Design of a line-of-sight fluorescence-based imaging diagnostic for classification of microbe species

Fluorescence imaging of certain biochemicals, including flavins and pyridine nucleotides, has utility in characterizing the metabolic state of tissue and in discriminating between microbial species. There is significant clinical utility in this class of imaging techniques but most measurements reported to date require specialized training and equipment rendering most implementations unsuitable for routine medical imaging. Here, a low-cost and robust imaging technique is designed using ultraviolet-induced fluorescence of pyridine nucleotides (primarily NADH) and flavins (primarily FAD) in microbial samples. The diagnostic is optimized to distinguish between different microbial species based on previously reported spectral data using a ratiometric imaging approach. A detailed performance analysis is provided that relates the measured fluorescence intensity ratio (FIR) to the relative concentration ratio of NADH to FAD using a simplified spectroscopic model. Analysis suggests the technique is sensitive to changes in the NADH/FAD concentration ratio over several orders of magnitude, with better than 10% FIR precision on a per-pixel basis for microbial smears as thin as 10 s of microns at a resolution of 30 mm−1 and exposures of 20 ms. Representative microbe samples from eight species were imaged to demonstrate the proposed technique. Results show that the FIR varies by an order of magnitude across different species but the intra-species variation is only ∼5% for the conditions used here. An additional imaging band may be necessary to classify species that contain red pigments or bacteriochlorophyll. Radiative trapping was discussed as a possible limitation of the technique, but no clear evidence for radiative trapping was observed here. Overall, the results suggest that the proposed approach is feasible for rapid, low-cost, and robust characterization of microbial samples.