[0001] This application claims the benefit of U.S. Provisional Application No. 63/327,289, filed April 04, 2022, which is incorporated herein by reference in its entirety.

BACKGROUND

[0002] Microfluidic platforms can be developed to screen and analyze small (e.g., picoliter - nanoliter) biological samples in large-scale nanowell arrays using acoustic actuation. For example, retrieval of the contents of individual wells can be achieved by acoustic actuation using an external focusing acoustic transducer.

SUMMARY

[0003] Recognized herein is an industry need for retrieval on demand of individual contents (e.g., single cells) from a single well in a large-scale nanowell array. Further, recognized herein is a solution that presents precise alignment of an acoustic beam with respect to a feature (e.g., a nanowell) in a microfluidic device.

[0004] 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, wherein said array of wells is in fluidic communication with a channel, and wherein said microfluidic device further comprises a cover, wherein said cover encloses said channel and said array of wells; (b) an acoustic transducer configured to apply one or more acoustic beams to a feature of said microfluidic device, wherein said acoustic transducer is not integrated with said microfluidic device, and wherein said one or more acoustic beams results in one or more echo signals; and (c) a signal collector configured to collect said one or more echo signals. In some embodiments, said acoustic transducer is not in contact with said microfluidic device. In some embodiments, said feature is a well. In some embodiments, said well has a volume of less than about one microliter. In some embodiments, said well has a volume of less than about two nanoliters. In some embodiments, said well has a volume of less than about one nanoliter. In some embodiments, said microfluidic device comprises at least 50,000 wells. In some embodiments, said microfluidic device comprises at least 75,000 wells. In some embodiments, said microfluidic device comprises at least 100,000 wells. In some embodiments, said cover is acoustically semi-transparent. In some embodiments, the system further comprises an actuator configured to translate the microfluidic device laterally or vertically with respect to the acoustic transducer. [0005] In some embodiments, said acoustic transducer is configured to operate at a frequency of about 1 megahertz (MHz) to about 50 MHz. In some embodiments, said acoustic transducer is configured to operate at a frequency of about 15 MHz to about 25 MHz. 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 operate at a frequency of at least 10 MHz. 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 is configured to apply said one or more acoustic beams to said microfluidic device via a coupling medium. In some embodiments, said coupling medium comprises water.

[0006] In some aspects, the present disclosure provides a method of analyzing a feature of a microfluidic device, the method comprising: (a) providing a microfluidic device comprising at least one feature, wherein said at least one feature comprises a liquid phase; (b) delivering one or more acoustic pulses to said microfluidic device, wherein said one or more acoustic pulses result in one or more echo signals; and (c) collecting said one or more echo signals. In some embodiments, the method further comprises providing one or more computer processors. In some embodiments, said one or more computer processors select said echo signals and interpret said echo signals to provide (1) the depth of said feature and (2) an image of said feature. In some embodiments, said one or more acoustic pulses are focused on said feature. In some embodiments, said feature comprises a microfluidic structure. In some embodiments, said microfluidic structure is a well.

[0007] In some aspects, the present disclosure provides a method for focusing an acoustic beam at a desired position within a microfluidic device comprising at least one feature, the method comprising: (a) using an acoustic transducer apply an acoustic beam to said at least one feature of said microfluidic device, wherein said acoustic transducer is not integrated with said microfluidic device; (b) scanning said microfluidic device with said acoustic beam to generate and collect echo signals, generating data from said echo signals and using said data to calculate the focus of said acoustic beam at said desired position. In some embodiments, the method further comprises using said data to adjust said acoustic beam focus to said desired position. In some embodiments, said desired position comprises a position in said at least one feature of said microfluidic device. In some embodiments, said desired position comprises a position of a well of said microfluidic device.

