CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63/327,287, filed April 04, 2022, which is incorporated herein by reference in its entirety.

BACKGROUND

[0002] Microfluidic platforms are being developed to screen and analyze small (e.g., picoliter - nanoliter) biological samples in large-scale nanowell arrays. Retrieving on demand the content (e.g., a single cell) from an individual nanowell in a large-scale nanowell array remains challenging.

SUMMARY

[0003] In some aspects, the present disclosure provides a system comprising: (a) a microfluidic device comprising an array of wells, wherein a well of said array of wells has a volume of less than about one microliter, and wherein said array of wells is in fluidic communication with a channel, wherein said microfluidic device further comprises a cover, wherein said cover encloses said channel and said array of wells; and (b) an acoustic transducer configured to apply an acoustic beam to said well of said microfluidic device, wherein said acoustic transducer is not integrated with said microfluidic device, wherein when said acoustic transducer applies said acoustic beam to said well, said well and said channel contain a liquid phase. In some embodiments, the system 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 control said acoustic transducer in pulsed mode. In some embodiments, the system further comprises an optical imaging device configured to image said well. In some embodiments, the system 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 operate said acoustic transducer in pulsed mode using pulses having a pulse width ranging from about 1 millisecond (ms) to about 100 ms. In some embodiments, said one or more computer processors are individually or collectively programmed to operate said acoustic transducer in pulsed mode using pulses having a pulse period ranging from about 10 to about 200 ms. In some embodiments, said one or more computer processors are individually or collectively programmed to actuate contents of said well using a sequence of pulses. In some embodiments, said sequence of pulses comprises a sequence of from 1 to 100 pulses. In some embodiments, said sequence of pulses are high-intensity pulses directed to said well. In some embodiments, said one or more computer processors are individually or collectively programmed to implement a method comprising: (a) receiving optical imaging data from the optical imaging device; (b) interpreting said imaging data to assess a content of said well; (c) selecting said well for content actuation based at least in part on (b);(d) causing said acoustic transducer to apply in a contactless manner said acoustic beam to said microfluidic device, 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 less than about two nanoliters. In some embodiments, said well has a volume less than about one nanoliter. In some embodiments, said microfluidic device comprises at least 5,000 of said wells. In some embodiments, said microfluidic device comprises at least 50,000 of said wells. In some embodiments, said microfluidic device comprises at least 500,000 of said wells. In some embodiments, said cover is acoustically low-absorbing. In some embodiments, at least one well of said array of wells comprises one or more cells. In some embodiments, at least one said well comprises one or more particles. In some embodiments, at least one said well comprises a single cell. In some embodiments, at least one said well comprises a single particle. In some embodiments, said acoustic transducer is configured to operate at a frequency of about 1 to about 30 mHz. In some embodiments, said acoustic transducer is configured to operate at a frequency of about 20 to about 30 mHz. In some embodiments, said acoustic transducer is configured to operate at a frequency of about 25 mHz. In some embodiments, said acoustic transducer is mounted on a moveable stage configured for lateral and/or vertical translation relative to said microfluidic device. In some embodiments, said acoustic transducer is configured to apply a focused acoustic beam on a spot having a size of about 25 micrometers (pm) to about 200 pm. In some embodiments, said acoustic transducer is configured to apply a focused acoustic beam on a spot having a size of about 50 pm to about 100 pm. In some embodiments, said acoustic transducer and said imaging device are arranged on opposite sides of the microfluidic device. In some embodiments, said acoustic transducer is situated at least 5 millimeters from said microfluidic device. In some embodiments, said acoustic transducer is configured to apply an acoustic beam to the microfluidic device via a coupling medium. In some embodiments, said coupling medium comprises water.

[0004] In some aspects, the present disclosure provides a method comprising: (a) providing a system comprising: (i) a microfluidic device comprising an array of wells, wherein a well of said array of wells has a volume of less than about one microliter, and wherein said array of wells is in fluidic communication with a channel, wherein said microfluidic device further comprises a cover, wherein said cover encloses said channel and said array of wells; and (ii) an acoustic transducer configured to apply an acoustic beam to said well of said microfluidic device, wherein said acoustic transducer is not integrated with said microfluidic device, wherein when said acoustic transducer applies said acoustic beam to said well, said well and said channel contain a liquid phase; and (b) using said acoustic transducer to apply an acoustic beam to said well of said microfluidic device.

