# Patent

## Title
Method for non-contact particle manipulation and control of particle spacing along an axis

## Abstract

One or more of the embodiments of the present invention provide for a method of non-contact particle manipulation and control of particle spacing along an axis which includes axial and radial acoustic standing wave fields. Particles are suspended in an aqueous solution, and this solution then flows into the cylindrical flow channel. While the solution flows through the flow channel, the outer structure of the flow channel is vibrated at a resonant frequency, causing a radial acoustic standing wave field to form inside the flow channel in the solution. These radial acoustic standing waves focus the particles suspended in the solution to the center axis of the cylindrical flow channel.
At the same time, a transducer is used to create an axial acoustic standing wave field in the flow channel parallel to the axis of the flow channel. This drives the particles, which are already being focused to the center axis of the flow channel, to nodes or anti-nodes of the axial standing wave at half-wavelength intervals, depending on whether the particles are more or less dense and more or less compressible than the surrounding fluid.

## Background
STATEMENT REGARDING FEDERAL RIGHTS
This invention was made with government support under Contract No. DE-AC52-06NA25396 awarded by the U.S. Department of Energy. The government has certain rights in the invention.


BACKGROUND OF INVENTION
The present invention relates to a method for controllably spacing particles along an axis. More specifically, the present invention relates to a method for manipulating particles and controlling their spacing along an axis without directly contacting the particles during droplet based flow cytometry.
Piezoelectric droplet generation devices used for inkjets have been adapted for use with coulter volume and optical sensing to perform the first sorting of biological cells. The basics of this system have remained mostly unchanged since then. A hydrodynamically focused sample stream containing particles is optically interrogated (for scatter or fluorescence) either just prior or just after the focused stream is ejected into the air. The ejected stream is vibrated through the use of piezoelectric transducers, which generate waves within the sample stream leading to droplet formation. This process can lead to very regularly and predictably spaced droplets which can then be charged and deflected to be either kept for further analysis or discarded. This is one common format generally referred to as flow cytometry or often as fluorescence activated cell sorting (FACS).
However, several factors limit the speed at which this process can take place. The maximum drop formation rate requires nearly 3.45 MPa to drive 2.5×105 drops per second, though practically, mechanical limitations of the flow cytometer as well as effects on cells restrict the pressure applied to the system, limiting the rate to 1.0×105 drops per second. Additionally, because the particle arrival rate is stochastic, the maximum sort rate is additionally limited to approximately 2.5×104 particles per second. Further, the uncertain position of the particles in the flow stream leads to empty droplets, droplets with multiple particles, and particles at the break-off point between one droplet and another, all of which can lead to aborted sorting events, and all of which greatly reduce the overall sort rate.
Particle sorters of this type have problems with large particles (particles of diameter greater than 1×10−4 meters) such as multicellular organisms, multicellular spheroids, and large microspheres typically used for combinatorial library synthesis. Large particle sorters generally needed larger flow channels and exit orifices to prevent the randomly arriving particles from clogging and to prevent particle effects on droplet break-offs. However, these larger flow channels restricted the linear velocity of the flow stream due to problems caused by increased turbulence. Also, as particles took up an increasing proportion of the exit orifice, droplet formation was disturbed, effectively limiting the size of particles to about 20% the size of the exit orifice or less. Combined, these factors restricted the sorting rate of large particles to roughly 1000 per second. Some work has been done with acoustic focusing of particles, though this has not corrected the problem of random particle arrival.
Some alternative methods have been attempted, such as a high pressure air jet that deflects the flow stream unless an analysis indicates that the particles in the stream should be kept at which point the air jet shuts down allowing the particles to pass through (though this method is limited to only about 15-1000 particles per second), a fluidic switching approach which is typically mechanical in nature (limited to only about 500 particles per second), and a laser ablation approach (though this method is no faster than current techniques leaves significant cell debris and is not suitable for sorting of microspheres).
Consequently, a need has long been felt for a method of assuring predictable arrival of particles to ensure accurate high speed sorting of large particles.
SUMMARY OF THE INVENTION
One or more of the embodiments of the present invention provide for a method of non-contact particle manipulation and control of particle spacing along an axis which includes axial and radial acoustic standing wave fields. Particles are suspended in an aqueous solution, and this solution then flows into the cylindrical flow channel. While the solution flows through the flow channel, the outer structure of the flow channel is vibrated at a resonant frequency, causing a radial acoustic standing wave field to form inside the flow channel in the solution. These radial acoustic standing waves focus the particles suspended in the solution to the center axis of the cylindrical flow channel.
At the same time, a transducer is used to create an axial acoustic standing wave field in the flow channel parallel to the axis of the flow channel. This drives the particles, which are already being focused to the center axis of the flow channel, to nodes or anti-nodes of the axial standing wave at half-wavelength intervals, depending on whether the particles are more or less dense and more or less compressible than the surrounding fluid. Once the particles have been focused and driven to half-wavelength positions along the center axis of the flow channel, the flow stream with particles spaced at definite intervals flows out of the flow channel. This allows for synchronization of droplet formation and particle arrival, such that a droplet is not created until a particle is present, or such that droplets always contain a particle. These droplets can then either be discarded or kept for further analysis.
Embodiments of the present invention may be combined with recent low cost flow cytometry developments in lasers (such as a self healing laser beam, a single laser focused to a line, multiple laser beams, and diode pumped solid-state lasers) and data acquisition systems (such as multianode photomultiplier tubes, fiber optics, avalanche photodiodes, low cost digital signal processing chips with onboard analogue-to-digital converters, and charge couple device technology), to create inexpensive low linear velocity parallel flow cytometry analyzers to maximize particle through-put. These embodiments of the present invention and assorted methods and devices can be combined with periodic droplet creation and new droplet-on-demand particle sorters (using focused piezo methods and flexural piezo methods) that are less limited by particle size to form less expensive large particle sorters.



