Development of microfluidic acoustic flow cytometry based on simultaneous ultrasound backscatter and photoacoustics for micron and sub-micron size objects

Vaskar Gnyawali

doi:10.32920/ryerson.14657958.v1

Abstract

I developed a flow cytometer based on simultaneous detection of ultra-high frequency ultrasound backscatter and photoacoustic waves from individual micron scale objects, such as, cells, microparticles, and microbubbles owing in a microuidic channel. Individual micron scale objects are ow focused through a focal zone, where both ultrasound and laser pulses focus, in a microchannel of a polydimethylsiloxane (PDMS) based microuidic device. At the focal zone, the objects are simultaneously insonified by ultrasound (center frequency 375 MHz) and irradiated by nanosecond laser (532 nm wavelength) pulses. The interactions generate ultrasound backscatter and photoacoustic signals from the individual objects, which are strongly dependent on their size, morphology, and biomechanical properties, such as the Young's modulus, and optical absorption properties. These parameters can be extracted by analyzing the unique spectral features of the detected signals. At frequencies less than 100 MHz, the signals from the micron scale objects do not contain these unique spectral signatures, thus higher frequencies are required. Cell analysis is the main application of interest using the acoustic flow cytometer. Combining ultrasound backscatter and photoacoustics results in sufficient information about a single cell that can be used for single cell analysis and for diagnostics applications. However, the usage of this system is not limited to biological cells. This system can also be used for analyzing individual microbubbles, which are used as ultrasound contrast agents. During my research, a novel microuidic technique is developed to generate microbubbles of desired sizes by shrinking microbubbles from O(100) _m by applying a suitable vacuum pressure. These shrunken bubbles of different sizes can be used as samples to validate the acoustic ow system for microbubble analysis.

Highlights

1.1 MotivationFlow cytometry is a commonly used technique in many clinical and biomedical laboratories for high throughput experimentation of single cells in a population.[1; 2] It is a crucial instrument for clinical diagnostics and hematology-related assays and is widely used in molecular biology, pathology, immunology, plant biology, and marine biology.[1; 3] Conventional flow cytometers are based on an optical approach, which uses scattering and fluorescence emission to provide information on size, granularity, and the expression level of chromophores bound on the cell surface and/or enclosed cell components
The acoustic flow cytometer is composed of a microfluidic device and fluid manipulation pumps; an ultrasonic system consisting of an ultra-high frequencies (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
The results show that the stability of hydrostatic pressure driven flow focusing is significantly better than the stability achieved by the syringe pumps

Summary

Introduction

1.1 MotivationFlow cytometry is a commonly used technique in many clinical and biomedical laboratories for high throughput experimentation of single cells in a population.[1; 2] It is a crucial instrument for clinical diagnostics and hematology-related assays and is widely used in molecular biology, pathology, immunology, plant biology, and marine biology.[1; 3] Conventional flow cytometers are based on an optical approach, which uses scattering and fluorescence emission to provide information on size, granularity, and the expression level of chromophores bound on the cell surface and/or enclosed cell components. The quality of the data generated when using fluorescent probes critically depends on sample preparation.[7] For some applications, such as gene expression and protein localization, higher fluorescence expression is required for higher optical resolution and sensitivity.[92; 93] On the other hand, impedance-based systems are only able to count and size cells and do not provide information of cell constituents.[9]. Due to these limitations, there is ongoing research in developing new flow cytometry systems. The flow profile is parabolic with velocity reaching its maximum at the center of the channel.[22]

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Conclusion