Enhanced porphyrin-based hypoxia imaging by temporal oversampling of delayed fluorescence signal.

Significance: Protoporphyrin IX (PpIX) delayed fluorescence (DF) is inversely related to the oxygen present in tissues and has potential as a novel biomarker for surgical guidance and real-time tissue metabolism assessment. Despite the unique promise of this technique, its successful clinical translation is limited by the low intensity emitted.

Aim: We developed a systematic study of ways to increase the PpIX DF signal through acquisition sampling changes, allowing optimized imaging at video rates.

Approach: To accomplish signal increase, time-gating signal compression was achieved through changes in pulse frequency and power density, using sampling rates that are faster than the decay rate of the signal. The increased signal yield was tested and validated in vitro and then demonstrated in vivo, with comparison to settings that sample the full lifetime emission decay.

Results: Results in vitro and in vivo demonstrated that optimized timing could increase the detected intensity by a factor of 7. The images showed results that were superior than when sampling the full DF lifetime decay.

Conclusions: The proposed timing optimization enhances PpIX-based DF real-time imaging of tissue hypoxia. By increasing sampling frequency and adjusting the acquisition gate and pulse width, the collected signal intensity improved sevenfold, demonstrated both in vitro and in vivo. The technique was shown to enable better visualization of small and anatomically challenging hypoxic structures. The improved target-to-background ratio and compatibility with pressure-enhanced sensing of tissue oxygen technique were demonstrated.