Patent Application
20180224367
Embodiments of the present invention relate to acoustic cytometry and more specifically to acoustic focusing hardware and implementations.
Note that the following discussion refers to a number of publications by author(s) and year of publication, and that due to recent publication dates certain publications are not to be considered as prior art vis-à-vis the present invention. Discussion of such publications herein is given for more complete background and is not to be construed as an admission that such publications are prior art for patentability determination purposes.
Flow cytometry is a powerful tool used for analysis of particles and cells in a myriad of applications primarily in bioscience research and medicine. The analytical strength of the technique lies in its ability to parade single particles (including bioparticles such as cells, bacteria and viruses) through the focused spot of light sources, typically a laser or lasers, in rapid succession, at rates exceeding thousands of particles per second. The high photon flux at this focal spot produces scatter of light by a particle and/or emission of light from the particle or labels attached to the particle that can be collected and analyzed. This gives the user a wealth of information about individual particles that can be quickly parleyed into statistical information about populations of particles or cells.
In traditional flow cytometry, particles are flowed through the focused interrogation point where a laser directs a laser beam to a focused point that includes the core diameter within the channel. The sample fluid containing particles is hydrodynamically focused to a very small core diameter of around 10-50 microns by flowing sheath fluid around the sample stream at a very high volumetric rate on the order of 100-1000 times the volumetric rate of the sample. This results in very fast linear velocities for the focused particles on the order of meters per second. This in turn means that each particle spends a very limited time in the excitation spot, often only 1-10 microseconds. When the linear flow of the hydrodynamic sheath fluid is stopped, the particles are no longer focused. Only resuming the hydrodynamic sheath fluid flow will refocus the particles. Further, once the particle passes the interrogation point the particle cannot be redirected to the interrogation point again because the linear flow velocity cannot be reversed. Still further, a particle cannot be held at the interrogation point for a user defined period of time for further interrogation because focusing is lost without the flow of the hydrodynamic sheath fluid. Because of the very high photon flux at the excitation point, flow cytometry is still a very sensitive technique, but this fast transit time limits the sensitivity and resolution that can be achieved. Often, greater laser power is used to increase the photon flux in an effort to extract more signal but this approach is limiting in that too much light can often photobleach (or excite to non-radiative states) the fluorophores being used to generate the signal and can increase background Rayleigh scatter, Raman scatter and fluorescence as well.
Slower flowing cytometry systems have been developed to push the limits of sensitivity and have shown detection limits down to the single molecule level. In one of these systems, it was shown that lower laser power (<1 mW) was actually preferable for single molecule detection of double stranded DNA fragments intercalated with fluorescent dyes. Because of the slow transit times (hundreds of microseconds to milliseconds), it was possible to get maximum fluorescence yield out of the dyes while reducing background, photobleaching and non-radiative triplet states with the lower laser power.
Slow flow hydrodynamic systems, while incredibly sensitive, are not in widespread use because fluidic dimensions are generally very small, which results in easy clogging and very limited sample throughput. In order to focus the sample stream to a core diameter small enough to maintain the uniform illumination and flow velocity required for precision particle measurement, the sheath must still be supplied in a very high volumetric ratio to the sample. In order to achieve a slow linear velocity, the volumetric sample rate must be extremely small. Therefore, to process appreciable numbers of events, the sample must be highly concentrated. If for example a relatively slow linear velocity of 1 centimeter per second is desired with a typical core diameter of about 10 microns, the sample must be delivered at about 0.05 microliters per minute. To process just 100 cells per second, the cell concentration must be 120,000 per microliter or 120 million per milliliter. This concentration requirement in turn makes clogging even more likely. The problem is further compounded by the tendency of many types of cells to clump in high concentration and to settle out and stick to surfaces when sample delivery rates are slow. The system created by Doornbos, circumvents the clogging problem by using a conventional flow cell with flow resistors to slow the flow, but he found it very difficult to control precise focused delivery of the sample. This method also does not eliminate the need for slow volumetric delivery and highly concentrated samples.
