Ultrasonic bubble remedy

Abstract

A system for measuring a sample contained in a vessel includes a transducer assembly configured to receive a plurality of electronic transmission signals and emit a corresponding plurality of transmitted acoustic signals toward the vessel and the sample, and configured to receive reflected acoustic signals from the vessel or sample and generate a corresponding plurality of received electronic signals; a signal transmission circuitry configured to generate and communicate the electronic transmission signals to the transducer assembly; a signal reception circuitry configured to receive the electronic reception signals from the transducer assembly; and a processor configured to control the signal transmission circuitry and receive information corresponding to the received electronic signals from the signal reception circuitry.

Description

[Background technology]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional No. 63 / 414,322, filed October 7, 2022, the entire contents of which are incorporated herein by reference.

[0002] Acoustic droplet ejection (ADE) is a technique that uses acoustic energy to move liquids without any physical contact. Some examples of ADE techniques are disclosed in U.S. Pat. No. 10,156,499 (incorporated herein by reference in its entirety). Acoustic energy (e.g., in the form of ultrasonic pulses) is emitted from a transducer toward a volume of liquid (hereinafter, "sample"). In some embodiments, a beam is focused on or near the upper surface of the sample, and acoustic energy is transferred to a portion of the sample, thereby moving this portion upward and away from the remainder of the sample (e.g., as a droplet). The sample may be contained within a well (also referred to herein as a vessel) of a plate (e.g., a 96- or 384-well microplate). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] U.S. Patent No. 10,156,499 Summary of the Invention [Means for solving the problem]

[0004] According to an embodiment, a system for measuring a sample contained within a vessel, the vessel including a bottom surface having a bottom bottom interface (BB) and a bottom top interface (TB), the system includes a transducer assembly configured to receive a plurality of electronic transmit signals and emit a corresponding plurality of transmitted acoustic signals toward the vessel and the sample, and to receive reflected acoustic signals from the vessel or the sample and generate a corresponding plurality of received electronic signals; signal transmission circuitry configured to generate and communicate the electronic transmit signals to the transducer assembly; and an electronic receiver. The system includes signal receiving circuitry configured to receive signals from the transducer assembly and a processor configured to control the signal transmission circuitry and receive information corresponding to the electronic received signals from the signal receiving circuitry, wherein the plurality of transmitted acoustic signals comprises a sequence of a first ping, a first bubble breaking signal, and a second ping, and the plurality of reflected acoustic signals comprises a first TB reflection corresponding to the first ping and a second TB reflection corresponding to the second ping, and the system is configured to emit the second bubble breaking signal based on a comparison of a characteristic of the first TB reflection with a characteristic of the second TB reflection. The characteristic of the first TB reflection may comprise a first peak amplitude, and the characteristic of the second TB reflection may comprise a second peak amplitude. The system may further be configured to emit the second bubble breaking signal if the first peak amplitude exceeds the second peak amplitude. The peak amplitude of the first ping and the peak amplitude of the second ping may be substantially the same. The total energy of the first bubble disruption signal may exceed the total energy of the first ping and may exceed the total energy of the second ping. The transducer assembly may be configured to focus acoustic energy at a first height when emitting the first ping, the first bubble disruption signal, and the second ping, and the transducer assembly may be configured to focus acoustic energy at a second height above the first height when emitting the second bubble disruption signal. The first height may be predetermined relative to the TB, and the second height may be substantially at the surface of the sample. The first height is between + / - 6 mm relative to the TB.The first bubble destruction signal may include a variable frequency. The energy of the second bubble destruction signal may be selected to prevent complete ejection of a droplet from the sample. The second bubble destruction signal may include a pre-conditioning signal selected to destroy the bubble and a post-conditioning signal selected to destroy the bubble. The pre-conditioning signal may be the bubble destruction signal. The transducer assembly may be configured to emit the second bubble destruction signal while focusing the acoustic energy substantially at the surface of the sample. The vessels may be included in a plate comprising a plurality of vessels containing a corresponding plurality of samples, each of the plurality of vessels may include a corresponding BB and TB, the system may further include at least one motor configured to move at least one of the plate or the transducer assembly so that the transducer assembly is positioned beneath each of the plurality of vessels, the system may further be configured to cause the transducer assembly to emit a sequence of a first ping, a first bubble-breaking signal, and a second ping for each of the plurality of vessels, the system may further be configured to cause the transducer assembly to emit a second bubble-breaking signal for a given of the plurality of vessels based on a comparison of a characteristic of the first TB reflection for the given vessel and a characteristic of the second TB reflection for the given vessel, the system may further be configured to emit the second bubble-breaking signal for a given of the plurality of vessels after the first ping, the first bubble-breaking signal, and the second ping have been emitted for each of the plurality of vessels.