[0005] In some aspects, the present disclosure provides a method of actuating contents of a microfluidic device comprising an array of wells, comprising: (a) using an optical imaging device to identify a well of said array of wells, wherein said well comprises at least one content of interest; and (b) using an acoustic transducer to apply one or more contactless acoustic pulses to said well comprising at least one content of interest, thereby actuating the contents of said well comprising at least one content of interest into an aqueous fluid-filled channel in fluid communication with said well, wherein said acoustic transducer is not integrated with said microfluidic device. In some embodiments, said one or more contactless acoustic pulses have a pulse width ranging from about 1 millisecond (ms) to about 100 ms. In some embodiments, said one or more contactless acoustic pulses have a pulse period ranging from about 10 ms to about 200 ms. In some embodiments, the method comprises applying from about 1 acoustic pulse to about 20 acoustic pulses. In some embodiments, the method further comprises providing one or more computer processors in communication with said imaging device. In some embodiments, the method further comprises: (a) using said one or more computer processors to cause said imaging device to image said array of wells, thereby receiving optical imaging data and interpreting said optical imaging data to assess the contents of said array of wells; and (b) selecting a well for content actuation and causing said acoustic transducer to apply in a contactless manner an acoustic beam to said microfluidic device, thereby actuating the contents of said well selected for content actuation. In some embodiments, said one or more wells has a volume of less than about two nanoliters (nL). In some embodiments, said one or more wells has a volume less than about one nL. In some embodiments, said array of wells comprises at least 5,000 said wells. In some embodiments, said array of wells comprises at least 50,000 said wells. In some embodiments, said array of wells comprises at least 500,000 said wells. In some embodiments, said acoustic pulses are applied at a frequency of about 1 to about 30 mHz. In some embodiments, said acoustic pulses are applied at a frequency of about 20 mHz to about 30 mHz. In some embodiments, said acoustic pulses are applied at a frequency of about 25 mHz. In some embodiments, said acoustic transducer is mounted on a moveable stage capable of lateral and/or vertical translation relative to the microfluidic device. In some embodiments, said acoustic transducer is configured to apply a focused acoustic beam on a spot having a size of about 25 pm to about 200 pm. In some embodiments, said acoustic transducer is configured to apply a focused acoustic beam on a spot having a size of about 25 pm to about 100 pm. In some embodiments, said acoustic transducer and said optical imaging device are arranged on opposite sides of said microfluidic device. In some embodiments, said acoustic transducer is situated at least 5 millimeters from said microfluidic device. In some embodiments, said acoustic transducer is configured to apply an acoustic beam to said microfluidic device via a coupling medium. In some embodiments, said coupling medium comprises water. In some embodiments, said content of interest comprises a particle, cell, biomolecule, color, or fluorescence. In some embodiments, said content of interest comprises a single cell or single particle. In some embodiments, said content of interest comprises a single cell or single particle having a desired property. In some embodiments, said content of interest comprises a single cell or single particle that includes a target of interest. 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.

[0006] 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

[0007] 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

[0008] 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:

[0009] FIG. 1 is a block diagram of an example of the presently disclosed acoustic retrieval system for generating an acoustic field that can be used to produce a localized fluid flow in the microfluidic device.

[0010] FIG. 2 is a schematic diagram illustrating an example of an acoustic retrieval process for actuating the contents of a nanowell in a single-phase microfluidic device for downstream processing.

[0011] FIG. 3 shows a computer system that is programmed or otherwise configured to implement methods provided herein.

DETAILED DESCRIPTION

[0012] 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.

[0013] Whenever the term “at least,” “greater than,” or “greater than or equal to” precedes the first numerical value in a series of two or more numerical 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.

[0014] 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. [0015] The present disclosure provides acoustic retrieval systems, microfluidic devices, and methods for using an externally applied acoustic field for selective retrieval of contents from the microfluidic device in the case where the fluid is a single phase (e.g., aqueous).

[0016] The present disclosure may provide an acoustic retrieval system. The present disclosure provides a microfluidic device. The present disclosure may provide a method for generating an acoustic field that can be used to produce a localized fluid flow in the microfluidic device containing a single fluid phase. The acoustic field may be generated by an external acoustic transducer that is coupled to the microfluidic device using a coupling medium.