BRIEF DESCRIPTION OF THE DRAWINGS
For a better understanding of the present invention, reference may be made to the accompanying drawings in which:
FIG. 1 illustrates a flow chart of the method for controllably spacing particles along an axis using only two acoustic standing wave fields.
FIG. 2 illustrates a flow chart of the method for controllably spacing particles along an axis using only two acoustic standing wave fields and a non-uniform electric field.
FIG. 3 illustrates a flow channel in which particles are spaced randomly.
FIG. 4 illustrates a flow channel that has been exposed to a radial acoustic standing wave field in which the particles have been focused to the center axis of the cylindrical flow channel.
FIG. 5 illustrates a flow channel that has been exposed to a radial acoustic standing wave field and an axial acoustic standing wave field, in which the particles have been focused to the center axis of the cylindrical flow channel and are uniformly spaced.
FIG. 6 illustrates a flow channel that has been exposed to a radial acoustic standing wave field, an axial acoustic standing wave field, and a non-uniform electric field, in which the particles have been focused to the center axis of the cylindrical flow channel and are more narrowly uniformly spaced than those shown in FIG. 5.
FIG. 7 illustrates a block diagram of the flow channel when only two acoustic standing wave fields are in use.
FIG. 8 illustrates a block diagram of the flow channel when two acoustic standing wave fields and a non-uniform electric field are in use.
FIG. 9 illustrates a flow chart of a feedback loop that corrects the frequency of the acoustic standing waves for changes in pressure/temperature.