[0008] In some aspects, the present disclosure provides a method of acoustic focus mapping of a microfluidic device, the method comprising: (a) scanning a microfluidic device in the direction normal to said microfluidic device to produce and collect echo signals; (b) monitoring said echo signals to determine when the focus is located at an interface of interest and interpolating said echo signals to determine a position at which to focus an acoustic beam. In some embodiments, the data from said echo signals measures amplitude. In some embodiments, the method further comprises determining said position at which to focus an acoustic beam when said amplitude of said echo signals is maximized. In some embodiments, the method further comprises further comprising using an interpolation algorithm to find other locations of interest in the microfluidic device. In some embodiments, the method further comprises generating a map of focus positions in the microfluidic device.

[0009] Another aspect of the present disclosure provides a non-transitory computer readable medium comprising machine executable code that, upon execution by one or more computer processors, implements any of the methods above or elsewhere herein.

[0010] Another aspect of 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.

[0011] 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

[0012] 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 FIGURES

[0013] 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:

[0014] FIG. 1 is a block diagram of an example of a presently disclosed echo imaging system for aligning the focus of an acoustic beam to individual features within a microfluidic device. [0015] FIG. 2 is a cross-sectional view of an example of a microfluidic structure and shows a process of determining the three-dimensional position of an acoustic focus using echo imaging of features inside the microfluidic structure.

[0016] FIG. 3 is a flow diagram illustrating an example of a method of using echo imaging to determine the position of a focused acoustic beam with respect to the location of features in a microfluidic device.

[0017] FIG. 4 is a plot showing an example of raw echo data averaged over 1000 pulses detected from the microfluidic device structure shown in FIG. 2.

[0018] FIG. 5 is a plot showing an example of a series of echo peaks that correspond to individual interfaces in the microfluidic device structure shown in FIG. 2.

[0019] FIG. 6 is a plot showing the variation of the amplitude of the third echo peak (see FIG. 5 (C)) as a function of the position of the z stage.

[0020] FIG. 7A is a plot showing an example of scanning a 100-pm-diameter well (e.g., nanowell feature) in the x direction.

[0021] FIG. 7B is a plot showing an example of scanning the 100-pm-diameter well in the y direction.

[0022] FIG. 8A shows a brightfield image of a 100-um-deep “L”-shape alignment marker in the channel of the microfluidic device.

[0023] FIG. 8B shows an image produced by the variation of the fourth echo amplitude measured in an x - y scan across the alignment marker of FIG. 8 A.

[0024] FIG. 9 shows a computer system that is programmed or otherwise configured to implement methods provided herein.

DETAILED DESCRIPTION

[0025] While various embodiments of the 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.

[0026] 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.

[0027] 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.

[0028] Other elements of the device, system and methods are possible, including the elements described in U.S. Patent 10,488,321B2, 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.

[0029] The present disclosure provides an echo imaging system, device, and methods for generating and using an acoustic echo pulse to precisely position a focused acoustic beam inside a microfluidic device.

[0030] The present disclosure provides an echo imaging system, device, and methods for generating and using an acoustic echo pulse to determine the three-dimensional position of an acoustic focus with respect to features in a microfluidic device. The acoustic echo pulse may be generated by an acoustic transducer that is coupled to the microfluidic device using a coupling medium.

[0031] The present disclosure provides an echo imaging system, device, and method for rapidly and accurately aligning the focus of an acoustic beam with respect to the location of individual features within a microfluidic device.

[0032] The present disclosure provides an echo imaging system, device, and method for aligning the focus of an acoustic beam to individual nanowell features in nanowell array within the microfluidic device.

[0033] The present disclosure provides an echo imaging system, device, and method for positioning the focus of the acoustic beam in three dimensions to retrieve the contents of individual nanowell features in a microfluidic nanowell device by acoustic actuation using high power pulses from the external acoustic focusing transducer. The relatively low power state of the echo pulses may be capable of not disturbing the microfluidic device or the biological sample components.

[0034] The present disclosure provides an echo imaging system, device, and method for unambiguous mapping of the best focus at any interface in the microfluidic device over the full lateral extent of the device features. An interface may, for example, be the top surface of the device or any internal interface such as the bottom of a channel.

