Time-resolved Raman Spectroscopy using a CMOS SPAD array to separate Raman and fluorescent signals

Many biological samples have a stronger fluorescent signal than Raman signal. Time-resolved Raman spectroscopy can separate Raman signals and fluorescent signals in the time-domain due to fluorescence occurring at longer time scales than Raman scattering [1]. Here, we separate Raman scattering and fluorescence by using a time-resolved 512 pixel single photon avalanche diode (SPAD) line sensor array with 512 pixels operating in time-correlated single photon counting (TCSPC) modality [2], configured as a spectrometer [3]. In this setup, the SPAD array measures the backscattered light from the sample as a function of time and wavelength. The arrival time of the photons are used in post processing to separate the Raman and fluorescence signal. The advantage of using a SPAD line array is that the integrated timing electronics allow for rapid and multiplexed single photon counting where numerous detectors measure the Raman and fluorescent signal. This increases possible count rate, therefore decreasing measurement time for practical application. In this work we typically use a measurement time of 30 s. In Fig. 1. we show the separation of Raman and fluorescence signal in olive oil. Here it can be seen that the Raman scattering occurs at earlier time-bins compared to the fluorescent signal which occurs at later time-bins. This method will now be applied to biological tissue samples. [1] F. Madonini et al., “Single photon avalanche diode arrays for time-resolved Raman spectroscopy,” Sensors, vol. 21, no. 13, p. 4287, Jun. 2021. doi:10.3390/s21134287 [2] E. P. McShane et al., “High resolution TCSPC imaging of diffuse light with a one-dimensional SPAD array scanning system,” Optics Express, vol. 30, no. 15, p. 27926, Jul. 2022. doi:10.1364/oe.461334 [3] C. S. Tye, et al., “Photon counting fibre optic distributed temperature sensing with a CMOS SPAD array,” Optics Express, vol. 32, no. 4, p. 6481, Feb. 2024. doi:10.1364/oe.506964