[0017] The present disclosure provides an acoustic retrieval system, microfluidic device, and method for using an acoustic field to produce a localized fluid flow in the microfluidic device that can be used to actuate on demand the contents of a single selected nanowell within a high- density array of nanowells in the device for downstream processing.

[0018] The acoustic retrieval system, device, and methods of the present disclosure may be used for analysis of a single cell (i.e., single cell analysis (SCA)), its contents, and/or selected biomolecules (e.g., secreted proteins or nucleic acids).

[0019] The acoustic retrieval system, device, and methods of the present disclosure may be used for analysis of a single cell in clinical applications such as screening for rare immune cells, for example B-cells and T-cells, in a sample.

[0020] The acoustic retrieval system, device, and methods of the present disclosure may be used for analysis of primary plasma cells from an immunized animal or human, hybridoma cells, genetically-modified Chinese Hamster Ovary (CHO) cells, and/or other cells that secrete antibodies.

[0021] Cells analyzed by the acoustic retrieval system, device, and methods of the present disclosure may incubated before, during, or after analysis. Cells analyzed by the acoustic retrieval system, device, and methods of the present disclosure may incubated before, during, or after analysis.

[0022] Additional reagents that specifically stain an antibody or other secreted molecule of cells analyzed by the acoustic retrieval system, device, and methods of the present disclosure can be pumped through the channel of the system or device. In such embodiments, staining reagents can then optionally be washed out by flowing a buffer solution or other wash solution through the channel.

[0023] The acoustic retrieval system, device, and methods of the present disclosure may be used for analysis of a single cell, its contents, and/or selected biomolecules in a synthetic biology application (e.g., yeast). [0024] The acoustic retrieval system, device, and methods of the present disclosure may be used for analysis of a single cell, its contents, and/or selected biomolecules in a drug discovery application.

System and Device

[0025] The present disclosure provides an acoustic retrieval system for generating and using an externally applied acoustic field to a microfluidic device for selective retrieval of contents from the microfluidic device.

[0026] Other elements of the device, system and methods are possible, including the elements described in U.S. Patent 10,488,321, entitled “Devices and Methods for High-Throughput Single Cell and Biomolecule Analysis and Retrieval in a Microfluidic Chip”, issued on November 26, 2019, the entire disclosure of which is incorporated herein by reference.

[0027] FIG. 1 provides an example of an acoustic retrieval system 100 for generating an acoustic field to produce a localized fluid flow in a microfluidic device. For example, the acoustic retrieval system 100 may include an external acoustic transducer 110 coupled to a microfluidic device 115. The microfluidic device may be configured to support automated high-throughput processes to isolate, screen, and/or retrieve single cells or biomolecules in a biological sample. For example, the localized fluid flow can comprise a cell culture media. [0028] The microfluidic device may comprise a first side and a second side, wherein the acoustic transducer can be positioned at either the first or second side. The microfluidic device may include an array of nanowells for compartmentalizing a biological sample into a plurality of subsamples for isolating, screening, and/or retrieving single cells or biomolecules in the sample. The microfluidic device may include wells.

Actuation

[0029] An example of using acoustic streaming of an aqueous carrier fluid to actuate the contents of an aqueous sample compartmentalized in a nanowell is described in FIG. 2.

[0030] The acoustic retrieval system 100 may include an optical imaging device 135 configured to an image microfluidic device 115. The microfluidic device 115 may comprise a first side and a second side, wherein an acoustic transducer may be positioned at the first side, and wherein the optical imaging device 135 may be positioned at the second side. The microfluidic device 115 may comprise a first side and a second side, wherein the acoustic transducer may be positioned at the second side, and wherein the optical imaging device 135 may be positioned at the first side. The optical imaging device 135 may be used to image one or more nanowells in a nanowell array in the microfluidic device 115 to identify wells that have specific contents of interest. [0031] The acoustic retrieval system 100 may further include a fluidic pump 140 for supplying a fluid into and/or out of the microfluidic device 115 via certain inlet or outlet ports. For example, the fluidic pump 140 may be used to introduce a cell suspension, biomolecules, particles (e.g., beads), processing reagents, and/or assay reagents into and/or out of the microfluidic device 115.

Wells

[0032] The microfluidic device may include an array of wells for compartmentalizing 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 nanowells, at least about 200,000 nanowells, 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.

[0033] 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. Cover

[0034] As depicted in FIG. 2, a channel 215 may be in communication with aqueous-filled nanowells 210 and enclosed by a top cover 220.