[0035] Focus mapping may be used to enable real time adjustment of the focus of the acoustic beam with respect to individual features over the full lateral extent of the features in the microfluidic device.

System and Device

[0036] FIG. 1 is a block diagram illustrating an example of an echo imaging system 100 for aligning the focus of an acoustic beam to individual features within a microfluidic device. The echo imaging system 100 may include an external acoustic transducer 110 coupled to a microfluidic device 115, wherein the microfluidic device may be configured to support automated processes to isolate, screen, and/or retrieve single cells or biomolecules in a biological sample.

[0037] 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.

[0038] An acoustic retrieval system 100 may include an optical imaging device 135 configured to image the microfluidic device 115. The microfluidic device 115 may comprise a first side and a second side, wherein the 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.

[0039] An 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 a microfluidic device 115.

Wells

[0040] 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.

[0041] 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. The nanowell may have a volume of about 1 nL.

Cover

[0042] As depicted in FIG. 2, a channel 215 may be an enclosed channel that is formed between the bottom substrate 210 and the top cover 225.

[0043] 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.

[0044] 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.

[0045] 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

[0046] 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. The three-dimensional position of the acoustic focus may be determined using echo imaging of features in the microfluidic device.

[0047] The acoustic transducer may be used to apply a focused acoustic beam to an individual nanowell in the microfluidic device. An 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 to the microfluidic device may be contactless and thereby limit the introduction of contaminants into the microfluidic device. The acoustic transducer may be configured such that the propagation direction of the acoustic beam is perpendicular to the plane of microfluidic device.

[0048] In some cases, the 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.

[0049] 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.

[0050] The acoustic transducer may be configured to apply a 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 the focused acoustic beam may be selected based on the density of nanowell features in the nanowell array of the microfluidic device.

[0051] The acoustic transducer may be coupled to the microfluidic device by immersion in a coupling medium that is acoustically low-absorbing. The coupling medium may be, for example, water.

Nanowell feature

[0052] As depicted in FIG. 2, arranged on a bottom substrate 210 and in communication with the channel 215 may be a nanowell feature (or a nanowell) 220. The nanowell feature may, for example, be a single nanowell feature in the array of nanowells. In one example, the nanowell feature may have a depth of at least about 1 pm to at least about 250 pm. The nanowell feature 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 feature may have a depth of at least about 5 pm to at least about 25 pm. The nanowell feature may have a depth of at least about 10 pm to at least about 20 pm.

[0053] The nanowell feature may be used to partition a biological sample into subsample volumes (e.g., picoliter - nanoliter subsample volume). The volume of the nanowell feature may be less than about 1 microliter (pL). The volume of the nanowell feature may be about 1 nanoliter (nL) or less. 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, or at most about 1 nL. The nanowell may have a volume of about 1 nL.

[0054] The nanowell feature may be used to retrieve a single cell (and/or its contents) from an aqueous biological sample. For example, the nanowell feature may be used to sequester a subsample volume from the aqueous biological sample wherein a cell and/or its contents, a bead (e.g., a bead functionalized to capture a target of interest), or a combination thereof is compartmentalized in an aqueous environment.

Pulse generator

[0055] An acoustic retrieval system may include a pulse generator. The pulse generator may be used to produce an electrical pulse at radio frequency (RF).

[0056] The pulse may have a frequency in the range of about 1 to about 30 MHz. The pulse may have 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, at least about 15 MHz, at least about 20 MHz, at least about 25 MHz, or at least about 30 MHz.

