequisite conditions of the method are that the shapes of both the observed Raman and fluorescence spectra remain largely unchanged during measurement and that their spectral coefficients can be reasonably estimated from the measurement conditions or measured data. Both proof-of-concept simulations using simulated data and experiments with polystyrene-based fluorescent beads demonstrated that the proposed method can extract Raman spectra by mitigating instrument-derived spectral variations. The application of the proposed method to a highly fluorescent polymer using both 532 and 785 nm excitation wavelengths demonstrates its capability to extract Raman spectral features across different optical configurations. This method broadens the application of Raman measurements under a fluorescence background and provides an additional strategy to enhance existing approaches. • Photobleaching during repeated spectral acquisition enables spectral extraction. • Instrument-derived spectral variations are suppressed in the extracted Raman spectrum. • No hardware modifications are required. • Compatible with conventional Raman instruments.