WO2024020348A2

WO2024020348A2 - Submersion transducer with adjustable focus

Info

Publication number: WO2024020348A2
Application number: PCT/US2023/070350
Authority: WO
Inventors: Daniel G. Stearns; William Whalen; David PEISLEY; Bahram Fathollahi; Richard Stearns
Original assignee: Acoustic Bio Inc.
Priority date: 2022-07-18
Filing date: 2023-07-17
Publication date: 2024-01-25
Also published as: WO2024020348A3

Abstract

The present disclosure provides a device comprising a transducer with adjustable focus. In some examples, the device comprises an acoustic transducer configured to provide an acoustic beam. The device may further comprise a lens mount acoustically coupled to the acoustic transducer. The lens mount may comprise a gap configured to contain a coupling fluid. The lens mount may be configured to receive the acoustic beam from the acoustic transducer. The device may further comprise a focusing lens acoustically coupled to the lens mount. The focusing lens may be configured to receive the acoustic beam from the lens mount. The focusing lens may be configured to focus the acoustic beam to the biological sample when the biological sample is in acoustic communication with the focusing lens.

Description

SUBMERSION TRANSDUCER WITH ADJUSTABEE FOCUS

CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63/368,727, filed July 18, 2022, which is incorporated herein by reference in its entirety.

BACKGROUND

[0002] Biological samples may be assayed for various applications, such as, for example, identification of therapeutics for various diseases or production of agricultural bioproducts. Biological cells may be assayed, including mammalian cells, fungi cells, or bacterial cells..

SUMMARY

[0003] Recognized herein is an industry -wide need for selectively actuating and displacing contents of wells in such arrays to make such contents available for further testing.

[0004] An aspect of the present disclosure provides a device for manipulating a biological sample, comprising: an acoustic transducer configured to provide an acoustic beam; and a lens mount acoustically coupled to said acoustic transducer, wherein said lens mount comprises gap configured to contain a coupling fluid, wherein said lens mount is configured to receive said acoustic beam from said acoustic transducer; and a focusing lens acoustically coupled to said lens mount, wherein said focusing lens is configured to (1) receive said acoustic beam from said lens mount and (2) focus said acoustic beam to said biological sample when said biological sample is in acoustic communication with said focusing lens. In some embodiments, said acoustic transducer is a plane-wave producing transducer. In some embodiments, said planewave producing transducer is an ultrasonic transducer. In some embodiments, said ultrasonic transducer is a non-focusing ultrasonic transducer. In some embodiments, said lens mount comprises one or more openings. In some embodiments, said lens mount comprises a set of switchable focusing lenses. In some embodiments, said switchable focusing lenses differ with respect to at least one property. The device of 7, wherein said at least one property is an F- number. In some embodiments, said F-number is at least about 1 to at least about 4. In some embodiments, said at least one property is a focal spot width. In some embodiments, said focal spot width is at least about 10 micrometer (pm) to at least about 500 pm. In some embodiments, said focal spot width is at least about 60 micrometer (pm) to at least about 240 pm. In some embodiments, said at least one property is depth of focus. In some embodiments, said depth of focus is at least about 100 micrometer (pm) to at least about 10000 pm. In some embodiments, said depth of focus is at least about 426 micrometer (pm) to be at least about 6816 pm. In some embodiments, said lens mount comprises one or more openings. In some embodiments, said lens mount comprises said one or more openings along the circumference of said focusing lens. In some embodiments, said lens mount comprises a lens replacement mechanism arranged to replaceable position a lens in a path of a wave produced by the transducer. In some embodiments, said lens replacement mechanism comprises a lens turret. In some embodiments, the lens turret houses a set of lenses; and each lens of the set of lenses has different focusing properties. In some embodiments, said different focusing properties comprise different F-numbers. In some embodiments, one or more computer processors operatively coupled to said acoustic transducer, wherein said one or more computer processors are individually or collectively programmed to control said acoustic transducer. In some embodiments, said one or more computer processors are individually or collectively programmed to actuate contents of one or more wells in a microfluidic device. In some embodiments, an optical imaging device configured to image said well. In some embodiments, one or more computer processors operatively coupled to said acoustic transducer and/or said optical imaging device, wherein said one or more computer processors are individually or collectively programmed to control said acoustic transducer and/or said optical imaging device. In some embodiments, said one or more computer processors are individually or collectively programmed to implement a method compnsing: receiving optical imaging data from the optical imaging device; interpreting said imaging data to assess a content of one or more wells in a microfluidic device; selecting said well for content actuation based at least in part on (b); causing said acoustic transducer to apply in a contactless maimer said acoustic beam to said well, thereby actuating the contents of said well. In some embodiments, at least one said well comprises a target, and wherein said one or more computer processors are individually or collectively programmed to use said optical imaging device to image said at least one well comprising said target. In some embodiments, said target is selected from the group consisting of particles, cells, and biomolecules. In some embodiments, said well has a volume of less than about 2 nanoliters (nL). In some embodiments, said well has a volume of less than about 1 nanoliter (nL). In some embodiments, said acoustic transducer is configured to operate at a frequency of at least about 1 megahertz (MHz) to at least about 30 MHz. In some embodiments, said acoustic transducer is configured to operate at a frequency of at least about 20 megahertz (MHz) to at least about 30 MHz. In some embodiments, said acoustic transducer is configured to operate at a frequency of at least about 25 megahertz (MHz). In some embodiments, said acoustic transducer is configured to apply an acoustic beam on a spot having a size of at least about 25 micrometers (pm) to at least about 200 pm. In some embodiments, said acoustic transducer is configured to apply an acoustic beam on a spot having a size of at least about 50 micrometers (pm) to at least about 100 pm. [0005] Another aspect of the present disclosure provides a method for manipulating a biological sample, comprising: activating a device comprising: an acoustic transducer that provides an acoustic beam; a lens mount acoustically coupled to said acoustic transducer, wherein said lens mount comprises a gap that contains a coupling fluid, wherein said lens mount receives said acoustic beam from said acoustic transducer; and a focusing lens acoustically coupled to said lens mount, wherein said focusing lens (1) receives said acoustic beam from said lens mount and (2) focuses said acoustic beam to said biological sample in acoustic communication with said focusing lens; and using said acoustic beam focused on said biological sample to manipulate said biological sample. In some embodiments, said acoustic transducer is a plane-wave producing transducer. In some embodiments, said gap further comprises a fluid when said acoustic transducer device is in operation. In some embodiments, said fluid comprises an aqueous liquid. In some embodiments, said fluid is a single-phase aqueous liquid. In some embodiments, said plane-wave producing transducer is an ultrasonic transducer. In some embodiments, said ultrasonic transducer is a non-focusing ultrasonic transducer. In some embodiments, said lens mount comprises one or more openings. In some embodiments, the lens mount comprises a set of switchable focusing lenses. In some embodiments, said switchable focusing lenses differ with respect to at least one property. In some embodiments, said at least one property is an F-number. In some embodiments, said F- number is at least about 1 to at least about 4. In some embodiments, said at least one property is a focal spot width. In some embodiments, said focal spot width is at least at least about 10 micrometers (pm) to at least about 500 pm. In some embodiments, said focal spot width is at least about 60 micrometers (pm) to at least about 240 pm. In some embodiments, said at least one property is depth of focus. In some embodiments, said depth of focus is at least about 100 micrometers (pm) to at least about 10000 pm. In some embodiments, said depth of focus is at least about 426 micrometers (pm) to be at least about 6816 pm. In some embodiments, said focusing lens comprises one or more openings. In some embodiments, said focusing lens comprises said one or more openings along the circumference of said focusing lens. In some embodiments, said lens mount comprises a lens replacement structure arranged to replaceable position a lens in a path of a wave produced by the transducer. In some embodiments, said lens replacement structure comprises a lens turret. In some embodiments, the lens turret houses a set of lenses; and each lens of said set of lenses has different focusing properties. In some embodiments, said different focusing properties comprise different F-numbers. In some embodiments, the method further comprises one or more computer processors operatively coupled to said acoustic transducer, wherein said one or more computer processors are individually or collectively programmed to control said acoustic transducer. In some embodiments, said one or more computer processors are individually or collectively programmed to actuate contents of one or more wells in a microfluidic device. In some embodiments, the method further comprises an optical imaging device configured to image said well. In some embodiments, the method further comprises one or more computer processors operatively coupled to said acoustic transducer device and/or said optical imaging device, wherein said one or more computer processors are individually or collectively programmed to control said acoustic transducer and/or said optical imaging device. In some embodiments, said one or more computer processors are individually or collectively programmed to implement a method comprising: receiving optical imaging data from the optical imaging device; interpreting said imaging data to assess a content of one or more wells in an array of wells; selecting said well for content actuation based at least in part on (b); causing said acoustic transducer to apply in a contactless manner said acoustic beam to said well, thereby actuating the contents of said well. In some embodiments, at least one said well comprises a target, and wherein said one or more computer processors are individually or collectively programmed to use said optical imaging device to image said at least one well comprising said target. In some embodiments, said target is selected from the group consisting of particles, cells, and biomolecules. In some embodiments, said well has a volume of less than about 2 nanoliters (nL). In some embodiments, said well has a volume of less than about 1 nanoliter (nL). In some embodiments, said acoustic transducer is configured to operate at a frequency of at least about 10 megahertz (mHz) to at least about 30 mHz. In some embodiments, said acoustic transducer is configured to operate at a frequency of at least about 20 megahertz (mHz) to at least about 30 mHz. In some embodiments, said acoustic transducer is configured to operate at a frequency of at least about 25 megahertz (mHz). In some embodiments, said acoustic transducer is configured to apply an acoustic beam on a spot having a size of at least about 25 micrometers (pm) to at least about 200 pm. In some embodiments, said acoustic transducer is configured to apply an acoustic beam on a spot having a size of at least about 50 micrometers (pm) to at least about 100 pm. In some embodiments, said acoustic transducer is situated at least about 5 millimeters (mm) from said biological sample. In some embodiments, said biological sample comprises a particle, cell, or a biomolecule. In some embodiments, said biological sample comprises a single cell or single particle. In some embodiments, said single cell or single particle has a desired property. In some embodiments, said single cell or single particle comprises a target of interest.