DETAILED DESCRIPTION OF THE INVENTION
FIG. 1 illustrates a flow chart of a method for controllably spacing particles along an axis using acoustic standing wave fields 100 according to an embodiment of the present invention. The first step in the process of controlling the spacing between particles is suspending the particles in an aqueous saline solution 110. Next, the step of the solution flowing into the flow channel 120 occurs, where the step of vibrating the channel walls at a resonant frequency 130 takes place. The vibration of the chamber walls at a resonant frequency causes a radial acoustic standing wave field to form, which performs the next step of focusing the particles to the center axis of the flow channel 140. At the same time, the step of forming an axial acoustic standing wave with a transducer 150 occurs, where the axial acoustic standing wave is parallel to the center axis of the flow channel. This axial acoustic standing wave causes the step of driving the particles to nodes or anti-nodes of the axial acoustic standing wave 160 to happen. At this point, the particles are spaced at uniform half-wavelength intervals along the center axis of the flow channel, and the final step of the solution flowing out of the flow channel for sorting 170 ensues.
One embodiment of a flow channel can be a cylindrical quartz capillary tube with an inner diameter of about approximately 200 μm, and the vibration of the chamber walls is driven by an acoustic flow cell with piezoelectric sources in contact with the outside of the flow channel. This acoustic flow cell can be driven at a frequency of about 5 MHz. The transducer that forms the axial acoustic standing wave field can also be an acoustic flow cell with piezoelectric sources in contact with the outside of the flow channel. The particles are focused to a stream with a diameter of about approximately 10 μm.
Alternatively, the solution could be something other than an aqueous saline solution. The flow channel may be a non-cylindrical shape, and may be made of a substance other than quartz, such as glass or plastic, and have a different diameter other than the 200 μm inner diameter discussed above, though not so large that the increasing diameter causes excessive turbulence. The acoustic standing wave fields may also be other than acoustic wave fields, such as magnetic field, electric fields, gravitational fields, and optical field. The frequency may be other than 5 MHz, but should be appropriately high to avoid cavitation—typically higher than 1 MHz. The focused stream of particles may also have a diameter of other than 10 μm depending on the size of the particles and the flow channel. Also, this method may be used in a parallel analysis situation, in which a plurality of flow channels run in parallel to accomplish the sorting in less time.
FIG. 2 illustrates a flow chart of a method for controllably spacing particles along an axis using acoustic standing wave fields and a non-uniform electric field 200 according to an embodiment of the present invention. The process can include the step of controlling the spacing between particles by suspending the particles in an aqueous saline solution 210. The process can also include the step of the solution flowing into the flow channel 220, where the step of vibrating the chamber walls at a resonant frequency 230 takes place. This vibration of the channel walls at a resonant frequency causes a radial acoustic standing wave field to form, which performs the step of focusing the particles to the center axis of the flow channel 240. The process can also include the step of forming an axial acoustic standing wave with a transducer 250, where the axial acoustic standing wave is parallel to the center axis of the flow channel. Simultaneously, the step of exposing the flow channel to a non-uniform electric field causing dielectrophoresis 260 occurs. The two steps of forming an axial acoustic standing wave with a transducer 250 and exposing the flow channel to a non-uniform electric field causing dielectrophoresis 260 cause the step of driving the particles to nodes or anti-nodes of the axial acoustic standing wave 270 to happen. These nodes and anti-nodes are more narrowly spaced than those occurring in FIG. 1, because the additional step of exposing the flow channel to a non-uniform electric field causing dielectrophoresis 270 allows for more precision in positioning the particles, and allows the axial standing waves to have a higher frequency and therefore a smaller wavelength. At this point, the particles are spaced at uniform half-wavelength intervals along the center axis of the flow channel, and the final step of the solution flowing out of the flow channel for sorting 280 ensues.