Sheathless, non-focusing flow cytometers have been developed but these instruments suffer from low sensitivity due to the need for a focal spot size that will excite particles throughout the channel. The spot size is reduced by using very small capillary channels but particles flow within the channel at variable rates according to the laminar flow profile that develops in the channel. This results in different transit times and coincidence of particles in the laser spot which both make analysis more difficult. Also, background cannot be reduced by spatially filtering optics that are designed to collect light from a tightly focused core stream. This limits sensitivity and resolution.
Other approaches have been demonstrated to manipulate particles using acoustic radiation pressure in a laboratory setting. These devices are planar devices modeled in Cartesian coordinates. Applying an acoustic field generates a quasi-one-dimensional force field that focuses particles into a ribbon in a rectangular chamber. For laminar flow, the resulting distribution of particles across the chamber places the particles in different velocity stream lines. Particles in different stream lines will not only be in different locations but they will also flow at different velocities. This in turn results in different residence times for particles at a location within the device. Planar focusing does not align particles in a manner suitable for use with flow cytometers.
An embodiment of the present invention comprises an acoustic focusing capillary further comprising a capillary coupled to at least one vibration source and the at least one vibration source possessing a groove. The capillary of this embodiment is preferably coupled to the vibration source at the groove. The groove preferably has an approximately same cross-sectional geometry as the capillary. The capillary can be circular, elliptical, oblate, or rectangular. The vibration source preferably comprises a piezoelectric material. In this embodiment, the groove preferably increases an acoustic source aperture of the capillary.
Another embodiment of the present invention comprises a method of manufacturing an acoustic focusing capillary. This embodiment further comprises providing a capillary and at least one vibration source, machining a groove into the vibration source, and coupling the at least one vibration source to the capillary at the groove. The groove preferably has an approximately same cross-sectional geometry as the capillary. The capillary can be circular, elliptical, oblate, or rectangular. The at least one vibration source preferably comprises a piezoelectric material. This embodiment can optionally comprise increasing the acoustic source aperture of the capillary.
In yet another embodiment of the present invention, an apparatus that hydrodynamically and acoustically focuses particles in a particle stream comprises a flow chamber, an outer confine of the flow chamber for flowing a hydrodynamic fluid therethrough, a central core of the flow chamber for flowing the particle sample stream therethrough, and at least one transducer coupled to the chamber producing acoustic radiation pressure. The transducer of this embodiment is preferably coupled to an outer wall of the flow chamber. The transducer can alternatively form a wall of the flow chamber.
A further embodiment of the present invention comprises a method of hydrodynamically and acoustically focusing a particle stream. This method preferably comprises flowing a sheath fluid into outer confines of a capillary, flowing a particle stream into a central core of the capillary, and applying acoustic radiation pressure to the particle stream within the sheath fluid. The particle stream of this method can be hydrodynamically focused and subsequently acoustically focused. Alternatively, the particle stream is acoustically focused and hydrodynamically focused simultaneously.
Another method of hydrodynamically and acoustically focusing particles is still a further embodiment of the present invention. This embodiment preferably comprises providing a fluid comprising particles therein, flowing a sheath fluid into outer confines of a flow chamber, flowing the fluid containing the particles into a central core of the flow chamber, and applying acoustic radiation pressure to the fluid comprising the particles. This embodiment can also comprise analyzing the particles.
One embodiment of the present invention comprises a method of aligning particles using acoustic radiation pressure. This embodiment preferably includes providing a fluid comprising particles therein, subjecting the fluid to acoustic radiation pressure, rotating the fluid 90 degrees, and subjecting the fluid to acoustic radiation pressure a second time to align the particles. This embodiment can also comprise analyzing the aligned particles.
Another embodiment of the present invention comprises a method of hydrodynamically and acoustically focusing particles in a fluid. This embodiment includes flowing a fluid comprising particles therein, subjecting the fluid to acoustic radiation pressure in one planar direction to acoustically focus the particles, and flowing a sheath fluid in a second planar direction thereby hydrodynamically focusing the fluid in the second planar direction to further focus the particles.
The present invention further includes methods for dislodging bubbles in a fluidic system. These methods comprise providing a fluid stream through a channel and resonating the channel at an acoustic frequency. These methods also include providing a fluid stream through a channel and vibrating the channel walls at a low frequency.
