Portable Multimodal Microscopy for Quantitative Malaria Diagnosis and Monitoring at the Point of Care

Malaria is a parasitic disease that affects millions around the globe annually and is one of the greatest public health challenges in human history. While much has been accomplished towards eradication of the disease, numerous challenges such as novel drug resistances underscore the need for improved diagnostic and monitoring technologies, especially for use in remote locations without existing healthcare infrastructure. This work proposes that a portable multimodal microscopy system may provide an effective tool to provide better diagnostic information and improve drug resistance monitoring efforts at the point-of-care. To accomplish this, two portable microscope prototypes were constructed that leverage combinations of brightfield, fluorescence, and cross-polarized imaging to provide information that may facilitate such advances. Sample preparation techniques were improved by optimizing microfluidic smear-generating cartridges for use with the portable microscopy system. These optimizations included the replacement of 1-millimeter thick slide glass substrates with 0.25-millimeter thick polycarbonate sheet material and the development of a one-step, in-tube fluorescence staining procedure. Polycarbonate was verified as a viable cartridge substrate by confirming its thermal stability, optical properties, and bonding potential throughout the manufacturing process. In-tube staining using Acridine Orange was shown to allow Plasmodium falciparum parasites to be distinguished from red blood cells and background when imaged in microfluidic smears. A novel aspheric lens fabrication technique was developed during the course of microscope development that leverages principles of fluid dynamics to create an exceptionally smooth concave parabolic surface in spinning polymers. When cured, it is possible to create plano-concave, plano-convex, and meniscus parabolic polymer lenses with infinitely variable radii of curvature. The theory and characterization of these lenses were explored along with the merits and drawbacks of the manufacturing technique. Two prototype portable microscopes were constructed, with the first utilizing a tri-modal brightfield, fluorescence, and cross-polarized approach to image malarial parasites in thin blood smears. The device can interface with a range of portable electronic devices. The microscope successfully resolved red blood cells using brightfield imaging, parasites using fluorescence imaging, and the malarial pigment hemozoin using cross-polarized imaging. It was eventually determined that cross-polarized imaging had little diagnostic or monitoring utility, and the modality was removed from the second-generation portable microscope. The second-generation microscope was optimized to utilize low-cost optical components to quantitatively measure parasitemia in thin blood smears. It is hypothesized that drug resistance monitoring may be possible at the point-of-care by using these measurements to calculate parasite clearance rates from the blood after treatment. The microscope was successful in achieving a linear correlation between its parasitemia measurements and those from a benchtop microscope with R2 = 0.939 and a limit of detection of 0.2 parasites per 100 red blood cells. These results indicate that the microscope is capable of quantitatively measuring parasitemia using thin blood smears, and further testing may prove it to be useful for in vivo data collection in remote locations