This combination system is constructed using a microfluidic channel design. It can have a planar structure with a lead zirconate titanate (PZT) mounting substrate as the generation mechanism for the ultrasonic manipulation field. A non-uniform electrode surface at the fluid/PZT interface creates the necessary field gradient for the dielectrophoresis, driven at the same frequency as the ultrasonic field. The frequency of acoustic excitation is sufficiently high so as to eliminate potential problems of electrolysis and deleterious transport by electrophoresis. Since the forces generated by the dielectrophoresis are relatively small, sorter electrodes typically have an elongated shape to maximize the time a particle experiences the sideward force and achieve the required displacement with a relatively low force. To improve the sorting speed, the sorter elements are elongated even more.
Alternatively, the solution could be something other than an aqueous saline solution. The acoustic standing wave fields may also be other than acoustic wave fields, such as magnetic field, electric fields, gravitational fields, and optical fields. The non-uniform electric field may be other than an electric field, such as an acoustic field, magnetic field, gravitational field, or optical field, but should further assist in particle spacing. The frequency may other than 5 MHz, but should be appropriately high to avoid cavitation—typically higher than 1 MHz. Also, this method may be used in a parallel analysis situation, in which a plurality of flow channels run in parallel to accomplish the sorting in less time.
FIG. 3 illustrates a prior art flow channel 300. The prior art flow channel 300 includes a quartz flow channel 310, an aqueous saline solution 320, and particles 330. The quartz flow channel 310 contains the aqueous saline solution 320, and the particles 330 are suspended in the aqueous saline solution 320.
In operation, the aqueous saline solution 320, in which the particles 330 are suspended, flows through the quartz flow channel 310. The particles 330, however, are randomly dispersed throughout the aqueous saline solution 320 because there are no outside forces acting upon this prior art flow channel 300 to position the particles 330 in any specific manner. Thus, the particles 330 flow into, through, and out of the quartz flow channel 310 without any uniformity as to where they are located within or the intervals at which they exit from the quartz flow channel 310.
FIG. 4 illustrates a flow channel upon which a radial acoustic standing wave field is acting 400 according to an embodiment of the present invention. The flow channel upon which an ultrasonic radial acoustic standing wave is acting 400 includes a quartz flow channel 310, an aqueous saline solution 320, and particles 330. The quartz flow channel 310 contains the aqueous saline solution 320, and the particles 330 are suspended in the aqueous saline solution 320.
In operation, the aqueous saline solution 320, in which the particles 330 are suspended, flows through the quartz flow channel 310. The radial acoustic standing wave field, produced by vibrating the quartz flow channel 310 at a resonant frequency, directs acoustic waves from the cylindrical quartz flow channel 310 radially inward such that all of the particles 330 are focused to the center axis of the cylindrical quartz flow channel 310. Thus, the particles 330 flow into, through, and out of the quartz flow channel 310 with uniformity as to where they are located within the quartz flow channel 310, but without uniformity as to the intervals at which they exit from the quartz flow channel 310.
Vibrating the quartz flow channel 310 has a further advantage—the extended source formed by the flow channel structure as the source aperture translates into longer residence times of the particles 330 within the acoustic positioning field. The longer residence times allow for the use of lower pressure levels to drive the acoustic focusing field, which alleviates the problem associated with cavitation in an ultrasonic field that could damage fragile particles 330.
FIG. 5 illustrates a flow channel upon which both a radial acoustic standing wave field and an axial acoustic standing wave field are acting 500 according to an embodiment of the present invention. The flow channel upon which both an ultrasonic radial acoustic standing wave and an ultrasonic axial acoustic standing wave field are acting 500 includes a quartz flow channel 310, an aqueous saline solution 320, and particles 330. The quartz flow channel 310 contains the aqueous saline solution 320, and the particles 330 are suspended in the aqueous saline solution 320.
In operation, the aqueous saline solution 320, in which the particles 330 are suspended, flows through the quartz flow channel 310. The radial acoustic standing wave field, produced by vibrating the quartz flow channel 310 at a resonant frequency, directs acoustic waves from the cylindrical quartz flow channel 310 radially inward such that all of the particles 330 are focused to the center axis of the cylindrical quartz flow channel 310. Thus, the particles 330 are no longer randomly positioned throughout the aqueous saline solution 320 in the quartz flow channel 310.
Further, the axial acoustic standing wave field, produced by a transducer at one end of the quartz flow channel 310, directs acoustic waves parallel to the axis of the cylindrical quartz flow channel 310. Particles 330 entrained within an acoustic standing wave field experience a time-averaged drift force of:




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if viscous and thermal conductivity are ignored, where the variables cf is the acoustic velocity in the fluid 320, cp is the acoustic velocity in a particle 330, p is the pressure of the axial wave at the location of a particle 330, v is the velocity of the axial wave at the location of a particle 330, ρp is the density of a particle 330, ρf is the density of the fluid 320, and R is the radius of a particle 330. From this formula, if particles 330 are more compressible and less dense than the surrounding fluid 320, they are driven to pressure anti-nodes along the center axis of the cylindrical quartz flow channel 310—acoustic traps. However, if particles 330 are denser and less compressible than the surrounding fluid 320, they are driven to pressure nodes along the center axis of the cylindrical quartz flow channel 310—more acoustic traps. The overall spacing of these particles 330 is therefore one half wavelength—on the order of between 100-1000 μm (though if these flow cells are used prior to hydrodynamic focusing, this spacing would increase slightly). Thus, the particles 330 flow into, through, and out of the quartz flow channel 310 with uniformity as to where they are located within and the intervals at which they exit from the quartz flow channel 310.

In the alternative, the quartz flow channel 310 may be made of a material other than quartz, such as glass or plastic. The overall spacing between the particles 330 may be other than 100-1000 μm, though it must be of such a distance as to be both viably analyzed and sorted at a rate comparable to or faster than current technologies.
FIG. 6 illustrates a flow channel upon which a radial acoustic standing wave field, an axial acoustic standing wave field, and a non-uniform electric field are acting 600 according to an embodiment of the present invention. The flow channel upon which a radial acoustic standing wave field, an axial acoustic standing wave field, and a non-uniform electric field are acting 600 includes a quartz flow channel 310, an aqueous saline solution 320, and particles 330. The quartz flow channel 310 contains the aqueous saline solution 320, and the particles 330 are suspended in the aqueous saline solution 320.
In operation, the aqueous saline solution 320, in which the particles 330 are suspended, flows through the quartz flow channel 310. The radial acoustic standing wave field, produced by vibrating the quartz flow channel 310 at a resonant frequency, directs acoustic waves from the cylindrical quartz flow channel 310 radially inward such that all of the particles 330 are focused to the center axis of the cylindrical quartz flow channel 310. Thus, the particles 330 are no longer randomly positioned throughout the aqueous saline solution 320 in the quartz flow channel 310.
Further, the axial acoustic standing wave field, produced by a transducer at one end of the quartz flow channel 310, directs acoustic waves parallel to the axis of the cylindrical quartz flow channel 310. Particles 330 entrained within an acoustic standing wave field experience a time-averaged drift force of:




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if viscous and thermal conductivity are ignored, where the variables cf is the acoustic velocity in the fluid 320, cp is the acoustic velocity in a particle 330, p is the pressure of the axial wave at the location of a particle 330, v is the velocity of the axial wave at the location of a particle 330, ρp is the density of a particle 330, ρf is the density of the fluid 320, and R is the radius of a particle 330. From this formula, if particles 330 are more compressible and less dense than the surrounding fluid 320, they are driven to pressure anti-nodes along the center axis of the cylindrical quartz flow channel 310—acoustic traps. However, if particles 330 are denser and less compressible than the surrounding fluid 320, they are driven to pressure nodes along the center axis of the cylindrical quartz flow channel 310—more acoustic traps.