[0006] Another aspect of the present disclosure provides a device for manipulating a biological sample, comprising: an acoustic transducer configured to provide an acoustic beam; and a focusing lens acoustically coupled to said lens mount wherein said device generates a higher- resolution image than that generated by a focusing transducer. In some embodiments, said ultrasonic transducer is a non-focusing ultrasonic transducer. In some embodiments, said acoustic transducer is a plane-wave producing transducer. In some embodiments, said planewave producing transducer is an ultrasonic transducer. In some embodiments, a lens mount located between said acoustic transducer and said focusing lens. In some embodiments, said lens mount comprises one or more openings. In some embodiments, said lens mount comprises a set of switchable focusing lenses. In some embodiments, said switchable focusing lenses differ with respect to at least one property. In some embodiments, said at least one property is an F-number. In some embodiments, said F-number is at least about 1 to at least about 4. In some embodiments, said at least one property is a focal spot width. In some embodiments, said focal spot width is at least about 10 micrometer (pm) to at least about 500 pm. In some embodiments, said focal spot width is at least about 60 micrometer (pm) to at least about 240 pm. In some embodiments, said at least one property is depth of focus. In some embodiments, said depth of focus is at least about 100 micrometer (pm) to at least about 10000 pm. In some embodiments, said depth of focus is at least about 426 micrometer (pm) to be at least about 6816 pm. In some embodiments, said focusing lens comprises one or more openings. In some embodiments, said focusing lens comprises said one or more openings along the circumference of said focusing lens. In some embodiments, said lens mount comprises a lens replacement mechanism arranged to replaceable position a lens in a path of a wave produced by the transducer. In some embodiments, said lens replacement mechanism comprises a lens turret. In some embodiments, the lens turret houses a set of lenses; and each lens of the set of lenses has different focusing properties. In some embodiments, one or more computer processors operatively coupled to said acoustic transducer, wherein said one or more computer processors are individually or collectively programmed to control said acoustic transducer. In some embodiments, said one or more computer processors are individually or collectively programmed to actuate contents of one or more wells in a microfluidic device. In some embodiments, the device further comprises an optical imaging device configured to image said well. In some embodiments, the device further comprises one or more computer processors operatively coupled to said acoustic transducer and/or said optical imaging device, wherein said one or more computer processors are individually or collectively programmed to control said acoustic transducer and/or said optical imaging device. In some embodiments, said one or more computer processors are individually or collectively programmed to implement a method comprising: receiving optical imaging data from the optical imaging device; interpreting said imaging data to assess a content of one or more wells in a microfluidic device; selecting said well for content actuation based at least in part on (b); causing said acoustic transducer to apply in a contactless manner said acoustic beam to said well, thereby actuating the contents of said well. In some embodiments, at least one said well comprises a target, and wherein said one or more computer processors are individually or collectively programmed to use said optical imaging device to image said at least one well comprising said target. In some embodiments, said target is selected from the group consisting of particles, cells, and biomolecules. In some embodiments, said well has a volume of less than about 2 nanoliters (nL). In some embodiments, said well has a volume of less than about 1 nanoliter (nL). In some embodiments, said acoustic transducer is configured to operate at a frequency of at least about 1 megahertz (MHz) to at least about 30 MHz. In some embodiments, said acoustic transducer is configured to operate at a frequency of at least about 20 megahertz (MHz) to at least about 30 MHz. In some embodiments, said acoustic transducer is configured to operate at a frequency of at least about 25 megahertz (MHz). In some embodiments, said acoustic transducer is configured to apply an acoustic beam on a spot having a size of at least about 25 micrometers (pm) to at least about 200 pm. In some embodiments, said acoustic transducer is configured to apply an acoustic beam on a spot having a size of at least about 50 micrometers (pm) to at least about 100 pm.

[0007] Another aspect of the present disclosure provides a method for imaging a biological sample, comprising: activating a device comprising: an acoustic transducer configured to provide an acoustic beam; and a focusing lens acoustically coupled to said lens mount using said device to generate a higher-resolution image than that generated by a focusing transducer. In some embodiments, said acoustic transducer is anon-focusing transducer. In some embodiments, said acoustic transducer is a plane-wave producing transducer. In some embodiments, the method further comprises a lens mount between said acoustic transducer and said focusing lens. In some embodiments, said lens mount comprises a gap. In some embodiments, said gap further comprises a fluid. In some embodiments, said fluid comprises an aqueous liquid. In some embodiments, said fluid is a single-phase aqueous liquid. In some embodiments, said plane-wave producing transducer is an ultrasonic transducer. In some embodiments, said lens mount comprises one or more openings. In some embodiments, the lens mount comprises a set of switchable focusing lenses. In some embodiments, said switchable focusing lenses differ with respect to at least one property. In some embodiments, said at least one property is an F-number. In some embodiments, said F-number is at least about 1 to at least about 4. In some embodiments, said at least one property is a focal spot width. In some embodiments, said focal spot width is at least at least about 10 micrometers (pm) to at least about 500 pm. In some embodiments, said focal spot width is at least about 60 micrometers (pm) to at least about 240 pm. In some embodiments, said at least one property is depth of focus. In some embodiments, said depth of focus is at least about 100 micrometers (pm) to at least about 10000 pm. In some embodiments, said depth of focus is at least about 426 micrometers (pm) to be at least about 6816 pm. In some embodiments, said focusing lens comprises one or more openings. In some embodiments, said focusing lens comprises said one or more openings along the circumference of said focusing lens. In some embodiments, said lens mount comprises a lens replacement structure arranged to replaceable position a lens in a path of a wave produced by the transducer. In some embodiments, said lens replacement structure comprises a lens turret. In some embodiments,: the lens turret houses a set of lenses; and each lens of said set of lenses has different focusing properties. In some embodiments, the method further comprises one or more computer processors operatively coupled to said acoustic transducer, wherein said one or more computer processors are individually or collectively programmed to control said acoustic transducer. In some embodiments, said one or more computer processors are individually or collectively programmed to actuate contents of a well in a microfluidic device. In some embodiments, the method further comprises an optical imaging device configured to image said well. In some embodiments, the method further comprises one or more computer processors operatively coupled to said acoustic transducer device and/or said optical imaging device, wherein said one or more computer processors are individually or collectively programmed to control said acoustic transducer and/or said optical imaging device. In some embodiments, said one or more computer processors are individually or collectively programmed to implement a method comprising: receiving optical imaging data from the optical imaging device; interpreting said imaging data to assess a content of said well; selecting said well for content actuation based at least in part on (b); causing said acoustic transducer to apply in a contactless manner said acoustic beam to said one or more wells, thereby actuating the contents of said well. In some embodiments, said well comprise a target, and wherein said one or more computer processors are individually or collectively programmed to use said optical imaging device to image said well comprising said target. In some embodiments, said target is selected from the group consisting of particles, cells, and biomolecules. In some embodiments, said well has a volume of less than about 2 nanoliters (nL). In some embodiments, said well has a volume of less than about 1 nanoliter (nL). In some embodiments, said acoustic transducer is configured to operate at a frequency of at least about 10 megahertz (mHz) to at least about 30 mHz. In some embodiments, said acoustic transducer is configured to operate at a frequency of at least about 20 megahertz (mHz) to at least about 30 mHz. In some embodiments, said acoustic transducer is configured to operate at a frequency of at least about 25 megahertz (mHz). In some embodiments, said acoustic transducer is configured to apply an acoustic beam on a spot having a size of at least about 25 micrometers (pm) to at least about 200 pm. In some embodiments, said acoustic transducer is configured to apply an acoustic beam on a spot having a size of at least about 50 micrometers (pm) to at least about 100 pm. In some embodiments, said acoustic transducer is situated at least about 5 millimeters (mm) from said biological sample. In some embodiments, said biological sample comprises a particle, cell, or a biomolecule. In some embodiments, said biological sample comprises a single cell or single particle. In some embodiments, said single cell or single particle has a desired property. In some embodiments, said single cell or single particle comprises a target of interest.

[0008] In some aspects, the present disclosure provides a system comprising one or more computer processors and computer memory coupled thereto. The computer memory comprises machine executable code that, upon execution by the one or more computer processors, implements any of the methods above or elsewhere herein.

[0009] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

INCORPORATION BY REFERENCE

[0010] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0012] FIG. l is a side view and a top view of an example of an acoustic transducer device that includes an ultrasonic transducer and a focusing lens. [0013] FIG. 2 is a schematic diagram of the acoustic transducer device showing an example of an acoustic beam profile as it exits the concave surface of the focusing lens.

[0014] FIG. 3 is a side view and a top view of an acoustic transducer device that includes the ultrasonic transducer and a lens turret for selecting the focusing properties of the acoustic transducer device.

[0015] FIG. 4 is a flow diagram of an example of a method for providing an acoustic transducer that may be optimized for a particular application.

[0016] FIG. 5 is an example of an acoustic retrieval process for actuating the contents of a nanowell in a microfluidic device for downstream processing.

[0017] FIG. 6 is a flow diagram illustrating an example of a method for selectively repositioning a target of interest in a well of a microfluidic device using an externally applied acoustic field or wave.

[0018] FIG. 7 shows a computer system 701 that is programmed or otherwise configured to control acoustic retrieval processes as disclosed herein.

[0019] FIG. 8 depicts an exemplary microfluidic device comprising an array of nanowells and an alignment marker.

[0020] FIG. 9 depicts an image of an alignment marker generated with an F2 transducer (A) versus that generated with an Fl transducer produced through methods as claimed herein (B).

[0021] FIG. 10 depicts an image of a nanowell array generated with an F2 transducer (A) versus that generated with an Fl transducer produced through methods as claimed herein (B).

DETAILED DESCRIPTION

[0022] While various embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed.

[0023] Whenever the term “at least,” “greater than,” or “greater than or equal to” precedes the first numencal value in a series of two or more numencal values, the term “at least,” “greater than” or “greater than or equal to” applies to each of the numerical values in that series of numerical values. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0024] Whenever the term “no more than,” “less than,” or “less than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “no more than,” “less than,” or “less than or equal to” applies to each of the numerical values in that series of numerical values. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

System and Device

[0025] The present disclosure provides an acoustic retrieval system. The system may comprise an acoustic transducer device. The acoustic transducer device may comprise a plane-wave generating transducer. The plane-wave generating transducer may be an ultrasonic transducer. The acoustic transducer device may comprise a focusing lens. The focusing lens and ultrasonic transducer may be lateral to each other. A lens mount may be placed between the focusing lens and the ultrasonic transducer. The lens mount may create a gap between the focusing lens and the ultrasonic transducer. The gap may comprise a fluid when the acoustic transducer device is in operation. The fluid may be an aqueous liquid. The fluid may be a single-phase aqueous liquid. The fluid may be water. The acoustic transducer device may generate and use an externally applied acoustic field. The field may be applied to one or more samples for selective retrieval of contents. The one or more samples may be in one or more wells. The one or more wells may be nanowells.

