Abstract
Whole-animal fluorescence imaging with recombinant or fluorescently-tagged pathogens or cells enables real-time analysis of disease progression and treatment response in live animals. Tissue absorption limits penetration of fluorescence excitation light, particularly in the visible wavelength range, resulting in reduced sensitivity to deep targets. Here, we demonstrate the use of an optical fiber bundle to deliver light into the mouse lung to excite fluorescent bacteria, circumventing tissue absorption of excitation light in whole-animal imaging. We present the use of this technology to improve detection of recombinant reporter strains of tdTomato-expressing Mycobacterium bovis BCG (Bacillus Calmette Guerin) bacteria in the mouse lung. A microendoscope was integrated into a whole-animal fluorescence imager to enable intravital excitation in the mouse lung with whole-animal detection. Using this technique, the threshold of detection was measured as 103 colony forming units (CFU) during pulmonary infection. In comparison, the threshold of detection for whole-animal fluorescence imaging using standard epi-illumination was greater than 106 CFU.
Highlights
Whole-animal optical imaging has found widespread use in the biological sciences
To significantly enhance detection sensitivity of fluorescent mycobacteria in the mouse lung, we present the integration of an optical fiber into a whole-animal imaging system for intravital excitation of a fluorescent target deep within an animal
We have developed a novel system to sensitively detect bacterial infection in living animals
Summary
Whole-animal optical imaging has found widespread use in the biological sciences. Fluorescence-based techniques have long been used to study both fixed and live biological specimens, as it allows for highly selective and specific detection of molecules even at low concentrations [1]. While traditional fluorescence microscopy requires the sacrifice of multiple animals at discrete time points, whole-animal imaging allows researchers to study disease progression or the efficacy of a therapeutic agent more precisely and with fewer animals than prior imaging techniques [2]. Whole-animal fluorescence imaging is used to study disease pathogenesis, develop novel probes, and measure response to new therapies [3]. To record a planar image of an internal fluorescent target in a small animal, whole-animal imaging systems can be configured either for epi-illumination, with the light source on the PLOS ONE | DOI:10.1371/journal.pone.0149932. To record a planar image of an internal fluorescent target in a small animal, whole-animal imaging systems can be configured either for epi-illumination, with the light source on the PLOS ONE | DOI:10.1371/journal.pone.0149932 February 22, 2016
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