Sizing biological cells using a microfluidic acoustic flow cytometer

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.

Determination of cell nucleus-to-cytoplasmic ratio using imaging flow cytometry and a combined ultrasound and photoacoustic technique: a comparison study

“…We have previously demonstrated that waveforms of US signals backscattered from individual biological cells are unaffected by cell nuclei since they are acoustically homogeneous at UHFs. 34 Indeed, the presence of a strongly scattering object within the cell would result in either one or two additional peaks in the time-domain signal depending on the size of the nucleus; however, such peaks were not observed in our experiments. While the US measurements are unaffected by the presence of the cell nucleus, the PA measurements are insensitive to the overall size of the cell.…”

“…We demonstrate that, while there were slight differences in the overall size of the cells predicted by the two techniques, good agreement between the two techniques was observed, especially for the calculated N:C ratio values. We have recently reported on an acoustic flow cytometer that sized cell diameter using only US methods 34 and have since incorporated a pulsed laser into our system to enable differentiation of RBCs and white blood cells via simultaneous US/PA detection. 43 Going forward, we plan on translating the presently reported N:C assessment technique to this high-throughput US/PA device to enable largescale investigations of cell population statistics, as well as the detection of cancer cells spiked into a blood sample.…”

“…Our previously reported spectral fitting algorithms required at least two spectral minima to fall within the bandwidth of the 400-MHz transducer 34 and were thus incapable of sizing spherical cells or organelles with diameter smaller than ∼3.5 μm when using US, or 7 μm when using PA. These limitations are acceptable when using US to determine cell size since most animal cells typically have diameters between 10 and 20 μm.…”

“…Cells were located using the optical objective and were translated into the confocal transducer/objective focal zone for measurement. While the location of the features in the power spectrum used for cell and nucleus sizing is robust to the spatial location of the cell within the acoustic detection volume, 34 centering the cell in the focal zone provides optimal signal-to noise ratio (SNR) in the time and frequency domains and increases the prominence of the spectral features. 34 A total of 200 US and 200 PA signals were acquired from each cell for signal averaging.…”

“…While the location of the features in the power spectrum used for cell and nucleus sizing is robust to the spatial location of the cell within the acoustic detection volume, 34 centering the cell in the focal zone provides optimal signal-to noise ratio (SNR) in the time and frequency domains and increases the prominence of the spectral features. 34 A total of 200 US and 200 PA signals were acquired from each cell for signal averaging. To prevent measurement of the same cell multiple times, the sample was manually translated in a raster pattern until measurements from 50 unique cells had been acquired.…”