Emerging optical technologies for fluorescence microscopy enable the use of low-cost lenses, filters, LEDs, and detectors, such as a cell-phone-integrated camera, in which the spatial resolution, quantum efficiency, and frame rates have improved to where high-quality imaging is practical.11,17 This has renewed the interest in the use of acridine orange (AO), a fluorescent vital dye that has been used in medical diagnostics since the 1950s for uses in diagnosing and detecting gynecological cancer, microorganisms in cerebrospinal fluid, and malaria.29–32 AO only stains nucleated cells and, therefore, does not require additional reagents (ex. RBC lysis and dilution reagents), in contrast to other POC systems. This is due to the amphipathic structure of AO that enables the molecules to pass through the cell and nuclear membranes and intercalate the major and minor grooves of DNA, leading to a strong nuclear to cytoplasmic contrast in living cells.33,34 The most promising advantage of staining whole blood with AO are the colorimetric features that can be extracted from leukocytes that are distinct to each cell population. AO () will yield a range of fluorescence emission (from 525 to 650 nm), contingent on intracellular environmental factors such as local pH.33,35 These local variations in intracellular pH are dependent on the presence of intracellular lysosomes, vesicles, and other components specific to the role that each leukocyte population plays; this enables differentiation and classification of each cell population based on its colorimetric features following AO staining.33,36 In Fig. 1, granulocytes represent the most acidic cell population containing many acidic vesicles resulting in the greatest red emission.35,36 The lymphocytes have minimal cytoplasm with the least acidic cellular content resulting in the greatest green emission. Monocytes have more cytoplasm but less acidic content and, thus, are between the emission spectrum of the other cell populations. Many POC devices utilize this colorimetric feature21,35 by comparing each cell’s red-to-green fluorescence intensity ratio (RG ratio).20,37,38 In postprocessing, a histogram plot of the frequency of RG ratios can reveal three peaks indicative of each cell population. The challenge in using this method is in the monocyte count because the peak can be too small to distinguish, creating issues in defining the dividing lines (cutoff values) between peaks. For instance, using a static approach where the cutoff values are constant for all tests, the peaks could shift slightly but enough to miscount the cells.