Acoustic transducer device

[0026] The present disclosure provides an acoustic transducer device for generating and using an externally applied acoustic field to one or more samples. The acoustic device may apply one or more acoustic beams to the one or more samples. The acoustic transducer may be applied to manipulate one or more samples. The acoustic field may be applied to the one or more samples for selective retrieval of contents from the one or more samples. The one or more samples may be in one or more wells. The one or more wells may be nanowells. The acoustic transducer may be mounted externally to a microfluidic device on a motorized stage, moveable in x, y, z directions to allow real time adjustment of the position of the acoustic transducer laterally and/or vertically relative to the microfluidic device. The acoustic transducer device may comprise a focusing transducer. The acoustic transducer device may comprise a non-focusing transducer.

[0027] The acoustic transducer may be used to apply a focused acoustic beam to an array of wells. The beam may be applied to one or more wells. The acoustic transducer may be mounted externally to the microfluidic device, such that no integration of the acoustic transducer within the microfluidic device is required. This setup may simplify and reduce the cost of fabrication of the microfluidic device. Further, external application of the acoustic beam the microfluidic device may be contactless and thereby limit the introduction of contaminants into the microfluidic device. [0028] The acoustic transducer may be configured such that the propagation direction of the acoustic beam is perpendicular to the plane of microfluidic device. In some cases, a focused acoustic beam may be delivered from the acoustic transducer at a distance from the microfluidic device. For example, the focused acoustic beam may be delivered from the acoustic transducer at a distance of about 0.25 centimeters (cm) to about 2 cm from the microfluidic device. The focused acoustic beam may be delivered from the acoustic transducer at a distance of at least about 0. 1 cm, at least about 0.2 cm, at least about 0.3 cm, at least about 0.4 cm, at least about 0.5 cm, at least about 0.6 cm, at least about 0.7 cm, at least about 0.8 cm, at least about 0.9 cm, at least about 1 cm, at least about 1.1 cm, at least about 1.2 cm, at least about 1.3 cm, at least about 1.4 cm, at least about 1.5 cm, at least about 1.6 cm, at least about 1.7 cm, at least about 1.8 cm, at least about 1.9 cm, at least about 2 cm, or more.

[0029] The acoustic transducer may be configured to operate at a frequency of about 1 to about 150 megahertz (MHz). The acoustic transducer may be configured to operate at a frequency of about 15 to about 25 MHz. The acoustic transducer may be configured to operate at a frequency of at least about 1 MHz, at least about 2 MHz, at least about 3 MHz, at least about 4 MHz, at least about 5 MHz, at least about 6 MHz, at least about 7 MHz, at least about 8 MHz, at least about 9 MHz, at least about 10 MHz, or more. The acoustic transducer may be configured to operate at a frequency of at least about 10 MHz, at least about 20 MHz, at least about 30 MHz, at least about 40 MHz, at least about 50 MHz, at least about 60 MHz, at least about 70 MHz, at least about 80 MHz, at least about 90 MHz, at least about 100 MHz, at least about 110 MHz, at least about 120 MHz, at least about 130 MHz, at least about 140 MHz, at least about 150 MHz, or more.

[0030] The acoustic transducer may be configured to apply the focused acoustic beam having a spot size in the range of about 50 micrometers (pm) to about 200 pm. The acoustic transducer may be configured to apply a focused acoustic beam having a spot size of at least about 50 pm, at least about 60 pm, at least about 70 pm, at least about 80 pm, at least about 90 pm, at least about 100 pm, at least about 110 pm, at least about 120 pm, at least about 130 pm, at least about 140 pm, at least about 150 pm, at least about 160 pm, at least about 170 pm, at least about 180 pm, at least about 190 pm, at least about 200 pm, or more. The spot size of a focused acoustic beam may be selected based on the density of nanowell features in a nanowell array of a microfluidic device.

[0031] The acoustic transducer may be coupled to a microfluidic device by immersion in a coupling medium. The coupling medium may be acoustically low-absorbing. The coupling medium may be water. [0032] The acoustic transducer device may include an ultrasonic transducer. The acoustic transducer device may operate at different ultrasonic frequencies. The ultrasonic transducer may be a non-focusing ultrasonic transducer.

[0033] The focusing properties (e.g., focal spot width and depth of focus) of the acoustic transducer device may be optimized for a particular application by selecting the ultrasonic frequency of the transducer and the appropriate F-number of the focusing lens. The disclosure provides a method for creating an acoustic transducer device that includes a non-focusing ultrasonic transducer coupled to an interchangeable focusing lens, wherein a plane-wave acoustic beam emitted by the transducer is focused to a spot defined by the curvature of the focusing lens. The focusing lens may, for example, be a plano-concave lens.

[0034] FIG. 1 a side view and a top view of an example of an acoustic transducer device 100 that includes an ultrasonic transducer and a focusing lens. Acoustic transducer device 100 may include a non-focusing ultrasonic transducer 110. Ultrasonic transducer 110 may be acoustically coupled to a focusing lens 115. Ultrasonic transducer 110 may be acoustically and physically coupled to focusing lens 115. Ultrasonic transducer 110 may be acoustically and physically coupled to focusing lens 115 by a lens mount 120. Ultrasonic transducer 110 may be configured to emit a plane wave acoustic beam. The plane wave acoustic beam may be focused to a spot defined by the curvature of focusing lens 115.

[0035] Ultrasonic transducer 110 may include a planar surface 125 for emitting a plane wave. Acoustic transducer device 100 may be configured to transmit an acoustic beam from ultrasonic transducer 110 to focusing lens 115. Focusing lens 115 may be a plano-concave lens.

Focusing lens 115 may include a lens planar surface 130 (parallel to ultrasonic transducer 110) and a lens concave surface 132.

[0036] In one embodiment, the acoustic transducer device may include an ultrasonic transducer and a single focusing lens having a selected F-number.

[0037] In one embodiment, the acoustic transducer device may include an ultrasonic transducer and a lens turret that includes multiple lenses with different F-numbers for selecting the focusing properties of the acoustic transducer.

[0038] In one embodiment, the acoustic transducer device may be coupled to a microfluidic device that is configured to support automated high-throughput processes to isolate, screen, and/or retrieve single cells or biomolecules in a biological sample.

Focusing Lens

[0039] The present disclosure provides systems and methods for acoustic retrieval comprising one or more focusing lens. A focusing lens 115 may be mounted via lens mount 120 aligned with planar front surface 125 of ultrasonic transducer 110 to provide a liquid flow gap 140. The size of liquid flow gap 140 may. for example, be from at least about 1 mm to at least about 5 mm. The size of liquid flow gap 140 may, for example, be at least about 1, 2, 3, 4, 5 mm or more. In one example, the size of liquid flow gap 140 is about 3 mm.

[0040] The present disclosure provides focusing lens with varying F-numbers. The F-number of focusing lens 115 is defined as the ratio of the diameter of the lens to the focal length. The F- number may be at least about 1, at least about 1.1, at least about 1.2, at least about 1.3, at least about 1.4, at least about 1.5, at least about 1.6, at least about 1.7, at least about 1.8, at least about 1.9, at least about 2, at least about 2. 1, at least about 2.2, at least about 2.3, at least about 2.4, at least about 2.5, at least about 2.6, at least about 2.7, at least about 2.8, at least about 2.9, at least about 3.0, at least about 3.1, at least bout 3.2, at least about 3.3, at least about 3.4, at least about 3.5, at least about 3.6, at least about 3.7, at least about 3.8, at least about 3.9, at least about 4.0, at least about 4.1, at least about 4.2, at least about 4.3, at least about 4.4, at least about 4.5, at least about 4.6, at least about 4.7, at least about 4.8, at least about 4.9, at least about 5.0, or more. The F-number may be about 1. The F-number may be about 2. The F- number may be about 3. The F-number may be about 4.

[0041] focal spot width to be adjusted from about 60 pm to about 240 pm, and the corresponding depth of focus to vary from about 426 pm to about 6816 pm.

[0042] The present disclosure provides focusing lens with varying focal spot widths. The focal spot width W is given by Fc/f where c is the speed of sound (1500 m/s in water), and f is the ultrasound frequency. The focal spot width may be at least about 10 pm to at least about 500 pm. The focal spot width may be at least about 10 pm, at least about 20 pm, at least about 30 pm, at least about 40 pm, at least about 50 pm, at least about 60 pm, at least about 70 pm, at least about 80 pm, at least about 90 pm, at least about 100 pm, at least about 110 pm, at least about 120 pm, at least about 130 pm, at least about 140 pm, at least about 150 pm, at least about 160 pm, at least about 170 pm, at least about 180 pm, at least about 190 pm, at least about 200 pm, at least about 210 pm, at least about 220 pm, at least about 230 pm, at least about 240 pm, at least about 250 pm, at least about 260 pm, at least about 270 pm, at least about 280 pm, at least about 290 pm, at least about 300 pm, at least about 310 pm, at least about 320 pm, at least about 330 pm, at least about 340 pm, at least about 350 pm, at least about 360 pm, at least about 370 pm, at least about 380 pm, at least about 390 pm, at least about 400 pm, at least about 410 pm, at least about 420 pm, at least about 430 pm, at least about 440 pm, at least about 450 pm, at least about 460 pm, at least about 470 pm, at least about 480 pm, at least about 490 pm, at least about 500 pm, or more. The focal spot width may be at least about 60 pm to at least about 240 pm.

[0043] The present disclosure provides focusing lens with varying depth of focus. The depth of focus is given by 7. 1WF, where width W is defined as above and where F-number F is defined as above. The depth of focus may be at least about 100 pm to at least about 10000 pm. The depth of focus may be at least about 100 pm, at least about 110 pm, at least about 120 pm, at least about 130 pm, at least about 140 pm, at least about 150 pm, at least about 160 pm, at least about 170 pm, at least about 180 pm, at least about 190 pm, at least about 200 pm, at least about 210 pm, at least about 220 pm, at least about 230 pm, at least about 240 pm, at least about 250 pm, at least about 260 pm, at least about 270 pm, at least about 280 pm, at least about 290 pm, at least about 300 pm, at least about 310 pm, at least about 320 pm, at least about 330 pm, at least about 340 pm, at least about 350 pm, at least about 360 pm, at least about 370 pm, at least about 380 pm, at least about 390 pm, at least about 400 pm, at least about 410 pm, at least about 420 pm, at least about 430 pm, at least about 440 pm, at least about 450 pm, at least about 460 pm, at least about 470 pm, at least about 480 pm, at least about 490 pm, at least about 500 pm, at least about 600 pm, at least about 700 pm, at least about 800 pm, at least about 900 pm, at least about 1000 pm, at least about 1500 pm, at least about 2000 pm, at least about 2500 pm, at least about 3000 pm, at least about 3500 pm, at least about 4000 pm, at least about 4500 pm, at least about 5000 pm, at least about 5500 pm, at least about 6000 pm, at least about 6500 pm, at least about 7000 pm, at least about 7500 pm, at least about 8000 pm, at least about 8500 pm, at least about 9000 pm, at least about 9500 pm, at least about 10000 pm, or more. The depth of focus may be at least about 426 pm to be at least about 6816 pm.