[0057] The pulse generator may produce a pulse of one or more cycles of the RF signal. Each pulse may comprise at least about 1 cycle to at least about 10 cycles. Each pulse may comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or more cycles. The pulse generator may produce a repetition of pulses in an echo imaging system. The repetition rate may be at least about 1 hertz (Hz) to at least about 100 kilohertz (kHz). The repetition rate may be at least about 1 Hz, at least about 2 Hz, at least about 3 Hz, at least about 4 Hz, at least about 5 Hz, at least about 6 Hz, at least about 7 Hz, at least about 8 Hz, at least about 9 Hz, at least about 10 Hz, at least about 20 Hz, at least about 30 Hz, at least about 40 Hz, at least about 50 Hz, at least about 60 Hz, at least about 70 Hz, at least about 80 Hz, at least about 90 Hz, at least about 100 Hz, at least about 200 Hz, at least about 300 Hz, at least about 400 Hz, at least about 500 Hz, at least about 600 Hz, at least about 700 Hz, at least about 800 Hz, at least about 900 Hz, at least about 1 kHz, at least about 2 kHz, at least about 3 kHz, at least about 4 kHz, at least about 5 kHz, at least about 6 kHz, at least about 7 kHz, at least about 8 kHz, at least about 9 kHz, at least about 10 kHz, at least about 20 kHz, at least about 30 kHz, at least about 40 kHz, at least about 50 kHz, at least about 60 kHz, at least about 70 kHz, at least about 80 kHz, at least about 90 kHz, at least about 100 kHz, or more. The repetition rate may be about 10 kHz.

[0058] An echo imaging system may include a pulse generator that may be used to produce a short electrical pulse at radio frequency (RF) (e.g., 1 to 5 cycles at 25 MHz) and an RF amplifier that may be used to amplify the short electrical pulse and send it to an acoustic transducer.

RF Amplifier

[0059] An RF amplifier may be used to amplify the electrical pulse produced by a pulse generator and send it to an acoustic transducer.

[0060] The RF amplifier may be used to amplify the electrical pulse produced by the pulse generator and send it to the 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 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 Device

[0061] 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.

[0062] 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.

[0063] 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.

Signal collector

[0064] An echo imaging system 100 may further include a signal collector. The signal collector may collect data over a time window. The signal collector may convert analog signals into digital signals. The signal collector may be a signal digitizer. The signal digitizer may be used to digitize one or more echo signals. The digitized echo signals may be transmitted to a computer 140 for analysis. The signal digitizer may, for example, be a commercially available signal digitizer such as a DynamicSignal cse 1222-4GS (available from Vitrek Corporation, Poway, CA). The signal digitizer may perform signal averaging over many echo pulses to increase the signal -to-noise ratio. An example of raw echo data averaged over 1000 pulses is described in FIG. 4.

Computer

[0065] An echo imaging system 100 may further include a computer 140. A signal digitizer may be used to collect data over a time window that is synchronized to the generation of the echo pulse signal. The trigger pulse may define the beginning of the time window.

[0066] FIG. 9 shows a computer system 901 that is programmed or otherwise configured to control acoustic retrieval processes as disclosed herein. The computer system 901 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 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.

[0067] The computer system 901 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.

[0068] The computer system 901 includes a central processing unit (CPU, also “processor” and “computer processor” herein) 905, which can be a single core or multi core processor, or a plurality of processors for parallel processing. The computer system 901 may also include a memory or memory location 910 (e.g., random-access memory, read-only memory, flash memory), electronic storage unit 915 (e.g., hard disk), communication interface 920 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 925, such as cache, other memory, data storage and/or electronic display adapters. The memory 910, storage unit 915, interface 920 and peripheral devices 925 are in communication with the CPU 905 through a communication bus (solid lines), such as a motherboard. The storage unit 915 can be a data storage unit (or data repository) for storing data. The computer system 901 can be operatively coupled to a computer network (“network”) 930 with the aid of the communication interface 920. The network 930 can be the Internet, an internet and/or extranet, or an intranet and/or extranet that is in communication with the Internet. The network 930 in some cases is a telecommunication and/or data network. The network 930 can include one or more computer servers, which can enable distributed computing, such as cloud computing. The network 930, in some cases with the aid of the computer system 901, can implement a peer-to- peer network, which may enable devices coupled to the computer system 901 to behave as a client or a server.