[0035] The cover may be acoustically low-absorbing. The cover may be low-absorbing due to its thickness. For example, the cover may be low-absorbing due to lower thickness. The cover may have a thickness of at least about 20 micrometers (pm) to at least about 1500 pm. The cover may have a thickness of 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 150 pm, at least about 200 pm, at least about 300 pm, at least about 350 pm, at least about 400 pm, at least about 450 pm, at least about 500 pm, at least about 550 pm, at least about 600 pm, at least about 650 pm, at least about 700 pm, at least about 750 pm, at least about 800 pm, at least about 850 pm, at least about 900 pm, at least about 950 pm, at least about 1000 pm, at least about 1100 pm, at least about 1200 pm, at least about 1300 pm, at least about 1400 pm, at least about 1500 pm, or more. The cover may have a thickness at least about 50 pm to at least about 1000 pm. The cover may have a thickness at least about 90 pm to at least about 1000 pm. The cover may have a thickness at least about 90 pm to at least about 190 pm.

[0036] The cover may be low-absorbing due to the properties of its material. Examples of properties of the material include, but are not limited to, the source of the material, the stiffness, and the transparency. For example, a cover with stiffer material may have higher power loss. A cover that is semi-transparent may be low-absorbing.

[0037] The thickness of the cover may be determined by various factors. For example, the thickness of the cover may be determined by the thickness of a microfluidic chip of the microfluidic device. Acoustic transducer

[0038] An acoustic transducer may be used to apply an acoustic beam to a microfluidic device. 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.

[0039] The acoustic transducer may be used to apply a focused acoustic beam to an individual nanowell in microfluidic device. 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. [0040] 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.

[0041] 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.

[0042] The acoustic transducer may be configured to apply the focused acoustic beam having a spot size in the range of about 25 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 25 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.

[0043] The acoustic transducer may be coupled to a microfluidic device by immersion in a coupling medium that is acoustically low-absorbing. The coupling medium may be water. Pulse generator [0044] An acoustic retrieval system may include a pulse generator. The pulse generator may be used to produce an electrical pulse at radio frequency (RF).

[0045] The pulse may be a tone burst of frequency in the range of about 1 to about 30 MHz. The pulse may be a tone burst of 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, at least about 15 MHz, at least about 20 MHz, at least about 25 MHz, or at least about 30 MHz.

[0046] The pulse may have a duration in the range of about 1 to about 100 ms. The pulse may have a duration of at least about 1 millisecond (ms), at least about 2 ms, at least about 3 ms, at least about 4 ms, at least about 5 ms, at least about 6 ms, at least about 7 ms, at least about 8 ms, at least about 9 ms, at least about 10 ms, at least about 20 ms, at least about 30 ms, at least about 40 ms, at least about 50 ms, at least about 60 ms, at least about 70 ms, at least about 80 ms, at least about 90 ms, at least about 100 ms, or more.

[0047] The pulse may be repeated a number of times. The pulse may be repeated by about 1 pulse to about 100 pulses. The pulse may be repeated by at least about 1 pulse, at least about 2 pulses, at least about 3 pulses, at least about 4 pulses, at least about 5 pulses, at least about 6 pulses, at least about 7 pulses, at least about 8 pulses, at least about 9 pulses, at least about 10 pulses, at least about 11 pulses, at least about 12 pulses, at least about 13 pulses, at least about 14 pulses, at least about 15 pulses at least about 16 pulses, at least about 17 pulses, at least about 18 pulses, at least about 19 pulses, at least about 20 pulses, at least about 30 pulses, at least about 40 pulses, at least about 50 pulses, at least about 60 pulses, at least about 70 pulses, at least about 80 pulses, at least about 90 pulses, at least about 100 pulses, or more. The pulse may be a sequence numbering from about 1 pulse to about 100 pulses. The pulse may be a sequence numbering from about 1 pulse to about 20 pulses.

[0048] The pulse sequence may have a period in the range of about 20 ms to about 100 ms. The pulse may be a sequence of such tone bursts with a period of at least about 20 ms, at least about 30 ms, at least about 40 ms, at least about 50 ms, at least about 60 ms, at least about 70 ms, at least about 80 ms, at least about 90 ms, at least about 100 ms, or more.

