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Fundamentals of Acoustic Cytometry
Publication Name:
Current Protocols in Cytometry
Unit Number:
UNIT 1.22
DOI:
10.1002/0471142956.cy0122s49
Print Publication Date:
July, 2009
Online Posting Date:
July, 2009 Abstract
Acoustic cytometry is a new technology that replaces or partly replaces hydrodynamic focusing of cells or particles with focusing derived from acoustic radiation pressure forces. It offers new possibilities for improving current flow cytometry assays and creating new ones. To take full advantage of these possibilities, it is necessary to understand the fundamental benefits and limitations of acoustic focusing as employed in flow cytometry analysis, either as a substitute for hydrodynamic focusing or in combination with it. Curr. Protoc. Cytom. 49:1.22.1-1.22.12. © 2009 by John Wiley & Sons, Inc.
Keywords: flow cytometry; acoustic focusing; sample preparation
Figures
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Figure 1.22.1Schematic of a line-driven capillary depicting tight single line focusing of particles in a flowing liquid using acoustic radiation pressure. -
Figure 1.22.2Illustration of an acoustic cytometer. Sample is drawn into the capillary, acoustically focused, analyzed, and transferred to waste. Similar to a conventional flow cytometer, the analysis stage includes a laser beam focused at the position of the particles in the optical cell. The scatter/fluorescent signal is conditioned by appropriate filters and dichroics before detection on PMTs. A control circuit is added to ensure the piezoelectric device is driven at the acoustic resonance frequency of the piezoelectric element/capillary. -
Figure 1.22.3Calculated acoustic force potential in the cross section of an acoustically-driven capillary. Particles with positive acoustic contrast are focused toward the potential trap in the center of the cross-section. -
Figure 1.22.4Calculated trajectories of different diameter microspheres as they travel along the axis of the acoustic focusing capillary. The vertical axis is the particle position relative to the capillary axis. The horizontal axis is the particle position along the length of the capillary. Sample flows from left to right at a rate of 1 ml/min. -
Figure 1.22.5Number of events versus peak area for 8 peak, 3.0-µm Sphereotech SPHERO rainbow calibration particles in an acoustic focusing cytometer running at 1 ml/min. Side scatter and FL1 coefficients of variation for the brightest peak at this flow rate are at 1.61% and 2.50%, respectively. As sample flow rate increases beyond a maximum optimal rate, coefficients of variation begin to degrade. -
Figure 1.22.6Micrographs of a blood sample diluted 200× in an optical cell 240 µm in diameter following an acoustic focusing capillary. (A) Acoustic field is OFF and the sample is unfocused. (B) Acoustic field is activated and the sample is focused into a rope-like structure of cells demonstrating the concentration effect. (C) The sample is diluted an additional 10×, showing single file alignment of cells. -
Figure 1.22.7Plot of number of events versus arrival interval at the interrogation laser for acoustically focused microspheres in flow at 1 ml/min. The black line is an exponential distribution. The experimental data (gray bars) closely matches this prediction. -
Figure 1.22.8(A) Schematic illustrating the sample squeezing effect of hydrodynamic focusing. The ratio of sample to sheath required to achieve a typical 10 to 30 µm core diameter is on the order of 1000:1 to 100:1. (B) A micrograph of fluorescent microspheres hydrodynamically focused using a sheath-to-sample ratio of 4:1. The core diameter (fluorescent streaks) is ~100 µm. (C) The same 4:1 sheath ratio as in B, but with acoustic focusing activated and dramatically assisting the hydrodynamic focus. -
Figure 1.22.9Plot of particle analysis rate versus initial sample concentration for different sample input rates and a maximum instrument rate of 10,000 particles/sec. The dashed and gray lines represent typical high and low conventional cytometer input rates of 100 µl/min and 10 µl/min. The solid black line represents a sample input rate of 3 ml/min, possible using the acoustic cytometer. (The calculations assume that samples with initial concentrations above the optimal concentration for maximum particle analysis rate are diluted to achieve acceptable coincidence while maintaining the optimal rate.) This figure shows that maximum instrument particle rates can be achieved over a large particle concentration range with proper up-front sample dilution and/or a range of sample input rates.
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