[0069] The CPU 905 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 910. The instructions can be directed to the CPU 905, which can subsequently program or otherwise configure the CPU 905 to implement methods of the present disclosure. Examples of operations performed by the CPU 905 can include fetch, decode, execute, and writeback.

[0070] The CPU 905 can be part of a circuit, such as an integrated circuit. One or more other components of the system 901 can be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).

[0071] The storage unit 915 can store files, such as drivers, libraries and saved programs. The storage unit 915 can store user data, e.g., user preferences and user programs. The computer system 901 in some cases can include one or more additional data storage units that are external to the computer system 901, such as located on a remote server that is in communication with the computer system 901 through an intranet or the Internet.

[0072] The computer system 901 can communicate with one or more remote computer systems through the network 930. For instance, the computer system 901 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 901 via the network 930. [0073] 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 901, such as, for example, on the memory 910 or electronic storage unit 915. 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 905. In some cases, the code can be retrieved from the storage unit 915 and stored on the memory 910 for ready access by the processor 905. In some situations, the electronic storage unit 915 can be precluded, and machine-executable instructions are stored on memory 910.

[0074] 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.

[0075] Aspects of the systems and methods provided herein, such as the computer system 901, 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.

[0076] 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.

[0077] The computer system 901 can include or be in communication with an electronic display 935 that comprises a user interface (LT) 940 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.

[0078] 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 905.

[0079] 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. [0080] 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.

Echo imaging

[0081] The three-dimensional position of an acoustic focus may be determined using echo imaging of features in the microfluidic device. As illustrated in FIG. 2, an echo imaging system 100 may include instrumentation to support echo imaging of features in the microfluidic device 115. For example, the echo imaging system 100 may include a pulse generator 120 that may be used to produce a short electrical pulse at radio frequency (RF). The echo imaging system may include an RF amplifier 125 that may be used to amplify the short electrical pulse and send it to an acoustic transducer 110.

[0082] The echo imaging system 100 may include an RF switch 130 that may be used to pass through a low-amplitude echo signal from an acoustic transducer 110 while blocking the high- amplitude incident pulse.

[0083] The echo imaging system 100 may further include a signal collector. In an example, the signal collector may be a signal digitizer 135. The signal digitizer may be used to digitize one or more echo signals. The digitized echo signals may be transmitted to a computer 140 for analysis. The signal digitizer can perform signal averaging over many echo pulses to increase the signal -to-noise ratio. An example of raw echo data averaged over 1000 pulses is described in FIG. 4.

[0084] The echo imaging system 100 may further include a computer 140. The signal digitizer may be used to collect data over a time window that is synchronized to the generation of the echo pulse signal. The trigger pulse defines the beginning of the time window.

[0085] FIG. 2 is a cross-sectional view of an example of a microfluidic structure 200 and shows a process of determining the three-dimensional position of an acoustic focus using echo imaging of features inside the microfluidic structure. For example, a microfluidic device of an echo imaging system may be based generally on a microfluidic structure 200. The microfluidic structure may include a bottom substrate 210 and a channel 215. The bottom substrate may, for example, be fabricated from a plastic, a cyclic olefin copolymer (COC), or a polydimethyl siloxane (PDMS) material. The channel 215 may be an enclosed channel that is formed between the bottom substrate 210 and the top cover 225.

[0086] The channel may be a flow channel for receiving and flowing a sample fluid and/or a processing fluid into and out of microfluidic structure. The channel may further include a network of inlet and outlet channels for receiving and flowing a sample fluid and/or a processing fluid into and out of the microfluidic structure. The top cover may, for example, be a thin film material that is semi-transparent to an acoustic beam. The term “semi-transparent” as used herein means low absorption, such as less than about 50% absorption. In an example, the top cover may be fabricated from a cyclic olefin copolymer (COC) material. The sample fluid may be an aqueous biological sample. The processing fluid may be an aqueous processing fluid. The processing fluid may be a non-aqueous processing fluid (e.g., an oil) that is immiscible with an aqueous sample.