[0049] The tone burst may also be chirped, corresponding to a changing frequency throughout the pulse duration. The frequency limits of the chirped tone burst may be in the range of at least about 1 to at least about 10 MHz around the central frequency. The frequency limits of the chirped tone burst may be 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, around the central frequency. RF Amplifier

[0050] An RF amplifier may be used to amplify the electrical pulse produced by a pulse generator and send it to an acoustic transducer. The acoustic transducer may be driven by an RF amplifier providing peak power in the range of at least about 5 watts (W) to at least about 30 W. The acoustic transducer may be driven by an RF amplifier providing peak power of at least about 5 watts W, at least about 6 W, at least about 7 W, at least about 8 W, at least about 9 W, at least about 10 W, at least about 15 W, at least about 20 W, at least about 25 W, at least about 30 W, or more.

Optical Imaging System

[0051] 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.

[0052] 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 1 IX, 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.

[0053] 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.

Acoustic Actuation for Sample Retrieval

[0054] An acoustic retrieval system may be used to produce a localized fluid flow in the microfluidic device that can be used to actuate, on demand, the contents of a single selected nanowell with a large, high-density array of nanowells in the device. The fluid in the microfluidic device may be a single liquid phase, and streaming induced by the acoustic field in the liquid phase may be used to actuate the contents of the nanowell. The liquid phase may be an aqueous phase. [0055] The use of acoustic actuation to retrieve contents from one or more specific nanowells in a nanowell array may provide a non-invasive method for isolating specific subsample components. Further, the use of acoustic actuation to retrieve content from a single nanowell on demand may have the advantages of being fully automated, high speed, and relatively low-cost as compared to, for example, the use of micro-manipulator robotics.

[0056] FIG. 2 illustrates an example of an acoustic retrieval process 200 for actuating the contents of a nanowell in a microfluidic device for downstream processing.

[0057] An array of nanowells 210 may be adjacent to a liquid-filled channel 215, wherein the channel 215 may be in communication with liquid-filled nanowells 210 and enclosed by a top cover 220. The liquid may be aqueous. The top cover may, for example, be formed of a thin film material.

[0058] Nanowells 210 may be loaded with a volume of sample fluid. For example, a fluidic pump 140 may be used to flow a sample aqueous fluid into the channel 215 to load a volume of sample fluid into each nanowell 210. A computer may be coupled to and programmed to control the operation of fluidic pump 140, as well as any valves that may be required for flowing fluid into, through and out of channel 215. The fluid may be aqueous.

[0059] The sample fluid in a nanowell 210 may contain a target 225 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. The target 225 may be an animal cell, such as a mammalian cell (e.g., a human cell). The target 225 may be a non-animal cell, such as a yeast cell or a bacterial cell. [0060] An external acoustic transducer 110 may be coupled to a microfluidic device 115. The microfluidic device may comprise a top cover 220. The microfluidic device 115 may be positioned such that the top cover 220 may face the acoustic transducer 110. Alternately, the bottom of the microfluidic device can be facing the transducer, and the top cover can face away from the transducer. The acoustic transducer 110 may be coupled to the microfluidic device 115 by immersion in a coupling medium 130 that is acoustically low-absorbing. For example, the coupling medium may be water. The acoustic transducer 110 may be controlled by a computer, e.g., to control the frequency, power, shape and timing of an acoustic pulse or pulse train generated using the acoustic transducer.

[0061] The acoustic transducer 110 may be used to apply a focused acoustic beam 230 to an individual nanowell 210 in a microfluidic device 115 for actuating the contents of the nanowell. The acoustic beam 230 may be focused with respect to the nanowell 210 in any manner that effects the actuation of the contents of the nanowell. [0062] The nanowell 210 may, for example, be selected based on an image analysis performed on the content of the well using an optical imaging device 135 to identify a target 225 of interest. In one example, the target 225 of interest is a single cell selected for retrieval and downstream processing and/or analysis. For example, a pulse generator 120 may be used to produce an electrical pulse or train of pulses at radio frequency. An RF amplifier 125 may be used to amplify the electrical pulse or pulse train produced by the pulse generator 120 and send it to the acoustic transducer 110.

