Acoustophoretic Characterization and Separation of Blood Cells in Acoustic Impedance Gradients

Single-cell phenotyping based on biophysical properties is a promising tool to distinguish cell types and their response to a given condition, and charting such properties also enables optimization of cell separations. Isoacoustic focusing, where cells migrate to their points of zero acoustic contrast in an acoustic impedance gradient, added the effective acoustic impedance of cells to the directory of biophysical properties that can be utilized to categorize or separate cells. This study investigates isoacoustic focusing in a stop-flow regime and shows how cells migrate towards their isoacoustic point. We introduce a numerical model that we use to estimate the acoustic energy density in acoustic impedance gradient media by tracking particles of known properties, and we investigate the effect of acoustic streaming. From the measured trajectories of cells combined with fluorescence intensity images of the slowly diffusing gradient, we read out the effective acoustic impedance of neutrophils and K562 cancer cells. Finally, we propose suitable acoustic impedance gradients that lead to a high degree separation of neutrophils and K562 cells in a continuous-flow configuration.