High-speed dual color fluorescence lifetime endomicroscopy for highly-multiplexed pulmonary diagnostic applications and detection of labeled bacteria.

We present a dual-color laser scanning endomicroscope capable of fluorescence lifetime endomicroscopy at one frame per second (FPS). The scanning system uses a coherent imaging fiber with 30,000 cores. High-speed lifetime imaging is achieved by distributing the signal over an array of 1024 parallel single-photon avalanche diode detectors (SPADs), minimizing detection dead-time maximizing the number of photons detected per excitation pulse without photon pile-up to achieve the high frame rate. This also enables dual color fluorescence imaging by temporally shifting the dual excitation lasers, with respect to each other, to separate the two spectrally distinct fluorescent decays in time. Combining the temporal encoding, to provide spectral separation, with lifetime measurements we show a one FPS, multi-channel endomicroscopy platform for clinical applications and diagnosis. We demonstrate the potential of the system by imaging SmartProbe labeled bacteria in samples of human lung using lifetime to differentiate bacterial fluorescence from the strong background lung autofluorescence which was used to provide structural information.