The particles 330 and the aqueous saline solution 320 in the quartz flow channel 310 are also exposed to a non-uniform electric field, which induces dielectrophoresis. The electric field is driven at the same frequency as the ultrasonic field, adding further driving force to position the particles 330 uniformly along the center axis of the cylindrical quartz flow channel 310. The combination of the axial acoustic standing wave field and the dielectrophoretic field allow for finer positioning of the particles 330 along the center axis of the cylindrical quartz flow channel 310, allowing for smaller uniform distances between particles 330 on the order of between 1-100 μm (though if these flow cells are used prior to hydrodynamic focusing, this spacing would increase slightly). Thus, the particles 330 flow into, through, and out of the quartz flow channel 310 with uniformity as to where they are located within and the intervals at which they exit from the quartz flow channel 310.
In the alternative, the quartz flow channel 310 may be made of a material other than quartz, such as glass or plastic. The overall spacing between the particles 330 may be other than 1-100 μm, though it must be of such a distance as to be viably analyzed and sorted at a rate comparable to or faster than current technologies.
FIG. 7 illustrates a block diagram of the flow channel assembly 700 according to an embodiment of the present invention. The flow channel assembly 700 of FIG. 7 includes a flow channel tube 710, a radial wave transducer 720, an axial wave transducer 730, an inlet tube 740, and an outlet port 750.
The radial wave transducer 720 is in contact with the flow channel tube 710 in such a manner that the vibrations of the radial wave transducer 720 cause the cylindrical walls of the flow channel tube 710 to vibrate in resonance. The axial wave transducer 730 is in contact with the flow channel tube 710 in such a manner that the vibrations of the axial wave transducer 730 cause an axial acoustic standing wave field to form that runs parallel to the center axis of the flow channel tube 710. The inlet tube 740 is attached to the flow channel tube 710 such that the flow stream of particles 330 suspended in an aqueous saline solution 320 flows from the inlet tube 740 into the flow channel tube 710. The outlet port 750 is attached to the flow channel tube 710 such that the flow stream of particles 330 suspended in an aqueous saline solution 320 flows out from the flow channel tube 710 through the outlet port 750.
In operation, the flow stream of particles 330 suspended in an aqueous saline solution 320 flows from the inlet tube 740 into the flow channel tube 710, which is made of quartz. While in the flow channel tube, the radial wave transducer 720, which is driven with a piezoelectric source, vibrates against the flow channel tube 710, causing the flow channel tube 710 to vibrate in resonance, which in turn causes a radial acoustic standing wave field to form inside the flow channel tube 710. At the same time, the axial wave transducer 730, which is driven with a piezoelectric source, vibrates against the flow channel tube 710, causing an axial acoustic standing wave field to form that runs parallel to the center axis of the flow channel tube 710. In combination, these wave fields focus and drive the particles 330 to half-wavelength spaced nodes and anti-nodes running along the center axis of the flow channel tube 710. The flow stream of particles 330 suspended in an aqueous saline solution 320, where the particles 330 are located at half-wavelength spaced nodes and anti-nodes running along the center axis of the flow channel tube 710 then exits the flow channel tube 710 through the outlet port 750.
In the alternative, the solution could be something other than an aqueous saline solution. The acoustic standing wave fields may also be other than acoustic wave fields, such as magnetic field, electric fields, gravitational fields, and optical fields. The flow channel may be a non-cylindrical shape, with multiple axial acoustic standing wave fields driving the particles to specific positions. The flow channel tube 710 may be a material other than quartz, such as glass or plastic.
FIG. 8 illustrates a block diagram of the flow channel assembly 800 according to an embodiment of the present invention. The flow channel assembly 800 of FIG. 8 includes a flow channel tube 810, a radial wave transducer 820, an axial wave transducer 830, a non-uniform active electrode surface 835, an inlet tube 840, and an outlet port 850.
The radial wave transducer 820 is in contact with the flow channel tube 810 in such a manner that the vibrations of the radial wave transducer 820 cause the cylindrical walls of the flow channel tube 710 to vibrate in resonance. The axial wave transducer 830 is in contact with the flow channel tube 810 in such a manner that the vibrations of the axial wave transducer 830 cause an axial acoustic standing wave field to form that runs parallel to the center axis of the flow channel tube 810. The non-uniform active electrode surface 835 is positioned at the axial wave transducer 830/aqueous saline solution 320 interface. The inlet tube 840 is attached to the flow channel tube 810 such that the flow stream of particles 330 suspended in an aqueous saline solution 320 flows from the inlet tube 840 into the flow channel tube 810. The outlet port 850 is attached to the flow channel tube 810 such that the flow stream of particles 330 suspended in an aqueous saline solution 320 flows out from the flow channel tube 810 through the outlet port 850.
