Separating fluorescence from Raman spectra using a CMOS SPAD TCSPC line sensor for biomedical applications

Time-resolved separation of Raman scattering from background fluorescence is demonstrated using a recently developed 512 pixel, 16.5 giga-events CMOS SPAD line sensor ¹ . The system is being developed with the aim of evaluating the suitability of liver tissue for transplant surgery, as these samples pose particular challenges to commercial Raman systems due to their high fluorescence emission across a wide spectral excitation range. Compared to previous work ² , the spectrometer operates without the use of time-gating, relying entirely on inpixel time-correlated single photon counting (TCSPC). Indeed, by employing the unique features of the sensor, such as on-chip histogramming and zoomable time resolution from 50ps to 6.4ns, the system is able to deliver both Raman and time-resolved fluorescence decay data. Time-resolved separation of Raman and fluorescence signals allows the spectrometer to be operated in the visible range (using a 532-nm pulsed laser), thus providing enhanced Raman scattering intensity compared with the use of a near-infrared laser, since scattering emission is proportional to λ-^{4 3} . The system is calibrated using a Neon calibration source and benchmarked using samples of pure distilled-water fluorescein, paracetamol and sesame oil in comparison with results from continuous wave excitation in a Renishaw InVia spectrometer. The Raman band of water at a Raman shift of 3000-3800 cm-1 is chosen to evaluate sensor performance because of its low intensity and its characteristic spectral profile which is readily compared with the literature ⁴ .