[0063] In operation, the acoustic beam 230 may travel through the coupling medium 130 and pass through the top cover 220 (e.g., a thin-film cover) and focus to a spot at a position within the channel 215. The acoustic beam 230 may pass through the bottom of the device and focus to a spot within a nanowell 210. The acoustic transducer 110 may be translated laterally to place the acoustic beam 230 at the position of the selected nanowell 210 in the channel 215. The acoustic transducer 110 may be translated vertically to adjust the width of the acoustic beam 230 at the position of the nanowell 210. Acoustic actuation of the contents of the nanowell 210 may be facilitated by pulsing the acoustic transducer 110 to create a transient, high-intensity acoustic field in a nanowell 210. Acoustic streaming may then occur to produce a localized flow of fluid into the selected nanowell 210. The fluid may be aqueous. For example, aqueous fluid flow may rebound from the bottom of the nanowell 210 and then be redirected up towards the channel 215, thereby sweeping out all or substantially all of the content of the nanowell 210. In another example, if the acoustic beam is incident from below the device, the aqueous fluid flow may be directed up towards the channel 215. Actuated material that includes the target 225 may be retrieved by flow to a designated collection point.

Wells

[0064] Each array of wells may contain identical volumes or non-identical volumes. The well may be a nanowell. The nanowell may be a single nanowell in an array of nanowells. 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.

[0065] The nanowell may have a depth of at least about 1 pm to at least about 250 pm. The nanowell may have a depth of at least about 1 pm, at least about 2 pm, at least about 3 pm, at least about 4 pm, at least about 5 pm, at least about 6 pm, at least about 7 pm, at least about 8 pm, at least about 9 pm, 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 150 pm, at least about 200 pm, at least about 250 pm, or more. The nanowell may have a depth of at least about 5 pm to at least about 25 pm. The nanowell may have a depth of at least about 10 pm to at least about 20 pm. RF Amplifier

[0066] The RF amplifier may provide peak power to an acoustic transducer. An acoustic transducer may be driven by an RF amplifier providing peak power in the range of at least about 5 W to at least about 30 W. The acoustic transducer may be driven by an RF amplifier providing peak power of at least about 5 watts W, at least about 6 W, at least about 7 W, at least about 8 W, at least about 9 W, at least about 10 W, at least about 15 W, at least about 20 W, at least about 25 W, at least about 30 W, or more.

[0067] The actuation parameters may include a pulse width of about 1 ms to about 100 ms. The pulse width may be at least about 1 ms, at least about 2 ms, at least about 3 ms, at least about 4 ms, at least about 5 ms, at least about 6 ms, at least about 7 ms, at least about 8 ms, at least about 9 ms, at least about 10 ms, at least about 20 ms, at least about 30 ms, at least about 40 ms, at least about 50 ms, at least about 60 ms, at least about 70 ms, at least about 80 ms, at least about 90 ms, at least about 100 ms, or more.

[0068] Actuation parameters may include a sequence of 1 to 20 pulses. Actuation parameters may include a sequence of at least about 1 pulse, at least about 2 pulses, at least about 3 pulses, at least about 4 pulses, at least about 5 pulses, at least about 6 pulses, at least about 7 pulses, at least about 8 pulses, at least about 9 pulses, at least about 10 pulses, at least about 11 pulses, at least about 12 pulses, at least about 13 pulses, at least about 14 pulses, at least about 15 pulses, at least about 16 pulses, at least about 17 pulses, at least about 18 pulses, at least about 19 pulses, at least about 20 pulses, or more. Actuation parameters may include a sequence of at most about 20 pulses, at most about 19 pulses, at most about 18 pulses, at most about 17 pulses, at most about 16 pulses, at most about 15 pulses, at most about 14 pulses, at most about 13 pulses, at most about 12 pulses, at most about 11 pulses, at most about 10 pulses, at most about 9 pulses, at most about 8 pulses, at most about 7 pulses, at most about 6 pulses, at most about 5 pulses, at most about 4 pulses, at most about 3 pulses, at most about 2 pulses, or at most about 1 pulse. The pulse may be a sequence of such tone pulses numbering from about 1 pulse to about 40 pulses, or about 1 pulse to about 20 pulses.

[0069] Actuation parameters may include a pulse period of at least about 10 ms to at least about 200 ms. Actuation parameters may include a pulse period of at least about 10 ms, at least about 20 milliseconds ms, at least about 30 ms, at least about 40 ms, at least about 50 ms, at least about 60 ms, at least about 70 ms, at least about 80 ms, at least about 90 ms, at least about 100 ms, at least about 110 ms, at least about 120 ms, at least about 130 ms, at least about 140 ms, at least about 150 ms, at least about 160 ms, at least about 170 ms, at least about 180 ms, at least about 190 ms, at least about 200 ms, or more.

