Stigmatic Microscopy Enables Low-Cost, 3D, Microscale Particle Imaging Velocimetry in Rehydrating Aqueous Two-Phase Systems

This paper describes the construction of a novel stigmatic microscope and image analysis algorithm to simultaneously analyze convective mixing both inside and outside of rehydrating μL-scale aqueous two-phase system (ATPS) droplets. Stigmatic microscopy is inexpensive and advantageous because it modifies the point-spread function of fluorescent particles to enable measurement of their 3D positions from single 2D images, without needing to take slices. In one application of the technique, the convection patterns captured clarify how different ATPS formulations succeed or fail to exclude cells for patterning. Particle flow traces reveal speed and directionality of circulation, indicating temporary eddies at the outer edge of the rehydrating droplet. In another application, the speed of circulation during rehydration was analyzed for different ATPS formulations and the results used to develop a new fast ELISA procedure. While this paper focuses on ATPS rehydration, the microscope and algorithm should be applicable to a broad range of microfluidic flows where microscale 3D convection is important.