Interfaces

[0087] As depicted in FIG. 2, the arrangement of a top cover 225, a channel 215, a feature 220, and a bottom substrate 210 in a microfluidic structure 200 may create several interfaces defined by the different components of the structure. For example, the microfluidic structure 200 may include five interfaces: a first and second interface at the top and bottom surfaces, respectively, of a top cover 225; a third interface at the bottom of a channel 215; a fourth interface at the bottom of a nanowell feature 220; and a fifth interface at the bottom of a substrate 210.

[0088] In the presence of a short acoustic pulse that is incident on the microfluidic structure, each interface may produce an echo that is separated in time by its distance (round trip) from and back to the acoustic transducer. For example, as depicted in FIG. 2, a shaded focused acoustic beam 230 is used to highlight an area of a microfluidic structure 200 that may be penetrated by the short acoustic pulse to produce an echo signal. The echo signals produced at each interface are indicated as Echo 1 through Echo 5, wherein Echo 1 is produced at the top surface of a top cover 225; Echo 2 is produced at the bottom surface of a top cover 225; Echo 3 is produced at the bottom of a channel 215; Echo 4 is produced at the bottom of nanowell feature 220; and Echo 5 is produced at the bottom of a substrate 210. Every interface may produce an echo signal, and these echo signals may be separated in time if the acoustic pulse is very short (e.g., in the range of 1 to 5 cycles). [0089] The echo signal may be used to provide with respect to one or more features of the microfluidic device one or more dimensions of the features (e.g., heights, distances, diameters, topographies). The position in z (normal to the plane of microfluidic structure 220) where the focus is located at a particular interface can be determined by maximizing the echo signal from that interface. The position of the focus in x-y can be determined by scanning an interface that corresponds to a feature of limited spatial extent to extract a two-dimensional image of the feature.

[0090] The microfluidic device can be fabricated from an optically transparent material to allow the content of the device to be imaged. Possible materials include polymeric substrates such as cyclic olefins (COC or COP), polymethylmethacrylate (PMMA), and polycarbonate (PC), or soft elastomers such as polydimethylsiloxane (PDMS), or glass and quartz. The channels of the microfluidic device can be sealed with a thin film cover that is semi-transparent to an acoustic beam in the frequency range of about 1 to about 30 MHz. Possible materials include COC, COP, PMMA, PC, and polyethylene terephthalate (PET).

[0091] Introduction of a cell suspension, biomolecules, particles, lysing buffer, amplification reagents, wash buffers, and carrier oil into and out of the device may be controlled by an external pump that interfaces with a manifold that can selectively control applied positive or vacuum pressure at specific inlet or outlet well on the device.

Determining the Position of a Focused Acoustic Beam

[0092] The present disclosure provides a method for focusing an acoustic beam at a desired position within a microfluidic device. The acoustic beam may be generated by an external transducer and coupled to the microfluidic device using a low absorption medium such as water. The three-dimensional position of the acoustic focus may be determined using echo imaging of features inside a device.

[0093] FIG. 3 is a flow diagram illustrating an example of a method 300 of using echo imaging to determine the position of a focused acoustic beam with respect to the location of a feature in a microfluidic device. The method may include, but is not limited to, the following steps.

[0094] At a step 310, an echo imaging system and microfluidic device that includes a nanowell array may be provided. For example, the echo imaging system 100 that includes an acoustic transducer 110 mounted on motorized x, y, z stages and microfluidic device 115 may be provided. The microfluidic device 115 may include a microfluidic structure 200 described with reference to FIG. 2.