An embodiment of the present invention is an apparatus that acoustically focuses particles into a quasi-planar arrangement in a fluid. This embodiment preferably comprises a capillary with an oblate cross-sectional geometry and at least one transducer coupled to the capillary. The capillary is preferably elliptical. This embodiment can further comprise an imager for imaging the particles.
Another embodiment of the present invention comprises a method for acoustically focusing particles into a quasi-planar arrangement in a fluid comprising particles. The method preferably comprises flowing the fluid comprising particles therein through a flow chamber comprising an oblate cross-sectional geometry and subjecting the fluid to acoustic radiation pressure. The cross-sectional geometry of the flow chamber is preferably elliptical. This embodiment can also include imaging the particles.
Objects, advantages and novel features, and further scope of applicability of the present invention will be set forth in part in the detailed description to follow, taken in conjunction with the accompanying drawings, and in part will become apparent to those skilled in the art upon examination of the following, or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.
The accompanying drawings, which are incorporated into and form a part of the specification, illustrate one or more embodiments of the present invention and, together with the description, serve to explain the principles of the invention. The drawings are only for the purpose of illustrating one or more preferred embodiments of the invention and are not to be construed as limiting the invention. In the drawings:
As used herein “a” means one or more.
As used herein “flow chamber” means a channel or capillary having a shape selected from rectangular, square, elliptical, oblate circular, round, octagonal, heptagonal, hexagonal, pentagonal, and triagonal. It is not necessary for the shape of the interior walls of the flow chamber be the same as the shape of the exterior walls. As a non-limiting example, a flow chamber can have an interior wall defined by a circular shape and the exterior wall defined by a rectangular shape. Additionally, the flow chamber can be part of a composite construction of materials and geometries wherein one of the above shapes defines the interior shape of the flow chamber.
As used herein “capillary” means a channel or chamber having a shape selected from rectangular, square, elliptical, oblate circular, round, octagonal, heptagonal, hexagonal, pentagonal, and triagonal. It is not necessary for the shape of the interior walls of the capillary be the same as the shape of the exterior walls. As a non-limiting example, a capillary can have an interior wall defined by a circular shape and the exterior wall defined by a rectangular shape.
One aspect of one embodiment of the present invention provides for ease in alignment during device fabrication and for larger acoustic source apertures. Another aspect of one embodiment of the present invention provides for a line-driven capillary with oblate cross-section to obtain quasi-planar particle concentration. Another aspect provides for planar particle concentration without particles contacting and/or staying in contact with the inner capillary wall. Another aspect provides for imaging applications where particles spread over a plane in a narrow depth of field. Another aspect provides for applying acoustic radiation pressure forces to assist in stabilizing standard hydrodynamic particle focusing systems. Yet another aspect provides for reduced sheath consumption in slow-flow hydrodynamic systems and to assist in particle focusing in planar systems (e.g. chip based systems). Still another aspect provides a method to dislodge bubbles from fluidic systems.
Line-driven capillaries are used to acoustically concentrate particles in a flowing stream within a capillary. Particles experience a time-averaged acoustic force resulting from acoustic radiation pressure.
Another aspect of one embodiment of the present invention provides a line-driven capillary system that both delivers stable acoustic signals within the capillary and possess consistent, repeatable electrical properties of the electromechanical circuit that drives the system.
In one embodiment of the present invention, line-driven capillary 10 is comprised of capillary 14 coupled to vibration source 16. Capillary 14 can be made from, but are not limited to glass, metal, plastic, or any combination thereof. Low loss materials are preferably particle concentrators and stainless steel is one of the better capillary materials. Vibration source 16 is preferably comprised of a piezoelectric material. Examples of piezoelectric materials include but are not limited to PZT, lithium niobate, quartz, and combinations thereof. Vibration source 16 can also be a vibration generator such as a Langevin transducer or any other material or device that is capable of generating a vibration or surface displacement of the capillary. Another aspect of the embodiment of the present invention comprises an acoustically focused line drive capillary that yields a larger acoustic source aperture than a standard line contact.
