Ultrasonic particle‐concentration for sheathless focusing of particles for analysis in a flow cytometer

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Abstract
Background: The development of inexpensive small flow cytometers is recognized as an important goal for many applications ranging from medical uses in developing countries for disease diagnosis to use as an analytical platform in support of homeland defense. Although hydrodynamic focusing is highly effective at particle positioning, the use of sheath fluid increases assay cost and reduces instrument utility for field and autonomous remote operations. Methods: This work presents the creation of a novel flow cell that uses ultrasonic acoustic energy to focus small particles to the center of a flowing stream for analysis by flow cytometry. Experiments using this flow cell are described wherein its efficacy is evaluated under flow cytometric conditions with fluorescent microspheres. Results: Preliminary laboratory experiments demonstrate acoustic focusing of flowing 10-μm latex particles into a tight sample stream that is ∼40 μm in diameter. Prototype flow cytometer measurements using an acoustic-focusing flow chamber demonstrated focusing of a microsphere sample to a central stream ∼40 μm in diameter, yielding a definite fluorescence peak for the microspheres as compared with a broad distribution for unfocused microspheres. Conclusions: The flow cell developed here uses acoustic focusing, which inherently concentrates the sample particles to the center of the sample stream. This method could eliminate the need for sheath fluid, and will enable increased interrogation times for enhanced sensitivity, while maintaining high particle-analysis rates. The concentration effect will also enable the analysis of extremely dilute samples on the order of several particles per liter, at analysis rates of a few particles per second. Such features offer the possibility of a truly versatile low-cost portable flow cytometer for field applications.