[0095] At a step 315, an external acoustic transducer can be coupled to the microfluidic device via a coupling medium. For example, the microfluidic device can be positioned such that its top cover is facing an acoustic transducer. The acoustic transducer may be coupled to a microfluidic device by immersion in a coupling medium. For example, the coupling medium may be water. [0096] At a step 320, the z position of a focus may be determined for each interface in the microfluidic device. The focus for each interface may be determined by scanning the z stage while collecting the echo signal from each interface. For example, a pulse generator may be used to produce a short electrical pulse at radio frequency. For example, the short electrical pulse may comprise 1 to 5 cycles at 25 megahertz (mHz). An RF amplifier may amplify a short pulse and send it to an acoustic transducer.

[0097] When a short acoustic pulse is incident on each interface of a microfluidic device, each interface produces an echo. For example, the microfluidic structure may include five interfaces that produce five echo signals that are separated in time by their distance (round trip) to an acoustic transducer. An RF switch may then be used to pass through the low-amplitude echo signal from each interface to a signal digitizer, while blocking a high amplitude incident pulse. The signal digitizer may then be used to digitize the echo signals from each interface. Several echo pulses may be used from each interface and processed by the signal digitizer to increase the signal-to-noise ratio. The z-direction position of a focus at a particular interface may be determined by maximizing the echo from that interface.

[0098] At a step 325, the position of the focus in the x-y direction may be determined for a certain feature in the microfluidic device. The location of a feature in an x-y plane may be determined by positioning the acoustic focus at an interface that defines the feature and monitoring an echo signal while scanning in the plane. For example, a pulse generator may be used to produce a short electrical pulse at a radio frequency. For example, the short electrical pulse may be produced for 1 to 5 cycles at 25 mHz. The RF amplifier can amplify the short pulse and send it to the acoustic transducer. The RF switch may be used to pass through a low- amplitude echo signal to the signal digitizer, while blocking a high amplitude incident pulse. The signal digitizer may be used to digitize the echo signal. Multiple pulses may be delivered to the microfluidic device and their echo signals processed by the signal digitizer to increase the signal-to-noise ratio.

[0099] At a step 330, the echo signals may yield scan results. The scan results may be combined to provide the information required for positioning the focal point of the acoustic beam at the desired feature.

Focus Mapping

[0100] The present disclosure provides an echo imaging system, device, and method for unambiguous mapping of the best focus at any interface in a microfluidic device over the full lateral extent of the features (e.g., an array of nanowells). The interface may, for example, be the top surface of the device or any internal interface of the microfluidic device, such as the bottom of a channel.

[0101] The present disclosure provides a method for scanning the microfluidic device in the z direction (normal to the microfluidic device) and monitoring the echo signals to determine when the focus is located at an interface of interest (based on the maximum echo signal) for numerous points in the x-y plane throughout the device.

[0102] Focus mapping may be used to enable real time adjustment to the focus of the acoustic beam with respect to individual features over the full lateral extent of the features in the microfluidic device. This may entail measuring the z position of the interface at a number of points on a grid and then interpolating to obtain the z position at any arbitrary point within the grid.

[0103] 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 : Echo system

[0104] As described in FIG. 1, a pulse generator can be used to produce a short electrical pulse at radio frequency (e.g., 1 to 5 cycles at 25 MHz) that is amplified and sent to the acoustic transducer. The acoustic transducer delivers an acoustic pulse to the microfluidic device, which generates echo signals. An RF switch passes through the low-amplitude echo signal while blocking the high-amplitude incident pulse. The echo signal is digitized and sent to a computer for analysis. The digitizer can perform signal averaging over many echo pulses to increase the signal-to-noise ratio.

Example 2: Raw echo data

[0105] Raw echo data over 1000 pulses in a microfluidic device system as described in FIG. 2 may be collected. FIG. 4 illustrates a plot showing an example of such raw data. The bottom substrate and the cover are composed of COC (cyclic olefin copolymer). The channel height is 188 pm and the well depth is 100 pm. Both the channel and well are filled with water. The excitation pulse is 1 cycle of 25 MHz RF. The echo pulses are generated at a repetition rate of 10 kHz, yielding an incident average power of 10 mW.