[0044] The focusing properties of the focusing lens may be optimized for a particular application by selecting the appropriate F-number or ultrasonic frequency.

[0045] The focusing lens may comprise various structural features for improved acoustic retrieval. Focusing lens 115 may, for example, be composed of a material that has low absorption at the operating ultrasonic frequency. For example, the focusing lens may be composed of a material that has low absorption at a frequency of at least about 2 MHz to at least about 30 MHz. The focusing lens may be composed of a material that has low absorption at a frequency of at least about 2 MHz, at least about 3 MHz, at least about 4 MHz, at least about 5 MHz, at least about 6 MHz, at least about 7 MHz, at least about 8 MHZ, at least about 9 MHz, at least about 10 MHz, at least about 11 MHz, at least about 12 MHz, at least about 13 MHz, at least about 14 MHz, at least about 15 MHz, at least about 16 MH, at least about 17 Hz, at least about 18 MHz, at least about 19 MHz, at least about 20 MHz, at least about 21 MHz, at least about 22 MHz, at least about 23 MHz, at least about 24 MHz, at least about 25 MHz, at least about 26 MHz, at least about 27 MHz, at least about 28 MHz, at least about 29 MHz, at least about 30 MHz, or more. The focusing lens may be composed of a material that has low absorption at a frequency of at least about 15 MHz to at least about 25 MHz. In an example, focusing lens 115 may be composed of a glass material. A focusing lens 115 may be coated with an anti -refl ection coating on lens planar front surface 130 and lens concave surface 132, thereby maximizing the acoustic power transmitted to a focal spot. The thickness of the antireflection coating may be at least about 1% to at least about 50% of the ultrasonic wavelength in the coating material. The thickness of the anti-reflection coating may be at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, or more of the ultrasonic wavelength in the coating material. The thickness of the anti-reflection coating may be at least about 25% of the ultrasonic wavelength in the coating material. The composition of the anti-reflection coating may be selected to be compatible with immersion in coupling medium. In an example, the coupling medium can be water. In an example, the anti-reflection coating is parylene.

[0046] Systems as described herein may comprise an acoustic transducer and a lens mount. Systems as described herein may comprise a non-focusing transducer and a lens mount. The lens mount may be acoustically coupled to the non-focusing transducer. The lens mount may comprise a gap configured to hold a liquid. The liquid may be a medium, such as water. A system comprising a non-focusing transducer acoustically coupled to a lens mount configured to hold a liquid medium, such as water, may have superior focusing abilities as compared to, for example, a focusing transducer. The system may have an F-number of at least about 0.5 to at least about 2. The F-number may be at least about 0.5, at least about 0.6, at least about 0.7, at least about 0.8, at least about 0.9, at least about 1, at least about 1.1, at least about 1.2, at least about 1.3, at least about 1.4, at least about 1.5, or more. The F-number may be at least about 1. The system may be used to actuate an acoustic beam to a well in an array of wells. The system may yield a narrower acoustic beam than that yielded by a focusing transducer. The system may provide more accuracy in actuating an acoustic beam to a well in an array of wells as compared to the accuracy provided by a focusing transducer. The system may provide a higher-resolution image of a nano well array than that generated by a focusing-transducer. The system may provide a higher-resolution image of a sample in an array of wells than that generated by a focusing-transducer. The system may provide a higher-resolution image of an alignment marker than that generated by a focusing-transducer.

Lens turret

[0047] The present disclosure provides systems and methods for acoustic retrieval comprising a lens turret. A lens turret may comprise two or more lenses. The two or more lenses may have the same F-number. The two or more lenses may have different F-numbers. Each lens in the lens turret may have an f-number in the range of about 1 to about 4. The F-number may be at least about 1, at least about 1.1, at least about 1.2, at least about 1.3, at least about 1.4, at least about 1.5, at least about 1.6, at least about 1.7, at least about 1.8, at least about 1.9, at least about 2, at least about 2.1, at least about 2.2, at least about 2.3, at least about 2.4, at least about 2.5, at least about 2.6, at least about 2.7, at least about 2.8, at least about 2.9, at least about 3.0, at least about 3. 1, at least bout 3.2, at least about 3.3, at least about 3.4, at least about 3.5, at least about 3.6, at least about 3.7, at least about 3.8, at least about 3.9, at least about 4.0, at least about 4. 1, at least about 4.2, at least about 4.3, at least about 4.4, at least about 4.5, at least about 4.6, at least about 4.7, at least about 4.8, at least about 4.9, at least about 5.0, or more. The F-number may be about 1. The F-number may be about 2. The F-number may be about 3. The F-number may be about 4.

[0048] As depicted in FIG. 3, lens turret 310 may be used for selecting the focusing properties of the acoustic transducer device in an acoustic retrieval system. Lens turret 310 may comprise a lens wheel 315 that houses multiple focusing lenses 320 (e.g., lenses 320a, 320b, 320c, and 320d) having different F-numbers. Lens turret 310 may be mounted next to ultrasonic transducer 110 by a turret mount 330. Turret mount 330 may be positioned to provide a liquid flow gap 335 between ultrasonic transducer 110 and a focusing lens 320. Liquid flow gap 335 may be configured to permit filling of the space between ultrasonic transducer 110 and the focusing lens 320 with a coupling medium. In an example, the coupling medium may be water. [0049] The lens turret may be manipulated to set different focus conditions. In an example, a focus condition may be focal spot width. In another example, a focus condition may be depth of focus. As depicted in FIG. 3, various focus conditions may be obtained by clocking lens turret 310 between the different lenses 320. The focal spot width may be set to at least about 10 pm to at least about 500 pm. The focal spot width may be at least about 10 pm, at least about 20 pm, at least about 30 pm, at least about 40 pm, at least about 50 pm, at least about 60 pm, at least about 70 pm, at least about 80 pm, at least about 90 pm, at least about 100 pm, at least about 110 pm, at least about 120 pm, at least about 130 pm, at least about 140 pm, at least about 150 pm, at least about 160 pm, at least about 170 pm, at least about 180 pm, at least about 190 pm, at least about 200 pm, at least about 210 pm, at least about 220 pm, at least about 230 pm, at least about 240 pm, at least about 250 pm, at least about 260 pm, at least about 270 pm, at least about 280 pm, at least about 290 pm, at least about 300 pm, at least about 310 pm, at least about 320 pm, at least about 330 pm, at least about 340 pm, at least about 350 pm, at least about 360 pm, at least about 370 pm, at least about 380 pm, at least about 390 pm, at least about 400 pm, at least about 410 pm, at least about 420 pm, at least about 430 pm, at least about 440 pm, at least about 450 pm, at least about 460 un, at least about 470 un, at least about 480 pun, at least about 490 pun, at least about 500 pun, or more. The focal spot width may be at least about 60 pm to at least about 240 pm. The depth of focus may be set to at least about 100 pm to at least about 10000 pm. The depth of focus may be at least about 100 pm, at least about 110 pm, at least about 120 pm, at least about 130 pm, at least about 140 pm, at least about 150 pm, at least about 160 pm, at least about 170 pm, at least about 180 pm, at least about 190 pm, at least about 200 pm, at least about 210 pm, at least about 220 pm, at least about 230 pm, at least about 240 pm, at least about 250 pm, at least about 260 pm, at least about 270 pm, at least about 280 pm, at least about 290 pm, at least about 300 pm, at least about 310 pm, at least about 320 pm, at least about 330 pm, at least about 340 pm, at least about 350 pm, at least about 360 pm, at least about 370 pm, at least about 380 pm, at least about 390 pm, at least about 400 pm, at least about 410 pm, at least about 420 pm, at least about 430 pm, at least about 440 pm, at least about 450 pm, at least about 460 pm, at least about 470 pm, at least about 480 pm, at least about 490 pm, at least about 500 pm, at least about 600 pm, at least about 700 pm, at least about 800 pm, at least about 900 pm, at least about 1000 pm, at least about 1500 pm, at least about 2000 pm, at least about 2500 pm, at least about 3000 pm, at least about 3500 pm, at least about 4000 pm, at least about 4500 pm, at least about 5000 pm, at least about 5500 pm, at least about 6000 pm, at least about 6500 pm, at least about 7000 pm, at least about 7500 pm, at least about 8000 pm, at least about 8500 pm, at least about 9000 pm, at least about 9500 pm, at least about 10000 pm, or more. The depth of focus may be at least about 426 pm to be at least about 6816 pm.

[0050] In one embodiment, the ultrasonic transducer 110 may operate at about 25 MHz and the F-numbers of the different lenses may be 1, 2, 3 and 4. The focal spot width in this embodiment may be adjusted from about 60 pm to about 240 pm, and the corresponding depth of focus may be adjusted from about 426 pm to about 6816 pm.

Lens mount

[0051] The present disclosure provides systems and methods for acoustic retrieval comprising a lens mount. A lens mount 120 may be configured to permit liquid to flow between focusing lens 115 and transducer device 110. The lens mount may have one or more openings to permit the liquid to flow between the focusing lens and the transducer device. For example, lens mount 120 may have a spider geometry that includes openings 122 arranged to permit liquid to flow between focusing lens 115 and ultrasonic transducer 110. In one embodiment, openings 122 may be arranged circumferentially in lens mount 120 to permit filling of the space between ultrasonic transducer 110 and focusing lens 115 with a coupling medium. Openings 122 may be any kind of opening that permits liquid to flow through gap 140. [0052] Focusing lens 115 may, for example, be coupled to lens mount 120 by a friction fits so that the lens can be easily interchanged, thereby permitting the selection of different focusing properties.

