Conventional Flow Cytometry vs. Acoustic Focusing
Keeping cells within a confined focal point is important for consistent excitation of the associated fluorophores as they pass through the tightly focused laser beam. However, as the sample rate is increased by raising the pressure behind the sample stream, the pressure differential between the sheath stream and the sample stream decreases, and the sample stream “core” widens. A wider sample core results in a broader distribution of cells as they transit through the laser, and thus fewer cells are accurately aligned with the laser focal point. To obtain optimal data from a conventional flow cytometer, with the lowest variability in signal detection, the instrument must be run at the lowest sample rate, which is typically 10–20 μL/min. Higher sample rates result in greater variability and less precise measurements. Acoustic focusing avoids this compromise in data and sample rates by uncoupling cell alignment from sheath flow.
A Breakthrough in Flow Cytometry Technology
The Attune™ Acoustic Focusing Cytometer incorporates many components of a conventional cytometer, including an optical cell for sample interrogation, lasers, and electronics for collecting fluorescence and scatter information. Syringe pumps are used in the Attune™ cytometer to deliver precise volumes of sample, and although delivery of sheath fluid is not required for focusing, the Attune™ cytometer uses a limited amount of fluid to prevent the sample from contacting the optical flow cell.
The resonance frequency required to maintain tight particle alignment in the acoustic focusing capillary is dependent on the capillary diameter and wall thickness. A capillary with an inner diameter of 300 μm, for example, requires 3 MHz for resonance, whereas a 600 μm capillary only requires ~1.5 MHz. Temperature and fluid properties can also affect the resonance of the capillary, so an electronic circuit is required to “tune” the capillary.
Figure 1. Schematic of an acoustic focusing capillary.
Acoustic Forces are Safe for Cells
Does Cell Size Make a Difference?
Acoustic Focusing With a Hydrodynamic Element
In addition, Attune™ Focusing Fluid performs the critical role of keeping the walls of the optical chamber clean by preventing sticky proteins, biomolecules, and reagents from making contact.
Advantages of the Attune™ Cytometer
By uncoupling cell alignment from hydrodynamic forces and sheath flow, the Attune™ cytometer allows rapid detection of rare events. The instrument is designed to collect up to 20,000,000 events per run, enabling detection of those rarest of rare events. Because there is minimal variation in results regardless of sample throughput rate, the Attune™ cytometer is ideal for detecting cell proliferation, where it is critical to precisely detect differences in fluorescence intensity between multiple cell populations (Figure 2). The Attune™ cytometer also provides clear resolution of difficult-to-detect signals—for example, from dim antigens such as ZAP-70—to help facilitate cell signaling studies.
Figure 2. Ten cell divisions identified with the Attune™ Acoustic Focusing Cytometer and the Molecular Probes® CellTrace™ Violet Cell Proliferation Kit. Human peripheral blood mononuclear cells were isolated from whole blood, stained with the CellTrace™ Violet Cell Proliferation Kit, and stimulated to proliferate in culture. Cells were then analyzed on the Attune™ cytometer at a flow rate of 25 μL/min. The histogram shows fluorescence intensity, with each peak representing one subsequent generation of proliferating cells.
Volumetric Throughput—Achieving Incredible Sample Rates
This feature also allows researchers to intentionally dilute samples to avoid washing by centrifugation. In the case of very low amounts of available sample, dilution to 1 mL or more can help preserve the sample while not significantly affecting data acquisition time. For example, a sample volume of 5 µL diluted 800-fold to a final volume of 4 mL can be processed in a few minutes at a run rate of 1 mL/min. Diluting a sample to this level prior to analysis with the Attune™ cytometer reduces background more than does a single round of centrifugation and can minimize or eliminate altogether the need for washing (and subsequent sample loss). For whole blood samples, this technique can be combined with a fluorescence trigger such as CD45 to perform sensitive, no-lyse, no-wash immunophenotyping without the variability and cell loss or degradation common to lysis and centrifugation sample prep.
The Future of Acoustic Focusing Cytometry
- Go to the Applied Biosystems web site and view the new acoustic focusing tutorial and learn more about the Attune™ Acoustic Focusing Cytometer.
- Kundt A, Lehmann O (1874) Annalen der Physik und Chemie (Poggendorff ’s Annalen) 153:1–11.
- Curtis HW, Stephans EJ (1982) IBM Technical Disclosure Bulletin 25(1).
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- Jonsson H, Nilsson A, Petersson F et al. (2005) Perfusion 20:39–43.
- Kaduchak G, Goddard G, Salzman G et al. (2008) US Patent 7,340,957.
- Learn More about the Attune™ Acoustic Focusing Cytometer
- a = particle radius
- β0 = compressibility of the surrounding fluid in the absence of particles;
- ρ0 = density of the surrounding fluid
- p = pressure
- v = velocity of the acoustic field
The time-averaged quantity is bracketed. Terms ƒ1 and ƒ2 determine how the mechanical properties of the particle differ from the medium, where:
The force acting on the particle is related to the gradient of the force potential (U) as follows: F = −∇U. Under acoustic forces, particles localize at a stable equilibrium where the potential U is at a minimum. The acoustic contrast of a particle is determined by the density and compressibility differences between it and the surrounding medium. The relative magnitudes of ƒ1 and ƒ2 determine the magnitude and the direction of the radiation force. For example, if a particle and the surrounding medium share the same density, then ƒ2 is zero and the acoustic contrast is due only to compressibility differences (ƒ1). If both ƒ1 and ƒ2 equal zero, then no acoustic contrast exists.
Cells inside the acoustic focusing capillary of the Attune™ cytometer experience a “standing wave” force that transports them to the minimum force potential at the axial center of the capillary. Nearly all particles and cells of biological origin have a positive acoustic contrast in aqueous buffer. A few materials, such as fat globules and air bubbles, carry a negative acoustic contrast with surrounding aqueous medium and migrate toward the walls of the capillary.