Abstract
We describe a new technique that combines ultrasound and microfluidics to rapidly size and count cells in a high-throughput and label-free fashion. Using 3D hydrodynamic flow focusing, cells are streamed single file through an ultrasound beam where ultrasound scattering events from each individual cell are acquired. The ultrasound operates at a center frequency of 375 MHz with a wavelength of 4 μm; when the ultrasound wavelength is similar to the size of a scatterer, the power spectra of the backscattered ultrasound waves have distinct features at specific frequencies that are directly related to the cell size. Our approach determines cell sizes through a comparison of these distinct spectral features with established theoretical models. We perform an analysis of two types of cells: acute myeloid leukemia cells, where 2,390 measurements resulted in a mean size of 10.0 ± 1.7 μm, and HT29 colorectal cancer cells, where 1,955 measurements resulted in a mean size of 15.0 ± 2.3 μm. These results and histogram distributions agree very well with those measured from a Coulter Counter Multisizer 4. Our technique is the first to combine ultrasound and microfluidics to determine the cell size with the potential for multi-parameter cellular characterization using fluorescence, light scattering and quantitative photoacoustic techniques.
Highlights
Flow cytometry is a high throughput technique used to count, size, and/or sort cells
High frequency pulse echo ultrasound in the 20–60 MHz range has been used to quantify tissue properties based on underlying tissue structure and biomechanical properties to aid in the diagnosis of diseases, such as liver fibrosis and cancer[30,31,32,33,34]
This study demonstrates the proof of concept that ultra-high frequency ultrasound can be integrated into a microfluidic device, and that the scattered ultrasound can be used to determine the size distribution of micrometer-sized particles and cell populations on a cell by cell basis
Summary
Flow cytometry is a high throughput technique used to count, size, and/or sort cells. The theory which models the scattering of sound waves from spherical objects was first developed in the 1950’s35 and refined over the several decades; the scattering behavior is well established[36,37,38,39] Using this scattering theory, we recently demonstrated that it is possible to determine the size of single cells using an acoustic microscope with ultrasound frequencies over 100 MHz40; this method was slow and laborious, requiring manual targeting of individual stationary cells, making it unsuitable for measuring large cell populations. This demonstrated that for the first time, pulse echo ultrasound can be used to rapidly size flowing micro-sized particles; the frequencies were too low and lacked the spectral resolution to characterize cells
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