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

Ettore Pedretti,Tushar R Choudhary,Robert R Thomson,Paul A Dalgarno,Nikola Krstajić,Alicia Megia-Fernandez,Mark Bradley,Robert K Henderson,Kevin Dhaliwal,Michael G Tanner,John M Girkin

doi:10.1364/boe.10.000181

Abstract

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 ex vivo samples of human lung using lifetime to differentiate bacterial fluorescence from the strong background lung autofluorescence which was used to provide structural information.

Highlights

Fiber based optical endomicrosopy (OEM) is a minimally invasive technique used in pulmonary medicine to access the alveolar space [1,2,3]
We present a dual-color laser scanning endomicroscope capable of fluorescence lifetime endomicroscopy at one frame per second (FPS)
We demonstrate the potential of the instrument by first using an example system of a multi-core fiber with 19 widely spaced cores with the distal end loaded with fluorescent microspheres, utilizing a 30,000 core imaging fiber to view microspheres on a slide, and viewing ex vivo human lung tissue to show the detection of bacteria against a strong spectrally similar

Summary

Introduction

Fiber based optical endomicrosopy (OEM) is a minimally invasive technique used in pulmonary medicine to access the alveolar space [1,2,3]. Fluorescence endomicroscopy, like all fluorescence imaging modalities, is typically limited to 2 or 3 channels restricted by the need to avoid spectral overlap, requiring tight bandwidth, filtering and imaging constraints These challenges are exacerbated in the lung tissue due to the high intensity green background autofluorescence generated from intrinsic elastin and collagen. We demonstrate 4 channel, one frame per second fluorescence imaging through a combination of dual channel FLIM and dual color, temporally dephased photon-counting This method of multiplexing colors in the time domain was described elsewhere [15], the parallel detection architecture applied here enables this technique at higher rates without photon pileup [21]. We demonstrate future clinical potential of the endomicroscope by imaging ex vivo human lung with labelled E.coli bacteria compared to healthy lung

Objective lens

Results and discussion

FLIM capability test with fluorescent microspheres

Ex vivo human lung tissue imaging with labeled bacteria

Conclusions

Preparation of the fluorescent beads for FLIM test