According to one embodiment of the present invention, groove 18 is machined into vibration source 16 into which capillary 14 is cradled, as illustrated in
Another aspect of one embodiment of the present invention provides ease of device construction.
Yet another aspect of one embodiment of the present invention provides for larger acoustic source aperture as compared to a true line-driven device.
Still another aspect of one embodiment of the present invention provides for repeatable acoustic/electrical performance.
Another aspect of one embodiment of the present invention provides for ease in alignment of capillary 14 with vibration source 16.
Still another aspect of one embodiment of the present invention provides for a larger glue surface upon which to attach a transducer.
Additionally, it is not necessary for the capillary to have a circular cross section. In one embodiment of the present invention, a square cross section groove in PZT is used. Capillaries can be constructed with many geometries including but not limited to elliptical, square, rectangular, general oblate shapes, as well as any cross sectional geometry.
Referring now to
For example, an acoustically driven capillary with an elliptical cross section is illustrated in
Here, a is the particle radius, β0 is the compressibility of the surrounding fluid, and ρ0 is the density of the surrounding fluid. The pressure and velocity of the acoustic field in the absence of the particle are described by p and v, respectively, and the brackets correspond to a time-averaged quantity. The terms f1 and f2 are the contrast terms that determine how the mechanical properties of the particle differ from the background medium. They are given by:
The subscript p corresponds to intrinsic properties of the particle. The force F acting on a particle is related to the gradient of the force potential by:
F=−vU
Particles are localized at positions where the potential U displays a minimum. (For a circular cross section capillary, a potential minimum is coincident with the axis of the capillary forming the particle trap in
The force potential U for an elliptical cross section capillary line-driven in a dipole type mode is illustrated in
Depending upon the aspect ratio of the ellipse, the force potential in orthogonal directions can be dramatically different. This is illustrated in
Results showing this effect are given in
Several characteristics of these types of modes exist:
Particles can be tightly focused either along major or minor axis of ellipse with ‘loose’ focusing along the orthogonal direction (selection of which axis is the weak focusing direction is mode dependent).
In the weakly focused dimension, enough force can exist to keep particles off the wall of the device.
Particles are confined to a plane which is conducive to imaging applications where it is necessary to place particles in a common plane especially when depth of focus is small.
Hydrodynamically-focused particle streams are used in flow cytometry as well as other areas where precisely aligned particles in a flowing core stream are required. Hydrodynamic focusing is traditionally employed in flow cytometry to focus particles into a tight stream for laser interrogation. A diagram of a hydrodynamically focused particle stream is illustrated in
where Q is the volumetric flow rate of the core stream and v is the velocity of the core stream. Note that larger volumetric sample delivery and/or lower velocities yield larger diameters of the core stream.
Hydrodynamically focused sample streams can suffer from instabilities of the central core stream position as a function of many factors. These can include but are not limited to nucleation of bubbles on the cell walls that alter stream lines, turbulence, and combinations thereof. It is advantageous to assist hydrodynamically focused systems with an external force that stabilizes the spatial position of the central core stream. An embodiment of the present invention comprises a device that uses multiple fluidic streams to steer the central core stream.
One embodiment of the present invention combines acoustic focusing of particles with hydrodynamic focusing. Acoustic focusing assists hydrodynamic focusing systems by stabilizing the absolute location of the particle stream against external forces. Acoustic focusing is also used to further tighten the focus of the particle stream within a hydrodynamically focused system where reduction in sheath fluid consumption or increase in sample throughput is desired without the loss of particle focus quality within the stream. This is particularly important for applications where the sample is dilute. A prime example is high speed sorting of “sticky” cells that must be kept at lower concentration to prevent aggregation. Another example is where reduction of sheath fluid is a priority without sacrificing particle focus. Furthermore, some systems that employ acoustic focusing may not want the sample to contact the walls. (For example, this will keep the build-up of proteins and small particles that are unaffected by acoustic radiation pressure off the capillary walls. These systems can use a slow, low-volume sheath to entrain the sample. Acoustic focusing can then be used to tightly focus the particles within the sample stream.)
