Ultrasonic-assisted hydrodynamic focusing for high resolution analysis and imaging in a flow cytometer

Flow cytometry analyzes individual cells at rates of 10 000’s per second. Historically, cells are accelerated to meters per second velocities and hydrodynamically focused into a narrow stream (∼10 microns) prior to the analysis volume (10’s picoliters). For sample concentrations used in immunology, the requirement of the narrow stream restricts the analysis rate to just a few hundred cells/second. If cellular imaging is included, the difficulty is increased further by requiring particles be positioned within the Depth of Focus (DOF) of a high-resolution imaging system. For instance, a 20ã objective with a 0.45 NA and 405 nm illumination has a DOF of only 5.6 microns. The present research demonstrates that combining ultrasonic radiation forces with hydrodynamic forces can greatly increase the throughput and resolution of a commercial high-resolution imaging flow cytometer. Experiments are conducted with cells traveling from 4 m/s to 8 m/s in hydrodynamic core sizes ranging from a few microns to over 58 microns. The data demonstrate a dramatic increase in cellular throughput, measurement resolution, and yield of in-focus images (from less than 50% to up to 100%) for fast-moving particles in hydrodynamic core streams with diameters exceeding many times the DOF.