In operation, the flow stream of particles 330 suspended in an aqueous saline solution 320 flows from the inlet tube 840 into the flow channel tube 810, which is made of quartz. While in the flow channel tube, the radial wave transducer 820, which is driven with a piezoelectric source, vibrates against the flow channel tube 810, causing the flow channel tube 810 to vibrate in resonance, which in turn causes a radial acoustic standing wave field to form inside the flow channel tube 810. At the same time, the axial wave transducer 830, which is driven with a piezoelectric source, vibrates against the flow channel tube 810, causing an axial acoustic standing wave field to form that runs parallel to the center axis of the flow channel tube 810. Concurrently, the non-uniform active electrode surface 835 creates the necessary field gradient for dielectrophoresis, driven at the same frequency as the ultrasonic field. In combination, these fields focus and drive the particles 330 to half-wavelength spaced nodes and anti-nodes running along the center axis of the flow channel tube 810. The flow stream of particles 330 suspended in an aqueous saline solution 320, where the particles 330 are located at half-wavelength spaced nodes and anti-nodes running along the center axis of the flow channel tube 810 then exits the flow channel tube 810 through the outlet port 850.
In the alternative, the solution could be something other than an aqueous saline solution. The acoustic standing wave fields may also be other than acoustic wave fields, such as magnetic field, electric fields, gravitational fields, and optical fields. The flow channel may be a non-cylindrical shape, with multiple axial acoustic standing wave fields driving the particles to certain positions. The flow channel tube 810 may be a material other than quartz, such as glass or plastic.
FIG. 9 illustrates a flow chart of a feedback loop 900 that corrects the frequency of the acoustic standing waves for changes in pressure/temperature according to an embodiment of the present invention. The feedback loop 900 begins with the step of exposing the flow channel to a radial acoustic standing wave fields at a frequency provided by a controller 910 to create a resonant vibration in the flow channel tube 710. Then, the step of reading pressure from within the flow cell tube with a pressure sensor 920 is performed to track temperature changes (and thus pressure changes) in the system. These temperature and pressure changes lead to changes in properties of the materials involved, such as changes in sound speed in fluid within the flow channel tube 710. Next, the step of sending the pressure readings to the controller 930 occurs, where the controller performs the step of using the pressure readings to provide a new frequency 940 to keep the flow channel tube 710 vibrating in resonance. The transducer and flow channel structure are chosen such that there is ample bandwidth to compensate for large temperature changes in the environment. The step of using the pressure readings to provide a new frequency 940 leads back to the original step of exposing the flow channel to a radial acoustic standing wave fields at a frequency provided by a controller 910 to create a resonant vibration in the flow channel tube 710, completing the feedback loop 900.
The controller may be one of a person, a mechanical device, an electric circuit, or a computer program. The sensor may be a temperature sensor as opposed to a pressure sensor.
One or more embodiments of the present invention, which focuses and uniformly spaces particles in a flow cytometer, greatly reduces the wasted time and effort dealing with stochastically arriving particles. The no longer uncertain positions of the particles in the flow stream avoid empty droplets, droplets with multiple particles, and particles at the break-off point between one droplet and another, all of which avoid aborted sorting events which greatly increase the overall sort rate. Further, the focusing of the particles allows larger particle to be sorted without increasing the size of the flow channel because there is less of a chance of the large particles to clog the flow channel. The smaller diameter flow channel leads to less turbulence, allowing for a higher linear velocity of the flow stream. One or more embodiments of the present invention assure predictable arrival of particles, allowing accurate high speed sorting of large particles.
While particular elements, embodiments, and applications of the present invention have been shown and described, it is understood that the invention is not limited thereto because modifications may be made by those skilled in the art, particularly in light of the foregoing teaching. It is therefore contemplated by the appended claims to cover such modifications and incorporate those features which come within the spirit and scope of the invention.

