PurposeRapid and accurate diagnosis of microbial keratitis (MK) could greatly improve patient outcomes. Here, we present the development of a rapid, accessible multicolour fluorescence imaging device (FluoroPi) and evaluate its performance in combination with fluorescent optical reporters (SmartProbes) to distinguish bacterial Gram status. Furthermore, we show feasibility by imaging samples obtained by corneal scrape and minimally invasive corneal impression membrane (CIM) from ex vivo porcine corneal MK models.MethodsFluoroPi was built using a Raspberry Pi single-board computer and camera, light-emitting-diodes (LEDs), and filters for white-light and fluorescent imaging, with excitation and detection of bacterial optical SmartProbes: Gram-negative, NBD-PMX (exmax488 nm); Gram positive, Merocy-Van (exmax590 nm). We evaluated FluoroPi with bacteria (Pseudomonas aeruginosaandStaphylococcus aureus) isolated from ex vivo porcine corneal models of MK by scrape (needle) and CIM with the SmartProbes.ResultsFluoroPi provides <1 µm resolution and was able to readily distinguish bacteria isolated from ex vivo models of MK from tissue debris when combined with SmartProbes, retrieved by both scrape and CIM. Single bacteria could be resolved within the field of view, with limits of detection demonstrated as 103to 104CFU/mL. Sample preparation prior to imaging was minimal (wash-free), and imaging and postprocessing with FluoroPi were straightforward, confirming ease of use.ConclusionsFluoroPi coupled with SmartProbes provides effective, low-cost bacterial imaging, delineating Gram-negative and Gram-positive bacteria directly sampled from a preclinical model of MK.Translational RelevanceThis study provides a crucial stepping stone toward clinical translation of a rapid, minimally invasive diagnostic approach for MK.