[0070] A computer may be programmed and used to control the delivery of acoustic pulses having, for example, a pulse width of about 1 to about 100 ms. A pulse width may be at least about 1 ms, at least about 2 ms, at least about 3 ms, at least about 4 ms, at least about 5 ms, at least about 6 ms, at least about 7 ms, at least about 8 ms, at least about 9 ms, at least about 10 ms, at least about 20 ms, at least about 30 ms, at least about 40 ms, at least about 50 ms, at least about 60 ms, at least about 70 ms, at least about 80 ms, at least about 90 ms, at least about 100 ms, or more. A pulse width may be at most about 100 ms, at most about 90 ms, at most about 80 ms, at most about 70 ms, at most about 60 ms, at most about 50 ms, at most about 40 ms, at most about 30 ms, at most about 20 ms, at most about 10 ms, at most about 9 ms, at most about 8 ms, at most about 7 ms, at most about 6 ms, at most about 5 ms, at most about 4 ms, at most about 3 ms, at most about 2 ms, at most about 1 ms, or less.

Acoustic beam

[0071] The actuation of the contents of a single nanowell may be achieved by using a high- resolution focused acoustic beam. The high-resolution beam may be generated by operating an acoustic transducer at a high frequency and small f-number.

[0072] The acoustic beam may have a spot size in the range of from about 25 pm to about 200 pm. The spot size may be at least about 25 pm, at least about 30 pm, at least about 35 pm, at least about 40 pm, at least about 45 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, or more.

Acoustic transducer

[0073] The acoustic transducer may be used to apply a focused acoustic beam to an individual nanowell in a microfluidic device for actuating the contents of the nanowell. The acoustic transducer may be operated in a frequency range of from about 1 to about 150 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.

[0074] A focal ratio, or an f-number, may be used to operate an acoustic transducer. The f- number may be in the range of about 1 to about 2. 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, or more. The f- number used to operate the acoustic transducer may be about 2. The f-number used to operate an acoustic transducer may denote a diameter of about 0.25 inches and a focal length of about 0.5 inches.

Computer

[0075] FIG. 3 shows a computer system 301 that is programmed or otherwise configured to control acoustic retrieval processes as disclosed herein. The computer system 301 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 various 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.

[0076] The computer system 301 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.

[0077] The computer system 301 includes a central processing unit (CPU, also “processor” and “computer processor” herein) 305, which can be a single core or multi core processor, or a plurality of processors for parallel processing. The computer system 301 may also include a memory or memory location 310 (e.g., random-access memory, read-only memory, flash memory), electronic storage unit 315 (e.g., hard disk), communication interface 320 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 325, such as cache, other memory, data storage and/or electronic display adapters. The memory 310, storage unit 315, interface 320 and peripheral devices 325 are in communication with the CPU 305 through a communication bus (solid lines), such as a motherboard. The storage unit 315 can be a data storage unit (or data repository) for storing data. The computer system 301 can be operatively coupled to a computer network (“network”) 330 with the aid of the communication interface 320. The network 330 can be the Internet, an internet and/or extranet, or an intranet and/or extranet that is in communication with the Internet. The network 330 in some cases is a telecommunication and/or data network. The network 330 can include one or more computer servers, which can enable distributed computing, such as cloud computing. The network 330, in some cases with the aid of the computer system 301, can implement a peer-to- peer network, which may enable devices coupled to the computer system 301 to behave as a client or a server.

[0078] The CPU 305 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 310. The instructions can be directed to the CPU 305, which can subsequently program or otherwise configure the CPU 305 to implement methods of the present disclosure. Examples of operations performed by the CPU 305 can include fetch, decode, execute, and writeback.

[0079] The CPU 305 can be part of a circuit, such as an integrated circuit. One or more other components of the system 301 can be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).

[0080] The storage unit 315 can store files, such as drivers, libraries and saved programs. The storage unit 315 can store user data, e.g., user preferences and user programs. The computer system 301 in some cases can include one or more additional data storage units that are external to the computer system 301, such as located on a remote server that is in communication with the computer system 301 through an intranet or the Internet.