## Claims

1. A uniform particle spacing method including:
suspending particles in an elongated fluid filled cavity where said fluid is flowing through and out said cavity;
exposing said cavity to a radial acoustic standing wave field, wherein said radial acoustic standing wave field focuses said particles to the center axis of said cavity; and
exposing said cavity to an axial acoustic standing wave field, wherein said axial acoustic standing wave field drives said particles to one of nodal and anti-nodal positions along the center axis of said cavity.

2. The uniform particle spacing method of claim 1 further including:
exposing said cavity to a non-uniform electric field, wherein said non-uniform electric field induces dielectrophoresis, wherein said dielectrophoresis further drives said particles one of nodal and anti-nodal positions along the center axis of said cavity.

3. The uniform particle spacing method of claim 2, wherein said non-uniform electric field is a created by a lead zirconate titanate transducer, wherein said transducer has a nonuniform active electrode surface at the transducer/fluid interface.

4. The uniform particle spacing method of claim 1, wherein said radial acoustic standing wave field is produced by vibrating the walls of said cavity at a resonant frequency.

5. The uniform particle spacing method of claim 4, wherein said vibration is created by a piezoelectric source.

6. The uniform particle spacing method of claim 1, wherein said axial acoustic standing wave field is created by a piezoelectric source.

7. The uniform particle spacing method of claim 1, wherein said nodes and anti-nodes are spaced at intervals of one-half the wavelength of said axial acoustic standing waves.

8. The uniform particle spacing method of claim 1, wherein said fluid is a saline solution.

9. The uniform particle spacing method of claim 1, wherein said cavity is a cavity in a shaft made of one of glass and quartz.

10. The uniform particle spacing method of claim 1, further including:
sensing changes in pressure inside said cavity, wherein said sensing occurs in a pressure sensor; and
adjusting the frequencies of said radial acoustic standing wave field and said axial acoustic standing wave field to compensate for said pressure changes.

11. A uniform particle spacing system including:
an elongated fluid filled cavity where said fluid is flowing through and out said cavity, wherein said cavity contains suspended particles, wherein a transducer is acoustically and communicably linked to said cavity and is adapted to vibrate to create a radial acoustic standing wave field, and wherein said radial acoustic standing wave field is adapted to focus said particles along the center axis of said cavity; and
a transducer acoustically and communicably linked to said cavity and is adapted to create an axial acoustic standing wave field, wherein said axial acoustic standing wave field is adapted to drive said particles to uniformly spaced nodes along the center axis of said cavity.

12. The uniform particle spacing system of claim 11 further including:
focusing a stream of particles with acoustic fields, wherein said stream of particles are focused to a diameter in the range of about 5×10−6 and 2×10−5 meters with interparticle spacing in the range of about 1×10−5 and 1×10−2 meters.

13. The uniform particle spacing system of claim 11 further including:
focusing a stream of particles with acoustic and dielectrophoretic fields, wherein said stream of particles are focused to a diameter in the range of about 5×10−6 and 2×10−5 meters with interparticle spacing in the range of about 1×10−7 and 1×10−3 meters.

14. A uniform particle spacing method including:
suspending particles in a solution, wherein said solution flows through a tube;
focusing said particles along the center axis of said tube with a first standing acoustic wave field; and
driving said particles to one of uniformly spaced nodal and anti-nodal positions along the center axis of said tube with a second standing acoustic wave field.

15. The uniform particle spacing method of claim 14 further including:
further driving said particles to one of nodal and anti-nodal positions along the center axis of said tube with a dielectrophoretic field.

16. The uniform particle spacing method of claim 14, wherein said first acoustic standing wave field is produced by vibrating the walls of said tube at a resonant frequency with a piezoelectric source.

17. The uniform particle spacing method of claim 14, wherein said second acoustic standing wave field is created by a piezoelectric source.

18. The uniform particle spacing method of claim 14, wherein said nodes and anti-nodes are spaced at intervals of one-half the wavelength of said second acoustic standing wave field.

19. The uniform particle spacing method of claim 14, further including:
sensing changes in pressure inside said tube, wherein said sensing occurs in a pressure sensor; and
adjusting the frequencies of said first acoustic standing wave field and said second acoustic standing wave field to compensate for said pressure changes.

20. A flow sorting method for uniform cell sorting comprising the steps of:
suspending particles in an elongated fluid filled cavity where said fluid is flowing through and out said cavity;
exposing said cavity to a radial acoustic standing wave field, wherein said radial acoustic standing wave field focuses said particles to the center axis of said cavity;
exposing said cavity to an axial acoustic standing wave field, wherein said axial acoustic standing wave field drives said particles to one of nodal and anti-nodal positions along the center axis of said cavity; and
hydrodynamically focusing a sample stream containing the particles and interrogating said sample stream containing the particles for scatter or fluorescence.