Abstract
We developed a label-free microfluidic acoustic flow cytometer (AFC) based on interleaved detection of ultrasound backscatter and photoacoustic waves from individual cells and particles flowing through a microfluidic channel. The AFC uses ultra-high frequency ultrasound, which has a center frequency of 375 MHz, corresponding to a wavelength of 4 μm, and a nanosecondpulsed laser, to detect individual cells. We validate the AFC by using it to count different color polystyrene microparticles and comparing the results to data from fluorescence-activated cell sorting (FACS). We also identify and count red and white blood cells in a blood sample using the AFC, and observe an excellent agreement with results obtained from FACS. This new label-free, non-destructive technique enables rapid and multi-parametric studies of individual cells of a large heterogeneous population using parameters such as ultrasound backscatter, optical absorption, and physical properties, for cell counting and sizing in biomedical and diagnostics applications.
Highlights
For many decades, flow cytometry has been one of the most effective and powerful approaches for rapidly analyzing the characteristics of single-cells in a large population[1,2,3]
Our technique builds upon previous combined US and PA static methodology[25], by integrating with microfluidics, to achieve high throughput label-free analysis of single cells in heterogeneous solutions. We first use this acoustic flow cytometer to differentiate and count different color polystyrene microparticles, and we find results consistent with those obtained from a commercial fluorescence-activated cell sorting (FACS) system. We show that this acoustic flow cytometer can identify and count red blood cells (RBCs) and white blood cells (WBCs), again showing good agreement with results from the commercial FACS system
The acoustic flow cytometer is composed of a microfluidic device and fluid manipulation pumps; an ultrasonic system consisting of an ultra-high frequency (UHF) transducer, ultrasonic pulse generator, and control tools; and an optical system consisting of a laser source and optical components such as lenses, mirrors, and light sources
Summary
Flow cytometry has been one of the most effective and powerful approaches for rapidly analyzing the characteristics of single-cells in a large population[1,2,3]. Impedance-based systems are only able to count and size cells and do not provide information of cell constituents[17] Due to these limitations, there is ongoing research in developing new flow cytometry approaches. Ning et al used photoacoustic microscopy at ultrasound frequency (35 MHz) to map in vivo blood vessel diameters, sO2, blood flow, and resolve individual red blood cells in the capillaries, by analyzing photoacoustic microscopic images[36] All of these in vivo examples demonstrate the utility of US and PA in cellular analysis. There is an unmet need for a label-free technique that rapidly probes suspended individual cells without the limitations of conventional flow cytometry approaches, such as the requirement of fluorescent-tagging and lack of a method to rapidly probe biomechanical cell properties
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