Wells

[0053] The present disclosure provides a microfluidic device comprising an acoustic transducer device. The microfluidic device may include an array of wells. The array of wells may compartmentalize a biological sample into one or more subsamples for isolating, screening, and/or retrieving single cells or biomolecules in the sample. The well may be a nanowell. The microfluidic device may include at least about 100 nanowells, at least about 1,000 nanowells, at least about 10,000 nanowells, at least about 100,000 nanowells, or at least about 1,000,000 nanowells. The microfluidic device may include at least about 100 nanowells, at least about 200 nanowells, at least about 300 nanowells, at least about 400 nanowells, at least about 500 nanowells, at least about 600 nanowells, at least about 700 nanowells, at least about 800 nanowells, or at least about 900 nanowells. The microfluidic device may include at least about 1,000 nanowells, at least about 2,000 nanowells, at least about 3,000 nanowells, at least about 4,000 nanowells, at least about 5,000 nanowells, at least about 6,000 nanowells, at least about 7,000 nanowells, at least about 8,000 nanowells, or at least about 9,000 nanowells. The microfluidic device may include at least about 10,000 nanowells, at least about 20,000 nanowells, at least about 30,000 nanowells, at least about 40,000 nanowells, at least about 50,000 nanowells, at least about 60,000 nanowells, at least about 70,000 nanowells, at least about 80,000 nanowells, or at least about 90,000 nanowells. The microfluidic device may include at least about 100,000 nano wells, at least about 200,000 nano wells, at least about 300,000 nanowells, at least about 400,000 nanowells, at least about 500,000 nanowells, at least about 600,000 nanowells, at least about 700,000 nanowells, at least about 800,000 nanowells, at least about 900,000 nanowells, or at least about 1,000,000 nanowells.

[0054] Each array of wells may contain identical volumes or non-identical volumes. The well may be a nanowell. The nanowell may have a volume of at most about 2 nanoliters (nL). The nanowell may have a volume of at most about 1 nL. The nanowell may have a volume of at most about 0. 1 nL, at most about 0.2 nL, at most about 0.3 nL, at most about 0.4 nL, at most about 0.5 nL, at most about 0.6 nL, at most about 0.7 nL, at most about 0.8 nL, at most about 0.9 nL, at most about 1 nL, at most about 1.1 nL, at most about 1.2 nL, at most about 1.3 nL, at most about 1.4 nL, at most about 1.5 nL, at most about 1.5 nL, at most about 1.6 nL, at most about 1.7 nL, at most about 1.8 nL, at most about 1.9 nL, or at most about 2 nL. A nanowell may have a volume of about 1 nL.

Optical Imaging Device [0055] Systems as disclosed herein may comprise an optical imaging device. An optical imaging device may incorporate bright field and fluorescence microscopy capabilities. The optical imaging device may be used to analyze a compartmentalized sample in one or more nanowells of the nanowell array. The optical imaging device may be configured with high magnification capabilities for high resolution imaging of single cells.

[0056] For example, the optical imaging system may be a fluorescence microscope with the capability to image a well array over a range of fluorescent wavelengths and in brightfield. For example, the microscope may include multiple illumination wavelengths and filter cubes to work at different fluorescent conditions. The microscope may include multiple objectives to provide magnifications in the range of at least about 2 to at least about 20X. The microscope may provide a magnification of at least about 2X, at least about 3X, at least about 4X, at least about 5X, at least about 6X, at least about 7X, at least about 8X, at least about 9X, at least about 10X, at least about 11X, at least about 12 X, at least about 13X, at least about 14X, at least about 15X, at least about 16X, at least about 17X, at least about 18X, at least about 19X, at least about 20X, or more.

[0057] The microscope may include a scientific CMOS camera to collect high resolution images over a large field of view at fast frame rates. The microscope may include fast and precise stages to move the microfluidic device and hence produce high resolution stitched images of the entire well array.

[0058] Methods as disclosed herein may further comprise providing one or more computer processors individually or collectively programmed to implement a method for acoustic retrieval. The method may comprise receiving optical imaging data from an optical imaging device. The method may further comprise interpreting said imaging data to assess a content of one or more wells in an array of wells. The method may further comprise selecting a well for content actuation based at least part on the interpretation of the data. The method may further comprise causing the acoustic transducer to apply a acoustic beam to said microfluidic device, thereby actuating the contents of said well.

[0059] Methods as disclosed herein may comprise selective acoustic retrieval of one or more samples in one or more wells. The one or more samples may be selected on the basis of one or more properties. The sample may selected from the group consisting of particles, cells, and biomolecules. The sample may be a single cell or a single particle. The sample may comprise a target of interest.

Computer

[0060] FIG. 7 shows a computer system 701 that is programmed or otherwise configured to control various systems and methods as disclosed herein. The computer system 701 can regulate various aspects of acoustic retrieval processes of the present disclosure. For example, the computer may be electronically coupled to various components of the present disclosure, such as the imaging device and acoustic transducer. The computer may be programmed to control vanous aspects of the acoustic retrieval processes as disclosed herein, such as imaging, focusing, processing image data, interpreting image data, generation of acoustic beams and pulses, properties of beams and pulses, flow of in the microfluidic device.

[0061] The computer system 701 can be an electronic device of a user or a computer system that is remotely located with respect to the electronic device. The electronic device can be a mobile electronic device.

[0062] The computer system 701 includes a central processing unit (CPU, also “processor” and “computer processor” herein) 705, which can be a single core or multi core processor, or a plurality of processors for parallel processing. The computer system 701 may also include a memory or memory location 710 (e.g., random-access memory, read-only memory', flash memory), electronic storage unit 715 (e.g., hard disk), communication interface 720 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 725, such as cache, other memory, data storage and/or electronic display adapters. The memory 710, storage unit 715, interface 720 and peripheral devices 725 are in communication with the CPU 705 through a communication bus (solid lines), such as a motherboard. The storage unit 715 can be a data storage unit (or data repository) for storing data. The computer system 701 can be operatively coupled to a computer network (“network”) 730 with the aid of the communication interface 720. The network 730 can be the Internet, an internet and/or extranet, or an intranet and/or extranet that is in communication with the Internet. The network 730 in some cases is a telecommunication and/or data network. The network 730 can include one or more computer servers, which can enable distributed computing, such as cloud computing. The network 730, in some cases with the aid of the computer system 701, can implement a peer-to- peer network, which may enable devices coupled to the computer system 701 to behave as a client or a server.

[0063] The CPU 705 can execute a sequence of machine-readable instructions, which can be embodied in a program or software. The instructions may be stored in a memory' location, such as the memory 710. The instructions can be directed to the CPU 705, which can subsequently program or otherwise configure the CPU 705 to implement methods of the present disclosure. Examples of operations performed by the CPU 705 can include fetch, decode, execute, and writeback. [0064] The CPU 705 can be part of a circuit, such as an integrated circuit. One or more other components of the system 701 can be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).

[0065] The storage unit 715 can store files, such as dnvers, libranes and saved programs. The storage unit 715 can store user data, e.g., user preferences and user programs. The computer system 701 in some cases can include one or more additional data storage units that are external to the computer system 701, such as located on a remote server that is in communication with the computer system 701 through an intranet or the Internet.

[0066] The computer system 701 can communicate with one or more remote computer systems through the network 730. For instance, the computer system 701 can communicate with a remote computer system of a user. Examples of remote computer systems include personal computers (e.g., portable PC), slate or tablet PC’s (e.g., Apple® iPad, Samsung® Galaxy Tab), telephones, Smart phones (e.g., Apple® iPhone, Android-enabled device, Blackberry®), or personal digital assistants. The user can access the computer system 701 via the network 730. [0067] Methods as described herein can be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system 701, such as, for example, on the memory 710 or electronic storage unit 715. The machine executable or machine readable code can be provided in the form of software. During use, the code can be executed by the processor 705. In some cases, the code can be retrieved from the storage unit 715 and stored on the memory 710 for ready access by the processor 705. In some situations, the electronic storage unit 715 can be precluded, and machine-executable instructions are stored on memory 710.

[0068] The code can be pre-compiled and configured for use with a machine having a processer adapted to execute the code, or can be compiled during runtime. The code can be supplied in a programming language that can be selected to enable the code to execute in a pre-compiled or as-compiled fashion.

[0069] Aspects of the systems and methods provided herein, such as the computer system 701, can be embodied in programming. Various aspects of the technology⁷ may be thought of as “products” or “articles of manufacture” typically in the form of machine (or processor) executable code and/or associated data that is earned on or embodied in a type of machine readable medium. Machine-executable code can be stored on an electronic storage unit, such as memory (e.g., read-only memory, random-access memory, flash memory ) or a hard disk. “Storage” type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server. Thus, another type of media that may bear the software elements includes optical, electrical and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links. The physical elements that carry such waves, such as wired or wireless links, optical links or the like, also may be considered as media bearing the software. As used herein, unless restricted to non-transitory, tangible “storage” media, terms such as computer or machine “readable medium” refer to any medium that participates in providing instructions to a processor for execution.

[0070] Hence, a machine readable medium, such as computer-executable code, may take many forms, including but not limited to, a tangible storage medium, a carrier wave medium or physical transmission medium. Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such as may be used to implement the databases, etc. shown in the drawings. Volatile storage media include dynamic memory, such as main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications. Common forms of computer-readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may read programming code and/or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0071] The computer system 701 can include or be in communication with an electronic display 735 that comprises a user interface (UI) 740 for providing, for example, information on the position or actuation parameters of an acoustic beam. The UI may provide information on the frequency of an acoustic beam. The UI may provide information on the sequence of pulses. The UI may provide information on the pulse period. The UI may provide information on the duration of pulses. The UI may provide information on the position of an acoustic beam. Examples of UI’s include, without limitation, a graphical user interface (GUI) and web-based user interface.

[0072] Methods and systems of the present disclosure can be implemented by way of one or more algorithms An algorithm can be implemented by way of software upon execution by the central processing unit 705.

[0073] Systems and methods as disclosed herein may further comprise one or more computer processors individually or collectively programmed to implement a method for acoustic retrieval. The one or more computer processors may be connected to an acoustic transducer. The one or more processors may be operatively coupled to the acoustic transducer, and may affect the acoustic transducer to supply one or more pulses to a sample, resulting in actuation of the sample. The one or more computer processors may receive optical imaging data from an optical imaging device. The imaging data may be interpreted to assess a content of one or more wells in an array of wells. A well may be selected for content actuation based at least part on the interpretation of the data. An acoustic transducer may then apply an acoustic beam to the well, thereby actuating the contents of the well.

