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High-speed wide-field fluorescence lifetime imaging for intraoperative tumor visualization and in vivo multiplexing

Murali Krishnamoorthy,Rahul Pal,Steven S. Hou,Hak Soo Choi,Brian Bacskai,A. Bogdanov,Kenneth K. Tanabe,Mark Varvares,Anand T. N. Kumar

Published 2025 in Biomedical Optics Express

ABSTRACT

We present a technique for fast wide-field fluorescence lifetime (FLT) imaging using simultaneous steady-state and time- (or frequency-) domain measurements acquired in a single shot and exploiting the theoretical dependence of fluorescence intensity on FLT. Using theory, simulations, and in vitro experiments, we show that the single-shot FLT (S-FLT) method can provide absolute FLTs at a higher signal-to-noise ratio compared to other high-speed FLT imaging techniques, without the need for sample-dependent system calibration or model training. We demonstrate that S-FLT can provide accurate tumor delineation in real-time during surgical resections in preclinical tumor models in vivo and in clinical patient specimens ex vivo. We also extend S-FLT for real-time multiplexing and demonstrate this technique for quantifying dynamic FLT changes in vitro and for multiplexing of organ-targeted NIR fluorophores in vivo. Our results suggest that S-FLT can enable real-time quantitative high-throughput studies in preclinical applications and accurate tumor margin delineation in real-time during surgeries.

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