Pile-up free fluorescence lifetime imaging with a SPAD-based single pixel camera

• Published in: Optics express Vol. 33; no. 11; p. 22296
• Main Authors: Farina, Serena, Ghezzi, Alberto, Labanca, Ivan, Acconcia, Giulia, D’Andrea, Cosimo, Farina, Andrea, Rech, Ivan
• Format: Journal Article
• Language: English
• Published: United States 02.06.2025
• ISSN: 1094-4087; 1094-4087
• DOI: 10.1364/OE.555297

In the last years, single-pixel imaging (SPI) has been extensively utilized in fluorescence lifetime imaging (FLIM) experiments. In this context, to attain high temporal resolution, time-correlated single photon counting (TCSPC) is typically adopted, with the major drawback of the pile-up phenomenon, that limits the photon count rate to 1–5% of the laser excitation rate. This clearly hinders the possibility of monitoring a wide variety of biological phenomena in real-time. In this paper, to the best of our knowledge, we apply for the first time to a single-pixel camera (SPC) a hardware-based method, that allows to completely avoid the onset of pile-up by matching the dead time of the employed single-photon avalanche diode (SPAD) detector to an integer multiple of the laser period. We therefore demonstrate that undistorted and high-fidelity lifetime maps can be acquired at count rates (40%) well above the classic pile-up limitation, even in the presence of computational imaging algorithms. These results are supported by a thorough analysis of both the raw data prior to the reconstruction process and the final reconstructed lifetime maps.