Operation of the ultrasonic transducer

[0074] The present disclosure provides systems and methods for acoustic retrieval. The system may comprise an acoustic transducer device. The acoustic transducer device may comprise a plane-wave generating transducer. The plane-wave generating transducer may be an ultrasonic transducer. The acoustic transducer device may comprise a focusing lens. The focusing lens and ultrasonic transducer may be lateral to each other. A lens mount may be placed between the focusing lens and the ultrasonic transducer. The lens mount may create a gap between the focusing lens and the ultrasonic transducer. The gap may comprise a fluid when the acoustic transducer device is in operation. The fluid may be a liquid. The fluid may be water. The acoustic transducer device may generate and use an externally applied acoustic field. The field may be applied to one or more samples for selective retrieval of contents. The one or more samples may be in one or more wells. The one or more wells may be nanowells. The acoustic transducer may be mounted externally to a microfluidic device on a motorized stage, moveable in x, y, z directions to allow real time adjustment of the position of the acoustic transducer laterally and/or vertically relative to the microfluidic device.

[0075] Systems and methods as described herein may comprise an acoustic transducer device. The acoustic transducer device may comprise an ultrasonic transducer. As depicted in FIG. 1, an ultrasonic transducer 110 may be configured to operate at a radio frequency (RF). An electrical pulse may be transmitted to ultrasonic transducer 110 via an RF input 145. Acoustic transducer device 100 may be used for generating an acoustic field or wave. The acoustic field or wave may be directed to produce a localized liquid flow in a device comprising a liquid. In operation, the plane wave acoustic beam emitted by ultrasonic transducer 110 may be focused to a spot defined by the curvature of focusing lens 115. FIG. 2 is a schematic diagram of the acoustic transducer device 100 showing an example of an acoustic beam profile 200 as it exits the concave surface of focusing lens 115. In operation, a plane-wave acoustic beam emitted from ultrasonic transducer 110 may travel through a coupling medium in liquid flow gap 140 and through focusing lens 115 and may be focused to a point as it exits the concave surface of focusing lens 115.

[0076] Systems and methods as described herein may comprise a focusing lens or a lens turret. As depicted in FIG. 3, lens turret 310 may be used for selecting the focusing properties of the acoustic transducer device in an acoustic retrieval system. Lens turret 310 may comprise a lens wheel 315 that houses multiple focusing lenses 320 (e.g., lenses 320a, 320b, 320c, and 320d) having different F-numbers. Lens turret 310 may be mounted next to ultrasonic transducer 110 by a turret mount 330. Turret mount 330 may be positioned to provide a liquid flow gap 335 between ultrasonic transducer 110 and a focusing lens 320. Liquid flow gap 335 may be configured to permit filling of the space between ultrasonic transducer 110 and the focusing lens 320 with a coupling medium.

[0077] Various properties of a focusing lens or a lens turret may be modulated. The property may be an F-number, a focal spot width, or a depth of focus.

[0078] The F-number may be in the range of about 1 to about 4. The F-number may be at least about 1, at least about 1.1, at least about 1.2, at least about 1.3, at least about 1.4, at least about

1.5, at least about 1.6, at least about 1.7, at least about 1.8, at least about 1.9, at least about 2, at least about 2. 1, at least about 2.2, at least about 2.3, at least about 2.4, at least about 2.5, at least about 2.6, at least about 2.7, at least about 2.8, at least about 2.9, at least about 3.0, at least about 3. 1, at least bout 3.2, at least about 3.3, at least about 3.4, at least about 3.5, at least about

3.6, at least about 3.7, at least about 3.8, at least about 3.9, at least about 4.0, at least about 4.1, at least about 4.2, at least about 4.3, at least about 4.4, at least about 4.5, at least about 4.6, at least about 4.7, at least about 4.8, at least about 4.9, at least about 5.0, or more. The F-number may be about 1. The F-number may be about 2. The F-number may be about 3. The F-number may be about 4. A lens turret may comprise two or more lenses. The two or more lenses may have the same F-number. The two or more lenses of the lens turret may have different F- numbers.

[0079] The focusing lens or lens turret may have various different focal spot widths. The focal spot width may be set to at least about 10 pm to at least about 500 pm. The focal spot width may be at least about 10 un, at least about 20 un, at least about 30 pun, at least about 40 pun, at least about 50 pun, at least about 60 pun, at least about 70 pun, at least about 80 pun, at least about 90 pun, at least about 100 pun, at least about 110 pun, at least about 120 pun, at least about 130 pim, at least about 140 pun, at least about 150 pun, at least about 160 pirn, at least about 170 pun, at least about 180 pun, at least about 190 pun, at least about 200 pun, at least about 210 pun, at least about 220 pun, at least about 230 pun, at least about 240 pun, at least about 250 pun, at least about 260 pun, at least about 270 pun, at least about 280 pun, at least about 290 pun, at least about 300 pun, at least about 310 pun, at least about 320 pun, at least about 330 pun, at least about 340 pun, at least about 350 pun, at least about 360 pun, at least about 370 pun, at least about 380 pun, at least about 390 pun, at least about 400 pun, at least about 410 pun, at least about 420 pun, at least about 430 pun, at least about 440 pun, at least about 450 pun, at least about 460 pun, at least about 470 pun, at least about 480 pun, at least about 490 pun, at least about 500 pun, or more. The focal spot width may be at least about 60 pm to at least about 240 pm.

[0080] The focusing lens or lens turret may have carious different depths of focus. The depth of focus may be set to at least about 100 pm to at least about 10000 pm. The depth of focus may be at least about 100 pm, at least about 110 pm, at least about 120 pm, at least about 130 pm, at least about 140 pm, at least about 150 pm, at least about 160 pm, at least about 170 pm, at least about 180 pm, at least about 190 pm, at least about 200 pm, at least about 210 pm, at least about 220 pm, at least about 230 pm, at least about 240 pm, at least about 250 pm, at least about 260 pm, at least about 270 pm, at least about 280 pm, at least about 290 pm, at least about 300 pm, at least about 310 pm, at least about 320 pm, at least about 330 pm, at least about 340 pm, at least about 350 pm, at least about 360 pm, at least about 370 pm, at least about 380 pm, at least about 390 pm, at least about 400 pm, at least about 410 pm, at least about 420 pm, at least about 430 pm, at least about 440 pm, at least about 450 pm, at least about 460 pm, at least about 470 pm, at least about 480 pm, at least about 490 pm, at least about 500 pm, at least about 600 pm, at least about 700 pm, at least about 800 pm, at least about 900 pm, at least about 1000 pm, at least about 1500 pm, at least about 2000 pm, at least about 2500 pm, at least about 3000 pm, at least about 3500 pm, at least about 4000 pm, at least about 4500 pm, at least about 5000 pm, at least about 5500 pm, at least about 6000 pm, at least about 6500 pm, at least about 7000 pm, at least about 7500 pm, at least about 8000 pm, at least about 8500 pm, at least about 9000 pm, at least about 9500 pm, at least about 10000 pm, or more. The depth of focus may be at least about 426 pm to be at least about 6816 pm. [0081] The focusing properties may be optimized for a particular application by selecting the appropriate F-number and ultrasonic frequency. Examples of the dependence of the focus properties on the ultrasonic frequency and lens F-number are shown in Table 1.

Table 1. Dependence of the focus properties on ultrasonic frequency and lens F-number

[0082] In one embodiment of the invention, the ultrasonic transducer 110 may be an Olympus part number V324-SU operating at 25 MHz and the plano-concave focusing lens 115 may be an Edmund Optics part number 45-380. This lens may have an F-number of 1 and may be composed of Coming N-SF11 glass. The lens may be coated on both sides with a parylene film of thickness 24 pm. Referring to Table 1, this configuration may yield a focal spot width of 60 pm and a depth of focus of 426 pm.

[0083] The present disclosure provides methods of acoustic retrieval. The method may comprise providing an acoustic transducer device. The device may comprise a plane-wave producing transducer. The plane-wave producing transducer may apply an acoustic beam to one or more samples. The plane-wave producing transducer may be acoustically coupled to a focusing lens. The focusing lens may manipulate the acoustic beam. The acoustic transducer device may further comprise a lens mount between the plane-wave producing transducer and the focusing lens. The lens mount may establish a gap between the plane-wave producing transducer and the focusing lens. The method may further comprise using the acoustic transducer device to apply an acoustic beam to the one or more samples. The gap may comprise a fluid when the acoustic transducer device is in operation. The fluid may comprise an aqueous liquid. The fluid may comprise a single-phase aqueous liquid. The plane-wave producing transducer may be an ultrasonic transducer. The ultrasonic transducer maybe a non-focusing ultrasonic transducer.

[0084] FIG. 4 provides a flow diagram of an example of a method 400 for providing an acoustic transducer that may be optimized for a particular application. Method 400 includes, but is not limited to, the following steps.

[0085] In a step 410, the focus conditions may be determined for a particular application. For example, the focus condition may be F-number, focal spot width, or depth of focus. The focusing conditions may be determined for a particular application by defining the focal spot width and depth of focus required for the application. In one embodiment, an acoustic transducer may be configured for acoustic actuation for sample retrieval from a device comprising a liquid. In one example, the acoustic transducer may be configured to support sample retrieval from a microfluidic device that includes an array of wells (e.g., a nanowell array).

[0086] In a step 415, a non-focusing ultrasonic transducer configured to operate over a range of ultrasonic frequencies and emit a plane wave acoustic beam may be provided. For example, ultrasonic transducer 110 that includes a planar surface 125 for emitting a plane wave acoustic beam may be provided.

[0087] In a step 420, a plano-concave focusing lens having an appropriate F-number for use at a certain ultrasonic frequency to focus the plane wave acoustic beam emitted by the ultrasonic transducer is provided. In one embodiment, a single focusing lens 115 having the appropriate F-number may be provided. The plane wave acoustic beam emitted by ultrasonic transducer 110 is focused to a spot defined by the curvature of the focusing lens 115.

[0088] In another embodiment, a lens turret 310 that includes a lens wheel 315 that houses multiple focusing lenses 320 having different F-numbers may be provided. Different focus conditions (i.e., focal spot width and depth of focus) may be obtained by simply clocking lens turret 310 between the different lenses 320.

[0089] At a step 425, the ultrasonic transducer and the focusing lens are coupled using an interchangeable lens mount to provide an acoustic transducer device that includes a liquid flow gap. For example, focusing lens 115 may be mounted via lens mount 120 aligned with planar front surface 125 of ultrasonic transducer 110 to provide a liquid flow gap 140. In another example, lens turret 310 may be mounted next to ultrasonic transducer 110 by a turret mount 330.