[0005] According to an embodiment, a method is provided for measuring a sample contained within a vessel, the vessel including a bottom surface having a bottom bottom interface (BB) and a bottom top interface (TB), the method comprising the steps of receiving a plurality of electronic transmit signals at a transceiver assembly; transmitting a plurality of transmitted acoustic signals by the transceiver assembly towards the vessel and the sample, the plurality of transmitted acoustic signals corresponding to the plurality of electronic transmit signals; receiving a plurality of reflected acoustic signals from the vessel or the sample at the transceiver assembly; generating a plurality of electronic receive signals by the transceiver assembly corresponding to the plurality of reflected acoustic signals; generating the plurality of electronic transmit signals by signal transmission circuitry; The method includes communicating a plurality of electronic transmit signals to a transducer assembly, receiving the plurality of electronic receive signals from the transducer assembly at signal reception circuitry, controlling the signal transmission circuitry with a processor, and receiving information corresponding to the electronic receive signals from the signal reception circuitry at the processor, wherein the plurality of transmitted acoustic signals comprises a sequence of a first ping, a first bubble breaking signal, and a second ping, and the plurality of reflected acoustic signals comprises a first TB reflection corresponding to the first ping and a second TB reflection corresponding to the second ping, and further includes emitting, by the transducer assembly, a second bubble breaking signal based on a comparison of a characteristic of the first TB reflection and a characteristic of the second TB reflection. The characteristic of the first TB reflection may comprise a first peak amplitude and the characteristic of the second TB reflection may comprise a second peak amplitude, and the method may further include emitting, by the transducer assembly, the second bubble breaking signal if the first peak amplitude exceeds the second peak amplitude. The peak amplitude of the first ping and the peak amplitude of the second ping may be substantially the same. The overall energy of the first bubble collapse signal may exceed the overall energy of the first ping and exceed the overall energy of the second ping.The method may further include using a transceiver assembly to focus acoustic energy at a first height when issuing the first ping, the first bubble destruction signal, and the second ping, and using the transceiver assembly to focus acoustic energy at a second height above the first height when issuing the second bubble destruction signal. The first height may be predetermined relative to the TB, and the second height may be substantially at the surface of the sample. The first height may be between + / - 6 mm relative to the TB. The first bubble destruction signal may include a variable frequency. The energy of the second bubble destruction signal may be selected to prevent complete ejection of a droplet from the sample. The second bubble destruction signal may include a pre-conditioning signal selected to destroy the bubble and a post-conditioning signal selected to destroy the bubble. The pre-conditioning signal may be the bubble destruction signal. The method may further include issuing the second bubble destruction signal while focusing acoustic energy substantially at the surface of the sample with the transceiver assembly. The vessels may be included in a plate with a plurality of vessels containing a corresponding plurality of samples, each of the plurality of vessels including a corresponding BB and TB, and the method may further include moving at least one of the plate or the transducer assembly using at least one motor so that the transducer assembly is positioned directly beneath each of the plurality of vessels, emitting, by the transducer assembly, a sequence of a first ping, a first bubble breaking signal, and a second ping for each of the plurality of vessels, and emitting, by the transducer assembly, a second bubble breaking signal for a given one of the plurality of vessels based on a comparison of a characteristic of the first TB reflection for the given vessel and a characteristic of the second TB reflection for the given vessel. The transducer assembly may further emit the second bubble breaking signal for the given one of the plurality of vessels after the first ping, the first bubble breaking signal, and the second ping have been emitted for each of the plurality of vessels. The non-transitory computer readable medium contains instructions that, when executed by a processor, can perform any one of the foregoing method embodiments.

