PROJECT SUMMARY Our objective is to develop a multi-stage microscale cyclone technology that will serve as an efficient and scalable platform for bioaerosol fractionation, enabling more effective studies of fundamental and applied viral aerobiology. The microcyclones will be designed to isolate selected aerosol size fractions collected from exhaled breath samples, enabling the distribution of respiratory virus within these samples to be evaluated with high size resolution. Significantly, the technology will be designed to overcome the limitations of existing aerobiological instruments by enhancing the dynamic size range, maximum number of collected fractions, resolution, throughput, and bioefficiency. The platform will take advantage of a high resolution stereolithography-based 3d printing technique to pattern large arrays of complex microcyclone structures in a monolithic substrate, with multiple arrays placed in series to allow selected aerosol size ranges to be isolated from the sample flow. The system will further support the integration of a hydrogel layer within each microcyclone array to allow the capture of live virus for infectivity studies. The microcyclone arrays will be combined with an established exhaled breath collection system and employed to study the distribution of influenza virus from infected subjects. To support these goals, individual microcyclone elements will be designed, fabricated, characterized, and optimized for isolating at least five distinct aerosol size fractions ranging from 200 nm to 10 µm, followed by the development of full microcyclone arrays integrating hundreds of individual separation elements in a single device. A set of cascaded arrays will be assembled into a reusable cartridge to enable collection of all target fractions within a single integrated unit, and the resulting cartridge will be interfaced with the existing system for high- volume exhaled breath collection (the G-II sampler developed in the Milton laboratory). The integrated instrument will be used to collect exhaled breath samples from at least 15 individuals with active influenza infection for the evaluation of virus distribution across all size fractions. The study results are expected to validate the technology as a powerful tool for enhancing our understanding of aerobiology and improving modeling, risk analysis, and mitigation strategies for a wide range of airborne diseases. Successful completion of these aims will offer a clear view of the potential of the microcyclone array technology for collection and high resolution fractionation of bioaerosols from exhaled breath samples. For future steps we envision optimizing bioefficiency of the technology to support culture of collected virus, scaling the arrays to support environmental sampling at higher flow rates, and extending application of the technology to the characterization of aerosolized bacteria and fungal spores.