Robotic Device

[0090] Systems as disclosed herein may include a robotic device. The acoustic transducer device may be coupled to the robotic device. The robotic device may be controllable to adjust a position of the acoustic transducer device in one, two or three dimensions. A computer, electronically coupled to and controlling the robotic device, may be programmed to control the robotic device and thereby control the position of the transducer device and thereby focus an acoustic beam from the transducer device to a position on the microfluidic device. Definitions

[0091] “Target” means any particulate or molecular material of interest that can be manipulated by a system of the invention, e.g., targets may be one or more, cells, cell components (e.g., organelles), biomolecules (e.g., nucleic acids or proteins), particles (e.g., beads).

[0092] “Sample” means any liquid that includes target material. A sample may also include other components, such as reagents, such as processing reagents and/or assay reagents. In some cases, the sample is an aqueous solution. In some cases, the sample is a biological liquid, such as blood, plasma, serum, urine, or cerebrospinal liquid, or a solution including a biological liquid.

[0093] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

EXAMPLES

[0094] Example 1: Acoustic Actuation for Sample Retrieval

[0095] The focusing ultrasonic transducer may be used to produce a localized liquid flow in a microfluidic device that can be used to actuate, on demand, the contents of a single selected nanowell within a large, high-density array of nanowells in the device. In this case, the liquid in the microfluidic device is a single aqueous phase, and streaming induced by the acoustic field in the aqueous phase is used to actuate the contents of the nanowell. An example of using acoustic streaming of an aqueous carrier liquid to actuate the contents of an aqueous sample compartmentalized in a nanowell is described with reference to FIG. 5.

[0096] FIG. 5 is an example of an acoustic retrieval process 500 for actuating the contents of a nanowell in a microfluidic device for downstream processing.

[0097] Acoustic retrieval process 500 may be controlled by a computer (not shown). The computer may be electronically coupled to various components of the invention, such as the ultrasonic transducer 110 and an imaging device (not shown). The computer may be programmed to control various aspects of the process, such as imaging, focusing, processing image data, interpreting image data, generation of acoustic beams and pulses, properties of acoustic beams and pulses, and/or flow of liquid in the microfluidic device.

[0098] Acoustic transducer device 100 may be coupled to a microfluidic device 505 by immersion in a coupling medium 510 that is acoustically low-absorbing (e.g., an aqueous medium, e.g., water). The ultrasonic transducer 110 may be controlled by the computer, e.g., to control the frequency, power, shape, and timing of an acoustic pulse generated using ultrasonic transducer 110.

[0099] Microfluidic device 505 may include an array of wells, such as nanowells 515. In some embodiments, nanowell 515 may have a volume of about 1 nanoliter or less.

[0100] The array of nanowells 515 may be adjacent to an aqueous-filled channel 520, wherein channel 520 provides a flow path along the aqueous-filled nanowells 515 and is enclosed by a top cover 525. Top cover 525 may, for example, be formed of glass or a thin film material.

[0101] Nanowells 515 may be loaded with a volume of sample aqueous liquid For example, a fluidic pump (not shown) may be used to flow a sample aqueous liquid into channel 520 to load a volume of sample aqueous liquid into each nanowell 515. The computer (not shown) may be programmed to control the operation of the fluidic pump, as well as any valves (not shown) that may be required for flowing liquid into, through and out of channel 520.

[0102] The sample liquid in a nanowell 515 may contain a target 530 of interest, such as a cell and/or its contents, an organelle, a bead (e.g., a bead functionalized to capture a target of interest), a biomolecule (such as a protein or nucleic acid), or a combination thereof suspended in an aqueous medium. In some embodiments, target 530 may be an animal cell, such as a mammalian cell (e.g., a human cell). In some embodiments, target 530 may be a non-animal cell, such as a yeast cell or a bacterial cell.

[0103] Acoustic transducer device 100 may be used to apply a focused acoustic beam 535 to an individual nano well 515 in microfluidic device 505 for actuating the contents of the nano well. In one example, acoustic transducer 100 may be operated at a frequency in the range of from about 15 MHz to about 25 MHz to produce a focused spot size of from about 60 pm to about 200 pm. The acoustic field generates a localized liquid flow in the individual nanowell 515 that sweeps the contents out into channel 520. The actuated material may be retrieved by flow to a designated collection point. [0104] The efficient actuation of the contents of an individual nanowell 515 benefits from precise matching of the acoustic focus to the well dimensions. For example, the nano wells 515 may have a cross-section ranging from about 25 pm to about 200 pm. The nanowells 515 may, for example, have a depth-to-width ratio in the range of from about 1 to about 4. In some embodiments, acoustic transducer device 300 that includes ultrasonic transducer 110 and a lens turret 310 for selecting the focusing properties of the acoustic transducer device may be used. [0105] The invention provides a method for using an externally applied acoustic field or wave for selectively repositioning a target of interest in a well of a microfluidic device for retrieval and downstream processing.

[0106] FIG. 6 is a flow diagram illustrating an example of a method 600 for selectively actuating a target of interest in a well of a microfluidic device using an externally applied acoustic field or wave. Method 600 may include, but is not limited to, the following steps. [0107] In a step 610, an acoustic transducer device and a microfluidic device that includes a well array are provided. For example, acoustic transducer device 100 and microfluidic device 505 are provided. Microfluidic device 505 may include an array of wells (e.g., a nanowells 515) adjacent to a channel (e.g., channel 520). Acoustic transducer device 100 may be coupled to microfluidic device 505 by immersion in a coupling medium (e.g., water).

[0108] In a step 615, a sample is partitioned into the wells of the microfluidic device. For example, a fluidic pump may be used to flow a sample aqueous liquid into channel 520 to load a volume of sample into each nanowell 515. The sample liquid in a nanowell 515 may contain a target of interest (e.g., target 530).

[0109] In a step 620, a well comprising a target of interest is identified. For example, an optical imaging device may be used to image one or more nanowells 515 in the nano well array in microfluidic device 505 to identify wells that have a target 530 of interest.

[0110] In a step 625, the acoustic transducer device is positioned to focus an acoustic beam on the identified well. For example, acoustic transducer device 100 may be positioned to focus the acoustic beam on a certain nanowell 515 in microfluidic device 505.

[OHl] In a step 630, the acoustic transducer device is activated to apply an acoustic beam to eject the target from the identified well. For example, ultrasonic transducer 110 is operated at a frequency in the range of from about 15 MHz to about 25 MHz to produce a focused spot size of from about 60 pm to about 200 pm. The acoustic field generates a localized liquid flow in the individual nanowell 515 that recovers the contents (e.g., target 530) out into channel 520. [0112] In a step 635, the ejected target is retrieved by flowing to a designated collection point. For example, a liquid flowed into and through channel 520 may be used to flow the actuated target 530 to the designated collection point for retrieval. [0113] Example 2: Comparison of Fl focusing immersion transducer versus F2 focusing immersion transducer

[0114] The acoustic beam-focusing capabilities of an Fl focusing transducer produced through methods as claimed herein were compared to those of an F2 focusing immersion transducer purchased through a supplier.

Methods

[0115] An Fl focusing immersion transducer was created from an F2 non-focusing immersion transducer (Olympus P/N V324-SU) combined with perylene coated lens (Edmund Optics PN 45-006). This Fl focusing immersion transducer was operated at 25 megahertz (mHz) to actuate a sample in a nanowell in an array in a microfluidic device. The nanowell array and an alignment marker in the array are shown in FIG. 8.

[0116] An F2 focusing transducer purchased from a supplier (Olympus P/N V324-SU-F0.50IN- PTF) was also operated at 25 mHz to actuate a sample in a nanowell in an array in a microfluidic device. The nanowell array and an alignment marker in the array are shown in FIG. 8

[0117] Results

[0118] Images of the alignment marker generated using the F2 and F l transducer (FIGS. 9A and 9B, respectively) show that the image generated with the Fl transducer appears sharper and in better focus. This indicates a narrower, more focused acoustic beam was generated using the Fl transducer than that generated using the F2 transducer.

[0119] Furthermore, images of the nanowell array taken by the F2 and Fl transducer (FIGS.

10A and 10B, respectively) show that the image generated by the Fl transducer is sharper than that generated by the F2 transducer.

[0120] Thus, transducers and methods as claimed herein generated narrower, more focusing acoustic beams and more focused images of nanowell arrays and samples in microfluidic devices.

[0121] While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the disclosure be limited by the specific examples provided within the specification. While the disclosure has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. Furthermore, it shall be understood that all aspects of the disclosure are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of disclosure and variables. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the disclosure. It is therefore contemplated that the disclosure shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A device for manipulating a biological sample, comprising:

(a) an acoustic transducer configured to provide an acoustic beam; and

(b) a lens mount acoustically coupled to said acoustic transducer, wherein said lens mount comprises gap configured to contain a coupling fluid, wherein said lens mount is configured to receive said acoustic beam from said acoustic transducer; and

(c) a focusing lens acoustically coupled to said lens mount, wherein said focusing lens is configured to (1) receive said acoustic beam from said lens mount and (2) focus said acoustic beam to said biological sample when said biological sample is in acoustic communication with said focusing lens.

2. The device of claim 1, wherein said acoustic transducer is a plane-wave producing transducer.

3. The device of claim 2, wherein said plane-wave producing transducer is an ultrasonic transducer.

4. The device of claim 3, wherein said ultrasonic transducer is a non-focusing ultrasonic transducer.

5. The device of claim 1, wherein said lens mount comprises one or more openings.

6. The device of claim 1 , wherein said lens mount comprises a set of switchable focusing lenses.

7. The device of claim 6, wherein said switchable focusing lenses differ with respect to at least one property.

8. The device of 7, wherein said at least one property is an F-number.

9. The device of claim 8, wherein said F-number is at least about 1 to at least about 4.

10. The device of claim 7, wherein said at least one property is a focal spot width.

11. The device of claim 10, wherein said focal spot width is at least about 10 micrometer (pm) to at least about 500 pm.

12. The device of claim 10, wherein said focal spot width is at least about 60 micrometer (pm) to at least about 240 pm.

13. The device of claim 7, wherein said at least one property is depth of focus.

14. The device of claim 13, wherein said depth of focus is at least about 100 micrometer (pm) to at least about 10000 pm.