[0081] The computer system 301 can communicate with one or more remote computer systems through the network 330. For instance, the computer system 301 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 301 via the network 330. [0082] 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 301, such as, for example, on the memory 310 or electronic storage unit 315. 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 305. In some cases, the code can be retrieved from the storage unit 315 and stored on the memory 310 for ready access by the processor 305. In some situations, the electronic storage unit 315 can be precluded, and machine-executable instructions are stored on memory 310. [0083] 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.

[0084] Aspects of the systems and methods provided herein, such as the computer system 301, can be embodied in programming. Various aspects of the technology 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 carried 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.

[0085] 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.

[0086] The computer system 301 can include or be in communication with an electronic display 335 that comprises a user interface (LT) 340 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.

[0087] 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 305.

[0088] A computer may be programmed and used to control the delivery of acoustic pulses having, for example, a pulse period of about 10 to about 200 ms. An acoustic pulse may have a period of at least about 10 ms, at least about 20 ms, at least about 30 ms, at least about 40 ms, at least about 50 ms, at least about 60 ms, at least about 70 ms, at least about 80 ms, at least about 90 ms, at least about 100 ms, at least about 110 ms, at least about 120 ms, at least about 130 ms, at least about 140 ms, at least about 150 ms, at least about 160 ms, at least about 170 ms, at least about 180 ms, at least about 190 ms, or at least about 200 ms.

[0089] A computer may be programmed and used to control the delivery of a sequence of acoustic pulses, e.g., a sequence of about 1 to about 20 pulses. A sequence of acoustic pulses may be at least about 1 pulse, at least about 2 pulses, at least about 3 pulses, at least about 4 pulses, at least about 5 pulses, at least about 6 pulses, at least about 7 pulses, at least about 8 pulses, at least about 9 pulses, at least about 10 pulses, at least about 11 pulses, at least about 12 pulses, at least about 13 pulses, at least about 14 pulses, at least about 15 pulses, at least about 16 pulses, at least about 17 pulses, at least about 18 pulses, at least about 19 pulses, at least about 20 pulses, or more. A sequence of acoustic pulses may be at most about 20 pulses, at most about 19 pulses, at most about 18 pulses, at most about 17 pulses, at most about 16 pulses, at most about 15 pulses, at most about 14 pulses, at most about 13 pulses, at most about 12 pulses, at most about 11 pulses, at most about 10 pulses, at most about 9 pulses, at most about 8 pulses, at most about 7 pulses, at most about 6 pulses, at most about 5 pulses, at most about 4 pulses, at most about 3 pulses, at most about 2 pulses, at most about 1 pulse, or less.

[0090] 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

Example 1 : Cell sorting

[0091] Immune cells are acquired and, subsequently, their supernatant is aspirated. The cells are then treated with 4% paraformaldehyde, incubated, and rinsed. Subsequently, 0.3% Triton®-X is used to permeabilize the cells. The cells are then washed and incubated with block buffer. After this incubation, the cells are washed and incubated with primary antibody. Following incubation, the cells are washed, treated with secondary antibody, and incubated. Subsequently, the cells are rinsed and stained with a DAPI solution. The cells are then loaded onto a semitransparent nanowell array as disclosed herein, and imaged with a fluorescent microscope. Cells deemed positive for the antigen of interest are identified. An acoustic transducer immersed in water applies an acoustic beam to each nanowell containing the targets of interest. The contents of positive microwells are thereby ejected towards a channel for retrieval.

[0092] Example 3: Acoustic retrieval of secreting cells. Cells known or believed to secrete a molecule of interest are loaded into a semi-transparent array of wells, as disclosed herein. In particular, primary plasma cells from an immunized animal or human, hybridoma cells, genetically-modified Chinese Hamster Ovary (CHO) cells, or other cells that secrete antibodies are loaded into the array.

Next, functionalized microspheres or reporter cells are loaded into the array of wells. Cells and other array contents are then incubated. Optionally, cell culture media may be flowed through the channel, as disclosed herein, during said incubation. Optical images of the contents of the nanowell arrays are captured at various intervals. In a preferred embodiment, optical image data is collected using fluorescence microscopy, as is known in the art. Additional reagents that specifically stain the antibody or other secreted molecule may then be pumped through the channel. In such embodiments, said staining reagents may then optionally be washed out by flowing a buffer solution or other wash solution through the channel.

Analysis of the optical image data collected is then performed to identify cells of interest. An acoustic transducer applies an acoustic beam to each of the cells of interest. The contents of each well of interest are thereby ejected towards a channel for retrieval.

[0093] 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.