[0006] According to an embodiment, a system for reducing bubbles in a sample contained in a container using an ultrasonic system having a transducer assembly, the container including a bottom surface having a bottom boundary (BB) and a bottom top boundary (TB), and a bubble in the sample is centered at the TB. The system includes a transducer assembly configured to align a focus of an acoustic energy beam, the focus height being substantially at an upper surface of the sample. The transducer assembly is further configured to emit a bubble disruption signal while the focus height is substantially at the upper surface of the sample, the bubble disruption signal having a peak amplitude selected to prevent complete ejection of a droplet from the sample. The system may be configured to perform at least one measurement to determine the peak amplitude of the bubble disruption signal. The peak amplitude may include a maximum amplitude substantially before the droplet will be completely ejected from the sample. The bubble destruction signal may further include a maximum amplitude determination signal selected to determine the maximum amplitude of the acoustic signal by processing a reflection of the maximum amplitude determination signal from the upper surface of the sample, where an acoustic signal having an amplitude greater than the maximum amplitude would cause a droplet to be ejected from the sample, and a bubble mitigation signal including a peak amplitude determined according to the maximum amplitude. The maximum amplitude determination signal may include a first disturbance signal selected to disturb the upper surface of the sample, a first measurement signal following the first disturbance signal, the first measurement signal being selected to be reflected by the upper surface of the sample, whereby reflection of the first measurement signal is processed to determine zero velocity droplets, a second disturbance signal following the first measurement signal, the second disturbance signal being selected to disturb the upper surface of the sample, the peak amplitude of the second disturbance signal exceeding the peak amplitude of the first disturbance signal, and a second measurement signal following the second disturbance signal, the second measurement signal being selected to be reflected by the upper surface of the sample, whereby reflection of the second measurement signal is processed to determine zero velocity droplets.The system may be further configured to emit a sequence of additional disturbance signals and additional measurement signals, the additional disturbance signals having increasing peak amplitudes. The bubble mitigation signal may include a plurality of signals, each having a peak amplitude determined according to a maximum amplitude. Each of the plurality of signals in the bubble mitigation signal may have a duration of at least 8 μs.

[0007] According to an embodiment, a method is provided for reducing bubbles in a sample contained in a container using an ultrasonic system having a transducer assembly, the container including a bottom surface having a bottom boundary (BB) and a bottom top boundary (TB), the bubble in the sample being centered at the TB, the method including: using the transducer assembly to focus an acoustic energy beam, the focal height being substantially at an upper surface of the sample; and using the transducer assembly to emit a bubble disruption signal while the focal height is substantially at the upper surface of the sample, the bubble disruption signal having a peak amplitude selected to prevent complete ejection of a droplet from the sample. The method may further include performing at least one measurement to determine the peak amplitude of the bubble disruption signal. The peak amplitude may include substantially a maximum amplitude before the droplet will be completely ejected from the sample. The bubble destruction signal may further include a maximum amplitude determination signal selected to determine the maximum amplitude of the acoustic signal by processing a reflection of the maximum amplitude determination signal from the upper surface of the sample, where an acoustic signal having an amplitude greater than the maximum amplitude would cause a droplet to be ejected from the sample, and a bubble mitigation signal including a peak amplitude determined according to the maximum amplitude. The maximum amplitude determination signal may include a first disturbance signal selected to disturb the upper surface of the sample, a first measurement signal following the first disturbance signal, the first measurement signal being selected to be reflected by the upper surface of the sample, whereby reflection of the first measurement signal is processed to determine zero velocity droplets, a second disturbance signal following the first measurement signal, the second disturbance signal being selected to disturb the upper surface of the sample, the peak amplitude of the second disturbance signal exceeding the peak amplitude of the first disturbance signal, and a second measurement signal following the second disturbance signal, the second measurement signal being selected to be reflected by the upper surface of the sample, whereby reflection of the second measurement signal is processed to determine zero velocity droplets.The method may further include a sequence of additional disturbance signals and additional measurement signals, the additional disturbance signals having increasing peak amplitudes. The bubble mitigation signal may include a plurality of signals, each having a peak amplitude determined according to a maximum amplitude. Each of the plurality of signals in the bubble mitigation signal may have a duration of at least 8 μs. A non-transitory computer-readable medium contains instructions that, when executed by a processor, may implement any one of the foregoing method embodiments.

[0008] According to an embodiment, a system is provided for determining the presence of air bubbles in a sample contained in a container in an ultrasound system, the container including a bottom surface having a bottom bottom interface (BB) and a bottom top interface (TB), the system comprising: an ultrasonic transducer assembly configured to emit a first ping having a first energy, and further configured to emit a bubble-breaking signal followed by a second ping having a second energy, and further configured to emit a higher-energy signal following the first ping and prior to the second ping, the higher-energy signal having energy greater than the first and second energies, the ultrasonic transducer assembly further configured to receive a first reflected signal from the first ping that is reflected from the TB, and further configured to receive a second reflected signal from the second ping that is reflected from the TB; and a processor configured to infer the presence of air bubbles in the sample when the peak amplitude of the first reflected signal exceeds the peak amplitude of the second reflected signal.

