Current methods to determine the bacterial burden in suspected ventilator-associated pneumonia (VAP) rely on the culture of BALF. The growth of colony forming units (CFU) above 42,43 or 44,45 is considered diagnostic of VAP. These are the accepted clinically relevant limits, and we have based our system on being able to perform at this limit of detection. As broncholavolar lavage involves the instillation and retrieval of fluid, a dilution effect is seen. The true bacterial burden in the infected distal lung is likely to be to .9 Therefore, to establish the suitability of the instrument for the lower limit of bacterial detection in the distal lung, we initially performed experiments in two scenarios whereby we used microspheres as surrogates for bacteria: (a) imaging of microspheres alone in black Eppendorf tubes (to avoid tube fluorescence) and (b) imaging of microspheres in ex vivo lung tissue placed in a well plate. 0.3% Inspeck™ microspheres (green Inspeck™ I-7219 and red Inspeck™ I-7224) were used as a fluorescence emission standard indicative of labeled bacteria. Green 0.3% Inspeck™ microspheres have equivalent emission to green OMI smartprobe7 labeled bacteria (Pseudomonas aeruginosa), and red 0.3% Inspeck™ microspheres are 1.5 times weaker than PKH red (Cellvue® Claret, Sigma-Aldrich) labeled bacteria (Pseudomonas aeruginosa). Blackened microtubes were filled with concentrations , , , and to derive the detection limit of the system without the lung tissue present. Tubes were centrifuged for 10 s before each experiment. Excised lung tissue was placed in well plates with 0.3% Inspeck™ microspheres deposited at the following concentrations: , , , and . Controls included excised lung tissue alone, empty microspheres (unstained control beads supplied with green Inspeck™ I-7219, Life Technologies) per milliliter in lung tissue, distilled water alone and lastly, empty microspheres per milliliter in distilled water. The controls were used to minimize false positive detections, especially in the green channel, where small parts of tissue could be mistaken for bacteria.