Abstract
SIGNIFICANCE: Fluorescence lifetime imaging (FLI) plays a pivotal role in enhancing our understanding of biological systems, providing a valuable tool for non-invasive exploration of biomolecular and cellular dynamics, both in vitro and in vivo. Its ability to selectively target and multiplex various entities, alongside heightened sensitivity and specificity, offers rapid and cost-effective insights. AIM: Our aim is to investigate the multiplexing capabilities of near-infrared (NIR) FLI within a scattering medium that mimics biological tissues. We strive to develop a comprehensive understanding of FLI's potential for multiplexing diverse targets within a complex, tissue-like environment. APPROACH: We introduce an innovative Monte Carlo (MC) simulation approach that accurately describes the scattering behavior of fluorescent photons within turbid media. Applying phasor analyses, we enable the multiplexing of distinct targets within a single FLI image. Leveraging the state-of-the-art single-photon avalanche diode (SPAD) time-gated camera, SPAD512S, we conduct experimental wide-field FLI in the NIR regime. RESULTS: Our study demonstrates the successful multiplexing of dual targets within a single FLI image, reaching a depth of 1 cm within tissue-like phantoms. Through our novel MC simulation approach and phasor analyses, we showcase the effectiveness of our methodology in overcoming the challenges posed by scattering media. CONCLUSIONS: This research underscores the potential of NIR FLI for multiplexing applications in complex biological environments. By combining advanced simulation techniques with cutting-edge experimental tools, we introduce significant results in the non-invasive exploration of biomolecular dynamics, to advance the field of FLI research.