An example of acoustically assisted hydrodynamic focusing is shown in
Referring now to
Measurements demonstrating acoustically assisted hydrodynamic focusing are illustrated in
Aspects of acoustically assisting hydrodynamically focused sample streams include but are not limited to:
There are many different arrangements where both acoustically focusing and hydrodynamically focusing can be advantageous.
The ability to use acoustically assisted hydrodynamic focusing of particles is also advantageous for applications in microfluidics. Hydrodynamic focused particle streams in microchannels, microfluidic chips, or other rectangular (or quasi-rectangular) channel geometrics can be enhanced by combining acoustic focusing and hydrodynamic focusing. In one embodiment of the present invention and to reduce sheath fluid consumption, one dimensional hydrodynamic focusing can be used, as illustrated in
Another embodiment of the present invention allows for serial acoustic focusing in microfluidic applications. Acoustic focusing applied to a flowing stream of particles in a quasi-rectangular cross section chamber focuses particles into a ribbon-like structure. In order to preserve the layered construction used in many microfluidic assemblies, it is advantageous to preserve the placement of the transducers in parallel planes. Thus, a method to focus particles into a narrow spatial configuration involves acoustically focusing particles into a plane, rotating the flow by 90 degrees, and then acoustically focusing again into the new orthogonal plane. The net result is a narrow spatial distribution of particles. When the transducer is used to excite a dipole type mode within the flow chamber, the result is particles narrowly focused about the central axis of the flow chamber.
In traditional flow cytometry, bubbles that adhere to walls of a fluidic system are problematic. They can interfere by moving laminar flow lines, affecting local reactions, deviating focused particle streams. For example, in flow cytometers, bubbles in a fluidic system can have the effect of moving the position of the hydrodynamically-focused sample stream. This movement appears as a misalignment of the optical system to the user and a recalibration is required. A technique to dislodge bubbles from nucleation sites in fluidic systems, especially microfluidic systems, is very desirable.
Acoustic radiation pressure has been shown to have a large effect on bubbles in fluids due to the large mismatch in density and compressibility between liquids and gases. Acoustic energy can be used to dislodge bubbles from fluidic system in several different ways.
In one embodiment of the present invention, a fluidic system is engineered such that when resonated at an appropriate acoustic frequency, bubbles experience a force that pulls them away from the wall and stabilizes their equilibrium position within the fluid stream that exists at the location of a pressure node (for bubbles driven at frequencies below their monopole resonance). The chamber is preferably driven acoustically with either an internal acoustic source or noninvasively with a source attached to an outer wall of the chamber. This is a robust method to dislodge bubbles from walls.
In another embodiment of the present invention, vibration of the channel walls at low frequencies is preferably used to dislodge the bubbles. By vibrating the wall as part of a structural resonance of the system, large surface displacements are achieved. (These displacements are typically larger at lower frequencies.) Large forces coupled with large displacements are preferably used to break the bond between the bubble and the chamber surface. The inertial forces coupled with localized fluid flows at the chamber wall surface are effective at bubble dislodgement.
The preceding examples can be repeated with similar success by substituting the generically or specifically described reactants and/or operating conditions of this invention for those used in the preceding examples.
Although the invention has been described in detail with particular reference to these preferred embodiments, other embodiments can achieve the same results. Variations and modifications of the present invention will be obvious to those skilled in the art and it is intended to cover in the appended claims all such modifications and equivalents. The entire disclosures of all references, applications, patents, and publications cited above and/or in the attachments, and of the corresponding application(s) are hereby incorporated by reference.
This application is a divisional of U.S. patent application Ser. No. 14/213,049, filed Mar. 14, 2014, which is a continuation of U.S. patent application Ser. No. 12/209,084 filed Sep. 11, 2008, now U.S. Pat. No. 8,714,014, issued May 6, 2014, which application claims priority to and the benefit of U.S. Provisional Patent Application Ser. No. 61/021,443, entitled “System and Method for Acoustic Focusing Hardware and Implementations”, to Kaduchak, filed on Jan. 16, 2008, and the specification thereof is incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 61021443 | Jan 2008 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14213049 | Mar 2014 | US |
| Child | 15888821 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 12209084 | Sep 2008 | US |
| Child | 14213049 | US |
