Sizing Cells Using Acoustic Flow Cytometry

We have developed an acoustic flow cytometer that combines ultrasound and microtluidics to rapidly size and count cells label-free in a high-throughput manner. Cells are hydrodynamically focused single file into a narrow stream through an ultrasound beam where ultrasound is scattered from each individual cell. The ultrasound operates at a center frequency of 375 MHz with a wavelength of 4 pm; in this regime, the power spectra of the backscattered ultrasound waves have distinct features at specific frequencies that are directly related to the cell size. Our approach can be used to determine the cell diameter by comparing these unique spectral features to established analytical solutions of ultrasound scattering. 3 mu m beads with known scattering properties were used to validate the technique. Then, we examined two cells lines with different average cell diameters; acute myeloid leukemia cells, where 2,390 measurements resulted in a mean diameter of 10.0 +/- 1.7 mu m, and HT29 colorectal cancer cells, where 1,955 measurements resulted in a mean diameter of 15.0 +/- 2.3 mu m. The resulting histograms showed anticipated distributions. Microfluidic based devices are commonly used for probing single cells, however, very few can determine the cell size. Our technique combines ultrasound and microtluidics to accurately determine the cell size on a cell-by-cell basis, with the potential for integration with fluorescence, light scattering and quantitative photoacoustic techniques for a multiparameter cellular characterization method.