[0106] The signal is processed to extract the echo pulses reflected by the individual interfaces in the device structure. For example, first a Hilbert transform is applied to filter the high frequency oscillations. Then a mild convolution filter is applied to smooth the data. Finally, the background is subtracted using a rolling ball filter. The output of the signal processing is a series of echo peaks that correspond to the individual interfaces in the device structure.

[0107] FIG. 5 is a plot showing an example of a series of echo peaks that correspond to the individual interfaces in the device structure. The first two peaks (A and B) are from the top and bottom of the COC cover (see FIG. 2, Echo 1 and Echo 2, respectively). The third peak (C) is from the bottom of the channel (Echo 3 in FIG. 2). The fourth peak (D) is from the bottom of the nanowell feature (Echo 4 in FIG. 2). The fifth peak (E) is from the bottom of the device (Echo 5 in FIG. 2).

[0108] The acoustic transducer may be mounted on motorized x, y, z stages to allow automated scans to be performed while monitoring the echo signal. The amplitude of the individual peaks is maximized when the acoustic focus is at the location of the interface. This amplitude data is used to find the best focus and alignment of the beam on features in the device.

[0109] The best focus for a particular interface may be determined by scanning the z stage while collecting the echo signal. Features in the plane of the device can be imaged by scanning the x and y stages while monitoring the echo peak from the bottom of the feature. FIG. 6 is a plot showing the variation of the amplitude of the third echo peak (see FIG. 5 (C)) as a function of the position of the z stage. As shown in FIG. 6, the echo amplitude has a maximum at 3930 pm. This is the z position at which the acoustic focus is located at the bottom of the channel.

[0110] The location of a feature in the x-y plane is determined by positioning the acoustic focus at the interface that defines the feature and monitoring the echo signal while scanning in the plane. FIG. 7A is a 700 showing an example of scanning a 100-pm-diameter well (e.g., nanowell feature) in the x direction. FIG. 7B is a plot 710 showing an example of scanning the 100-pm-diameter well in the y direction. Referring now to FIG. 7A, the fourth echo peak (see FIG. 5 (D)) corresponds to the bottom of the well. The variation of the amplitude of the fourth echo peak as the x stage is scanned indicates the location of the well along the x-axis. In this example, the center of the well is at the x stage location of 240 pM. A similar scan along the y- axis yields the results shown in FIG. 7B. As shown in FIG. 7B, the y-stage location of the center of the well is 245 pm.

[0111] Referring now to FIG. 6, FIG. 7A, and FIG. 7B, the data show that the combination of the scan results provide the information necessary to position the focal point of the acoustic beam precisely at the center of the well, as shown schematically in FIG. 2.

[0112] The present disclosure provides unambiguous mapping of the best focus at any interface in a microfluidic device over the full lateral extent of the features. For example, the maximum echo signal for a peak (C) in plot 500 of FIG. 5 corresponds to the bottom of the channel (Echo 3 in FIG. 2), when the focus of the acoustic beam is located at the water-channel bottom interface. In focus mapping, the z position can be measured for several different x-y positions around the perimeter of the channel. Then a focus map can be generated by interpolation of the measured z positions over an x-y grid. In this example, the focus map provides an unambiguous measurement of the position of the channel floor. The focus map can be used to make corrections to the z position of the acoustic transducer in real time as it is scanned in the x and y directions, to keep the focus at the bottom of the channel.

[0113] It may also be useful to perform scans using the x-y stages to image features that might have more complex shapes. As an example, FIG. 8A shows a brightfield image of an “L”-shape alignment marker which is a feature in the channel of the microfluidic device having a 100 pm depth. The variation of the fourth echo amplitude coming from the bottom of this feature produces the image shown in FIG. 8B. The centroid of this marker shape is located at the inside comer, where the red crosshair is positioned in FIG. 8A. The echo image is analyzed to find the centroid in x-y stage coordinates and thereby precisely position the focus of the acoustic beam on the crosshair.

[0114] 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.