15. The device of claim 13, wherein said depth of focus is at least about 426 micrometer (pm) to be at least about 6816 pm. The device of claim 1, wherein said lens mount comprises two or more openings. The device of claim 16, wherein said lens mount comprises said two or more openings along the circumference of said focusing lens. The device of claim 1, wherein said lens mount comprises a lens replacement mechanism arranged to replaceable position a lens in a path of a wave produced by the transducer. The device of claim 18, wherein said lens replacement mechanism comprises a lens turret. The device of claim 19, wherein:

(a) the lens turret houses a set of lenses; and

(b) each lens of the set of lenses has different focusing properties. The device of claim 20, wherein said different focusing properties comprise different F- numbers. The device of claim 1, wherein said acoustic transducer is configured to operate at a frequency of at least about 1 megahertz (MHz) to at least about 30 MHz. The device of claim 1, wherein said acoustic transducer is configured to operate at a frequency of at least about 20 megahertz (MHz) to at least about 30 MHz. The device of claim 23, wherein said acoustic transducer is configured to operate at a frequency of at least about 25 megahertz (MHz). The device of claim 1, wherein said acoustic transducer is configured to apply an acoustic beam on a spot having a size of at least about 25 micrometers (pm) to at least about 200 pm. The device of claim 1, wherein said acoustic transducer is configured to apply an acoustic beam on a spot having a size of at least about 50 micrometers (pm) to at least about 100 pm. A method for manipulating a biological sample, comprising:

(a) activating a device comprising: i. an acoustic transducer that provides an acoustic beam; ii. a lens mount acoustically coupled to said acoustic transducer, wherein said lens mount comprises a gap that contains a coupling fluid, wherein said lens mount receives said acoustic beam from said acoustic transducer; and iii. a focusing lens acoustically coupled to said lens mount, wherein said focusing lens (1) receives said acoustic beam from said lens mount and (2) focuses said acoustic beam to said biological sample in acoustic communication with said focusing lens; and

(b) using said acoustic beam focused on said biological sample to manipulate said biological sample. The method of claim 27, wherein said acoustic transducer is a plane-wave producing transducer. The method of claim 27, wherein said gap further comprises a fluid when said acoustic transducer device is in operation. The method of claim 29, wherein said fluid comprises an aqueous liquid. The method of claim 30, wherein said fluid is a single-phase aqueous liquid. The method of claim 28, wherein said plane-wave producing transducer is an ultrasonic transducer. The method of claim 32, wherein said ultrasonic transducer is a non-focusing ultrasonic transducer. The method of claim 27, wherein said lens mount comprises one or more openings. The method of claim 27, wherein the lens mount comprises a set of switchable focusing lenses. The method of claim 35, wherein said switchable focusing lenses differ with respect to at least one property. The method of claim 36, wherein said at least one property is an F-number. The method of claim 37, wherein said F-number is at least about 1 to at least about 4. The method of claim 36, wherein said at least one property is a focal spot width. The method of claim 39, wherein said focal spot width is at least at least about 10 micrometers (pm) to at least about 500 pm. The method of claim 39, wherein said focal spot width is at least about 60 micrometers (pm) to at least about 240 pm. The method of claim 36, wherein said at least one property is depth of focus. The method of claim 42, wherein said depth of focus is at least about 100 micrometers (pm) to at least about 10000 pm. The method of claim 42, wherein said depth of focus is at least about 426 micrometers (pm) to be at least about 6816 pm. The method of claim 27, wherein said lens mount comprises one or more openings. The method of claim 27, wherein said lens mount comprises said one or more openings along the circumference of said focusing lens. The method of claim 27, wherein said lens mount comprises a lens replacement structure arranged to replaceable position a lens in a path of a wave produced by the transducer. The method of claim 47, wherein said lens replacement structure comprises a lens turret. The method of claim 48, wherein:

(a) the lens turret houses a set of lenses; and

(b) each lens of said set of lenses has different focusing properties. The method of claim 49, wherein said different focusing properties comprise different F-numbers. The method of claim 27, wherein said acoustic transducer is configured to operate at a frequency of at least about 10 megahertz (mHz) to at least about 30 mHz. The method of claim 27, wherein said acoustic transducer is configured to operate at a frequency of at least about 20 megahertz (mHz) to at least about 30 mHz. The method of claim 27, wherein said acoustic transducer is configured to operate at a frequency of at least about 25 megahertz (mHz). The method of claim 27, wherein said acoustic transducer is configured to apply an acoustic beam on a spot having a size of at least about 25 micrometers (pm) to at least about 200 pm. The method of claim 27, wherein said acoustic transducer is configured to apply an acoustic beam on a spot having a size of at least about 50 micrometers (pm) to at least about 100 pm. The method of claim 27, wherein said acoustic transducer is situated at least about 5 millimeters (mm) from said biological sample. The method of claim 27, wherein said biological sample comprises a particle, cell, or a biomolecule. The method of claim 57, wherein said biological sample comprises a single cell or single particle. The method of claim 58, wherein said single cell or single particle has a desired property. The method of claim 58, wherein said single cell or single particle comprises a target of interest. A device for manipulating a biological sample, comprising:

(a) an acoustic transducer configured to provide an acoustic beam; and

(b) a focusing lens acoustically coupled to said lens mount wherein said device generates a higher-resolution image than that generated by a focusing transducer. The device of claim 61, wherein said acoustic transducer is a plane-wave producing transducer. The device of claim 62, wherein said plane-wave producing transducer is an ultrasonic transducer. The device of claim 63, wherein said ultrasonic transducer is a non-focusing ultrasonic transducer. The device of claim 61, further comprising a lens mount located between said acoustic transducer and said focusing lens. The device of claim 65, wherein said lens mount comprises one or more openings. The device of claim 65, wherein said lens mount comprises a set of switchable focusing lenses. The device of claim 67, wherein said switchable focusing lenses differ with respect to at least one property. The device of claim 68, wherein said at least one property is an F-number. The device of claim 69, wherein said F-number is at least about 1 to at least about 4. The device of claim 68, wherein said at least one property is a focal spot width. The device of claim 71, wherein said focal spot width is at least about 10 micrometer (pm) to at least about 500 pm. The device of claim 71, wherein said focal spot width is at least about 60 micrometer (pm) to at least about 240 pm. The device of claim 68, wherein said at least one property is depth of focus. The device of claim 74, wherein said depth of focus is at least about 100 micrometer (pm) to at least about 10000 pm. The device of claim 74, wherein said depth of focus is at least about 426 micrometer (pm) to be at least about 6816 pm. The device of claim 65, wherein said lens mount comprises a lens replacement mechanism arranged to replaceable position a lens in a path of a wave produced by the transducer. The device of claim 77, wherein said lens replacement mechanism comprises a lens turret. The device of claim 78, wherein:

(a) the lens turret houses a set of lenses; and

(b) each lens of the set of lenses has different focusing properties. The device of claim 61, wherein said acoustic transducer is configured to operate at a frequency of at least about 1 megahertz (MHz) to at least about 30 MHz. The device of claim 61, wherein said acoustic transducer is configured to operate at a frequency of at least about 20 megahertz (MHz) to at least about 30 MHz. The device of claim 61, wherein said acoustic transducer is configured to operate at a frequency of at least about 25 megahertz (MHz). The device of claim 61, wherein said acoustic transducer is configured to apply an acoustic beam on a spot having a size of at least about 25 micrometers (pm) to at least about 200 pm. The device of claim 61, wherein said acoustic transducer is configured to apply an acoustic beam on a spot having a size of at least about 50 micrometers (pm) to at least about 100 pm. A method for imaging a biological sample, comprising:

(a) activating a device comprising: i. an acoustic transducer configured to provide an acoustic beam; and ii. a focusing lens acoustically coupled to said lens mount

(b) using said device to generate a higher-resolution image than that generated by a focusing transducer. The method of claim 85, wherein said acoustic transducer is a non-focusing transducer. The method of claim 85, wherein said acoustic transducer is a plane-wave producing transducer. The method of claim 85, further comprising a lens mount between said acoustic transducer and said focusing lens. The method of claim 88, wherein said lens mount comprises a gap. The method of claim 89, wherein said gap further comprises a fluid. The method of claim 90, wherein said fluid comprises an aqueous liquid. The method of claim 91, wherein said fluid is a single-phase aqueous liquid. The method of claim 87, wherein said plane-wave producing transducer is an ultrasonic transducer. The method of claim 88, wherein said lens mount comprises one or more openings. The method of claim 94, wherein said lens mount comprises said one or more openings along the circumference of said focusing lens. The method of claim 88, wherein the lens mount comprises a set of switchable focusing lenses. The method of claim 96, wherein said switchable focusing lenses differ with respect to at least one property. The method of claim 97, wherein said at least one property is an F-number. The method of claim 98, wherein said F-number is at least about 1 to at least about 4.. The method of claim 97, wherein said at least one property is a focal spot width.. The method of claim 100, wherein said focal spot width is at least at least about 10 micrometers (pm) to at least about 500 pm. . The method of claim 100, wherein said focal spot width is at least about 60 micrometers (pm) to at least about 240 pm. . The method of claim 97, wherein said at least one property is depth of focus.. The method of claim 103, wherein said depth of focus is at least about 100 micrometers (pm) to at least about 10000 pm. . The method of claim 103, wherein said depth of focus is at least about 426 micrometers (pm) to be at least about 6816 pm. . The method of claim 88, wherein said lens mount comprises a lens replacement structure arranged to replaceable position a lens in a path of a wave produced by the transducer. . The method of claim 106, wherein said lens replacement structure comprises a lens turret. . The method of claim 107, wherein:

(a) the lens turret houses a set of lenses; and

(b) each lens of said set of lenses has different focusing properties. . The method of claim 85, wherein said acoustic transducer is configured to operate at a frequency of at least about 10 megahertz (mHz) to at least about 30 mHz.. The method of claim 85, wherein said acoustic transducer is configured to operate at a frequency of at least about 20 megahertz (mHz) to at least about 30 mHz.. The method of claim 85, wherein said acoustic transducer is configured to operate at a frequency of at least about 25 megahertz (mHz). . The method of claim 85, wherein said acoustic transducer is configured to apply an acoustic beam on a spot having a size of at least about 25 micrometers (pm) to at least about 200 pm. . The method of claim 85, wherein said acoustic transducer is configured to apply an acoustic beam on a spot having a size of at least about 50 micrometers (pm) to at least about 100 pm. . The method of claim 85, wherein said acoustic transducer is situated at least about 5 millimeters (mm) from said biological sample.

. The method of claim 114, wherein said biological sample comprises a particle, cell, or a biomolecule. . The method of claim 114, wherein said biological sample comprises a single cell or single particle. . The method of claim 116, wherein said single cell or single particle has a desired property. . The method of claim 116, wherein said single cell or single particle comprises a target of interest.