[0009] According to an embodiment, a method is provided for determining the presence of gas bubbles in a sample contained in a container in an ultrasound system having an ultrasound transducer assembly and a processor in communication with the ultrasound transducer assembly, the container including a bottom surface having a bottom bottom interface (BB) and a bottom top interface (TB), the method including the steps of: issuing a first ping from the ultrasound transducer assembly having a first energy; issuing a bubble breaking signal from the ultrasound transducer assembly followed by a second ping having a second energy; and issuing a signal from the ultrasound transducer assembly to break up the first ping. Subsequently and prior to the second ping, emitting a higher energy signal, the higher energy signal having an energy greater than the first energy and the second energy, receiving, by an ultrasonic transducer assembly, a first reflected signal from the first ping that is reflected from the TB, receiving, by the ultrasonic transducer assembly, a second reflected signal from the second ping that is reflected from the TB, and inferring, by a processor, the presence of an air bubble in the sample when the peak amplitude of the first reflected signal exceeds the peak amplitude of the second reflected signal. A non-transitory computer-readable medium may contain instructions that, when executed by a processor, perform any of the foregoing method embodiments.

[0010] According to an embodiment, a method for performing sonoporation on a sample, including cells, contained in a vessel, the vessel including a bottom surface having a bottom bottom interface (BB) and a bottom top interface (TB). The method includes transmitting a ping toward the sample by a transducer assembly configured to transmit and receive acoustic signals; receiving a reflected signal at the transducer assembly, the reflected signal including reflections from the ping, from the TB, and from the surface of the sample; measuring the energy of the reflected signal using a processor; and estimating, using the processor, a volume of bubbles based on the energy of the reflected signal. The estimating the volume of bubbles may be based at least in part on the energy of reflections from the TB in the reflected signal. The estimating the volume of bubbles may be based at least in part on the energy of reflections from the surface of the sample in the reflected signal. The estimating the volume of bubbles may be based at least in part on the energy of reflections from the surface of the sample in the reflected signal. The energy of the reflected signal may correspond to the peak amplitude of the reflected signal for a given reflection. The method may further include transmitting, with the transducer assembly, a bubble-breaking signal configured to break at least a portion of the bubbles; transmitting, with the transducer assembly, a second ping toward the sample; receiving, with the transducer assembly, a second reflected signal in response to the second ping, the second reflected signal including a reflection from the TB and a reflection from a surface of the sample; measuring, with a processor, the energy of the second reflected signal; and estimating, with the processor, a volume of the second bubbles based on the energy of the second reflected signal. Estimating the volume of the second bubbles may be based, at least in part, on the energy of the reflection from the TB in the second reflected signal. Estimating the volume of the bubbles may be based, at least in part, on the energy of the reflection from the surface of the sample in the reflected signal.The step of estimating the volume of bubbles may be based, at least in part, on energy of reflection from the surface of the sample in the reflected signal. The method may further include transmitting a second bubble destruction signal configured to destroy at least some of the bubbles based on the volume of the second bubbles. The second bubble destruction signal may be different from the bubble destruction signal. The second bubble destruction signal may be determined by a processor, at least in part, based on the volume of the second bubbles. The method may further include performing transfection on cells in the sample. The method may further include predicting efficiency of transfection based, at least in part, on the volume of the bubbles. The system may be configured to perform any one of the aforementioned method embodiments. A non-transitory computer-readable medium contains instructions that, when executed by a processor, may perform any one of the aforementioned method embodiments. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 shows a representation of an ADE system, including a cross-sectional view of a reservoir plate containing multiple reservoirs (or wells) that hold individual samples, a receiving plate, a transducer assembly, and a block diagram of the electronic circuitry.

[0012] [Figure 2] FIG. 2 shows a block diagram of the transducer assembly.

[0013] [Figure 3] FIG. 3 shows a representation of the movement of the transducer assembly relative to the container plate when ADE is performed on multiple samples.

[0014] [Figure 4] FIG. 4 shows a top view of a reservoir plate with multiple reservoirs.

[0015] [Figure 5]FIG. 5 shows a top view of multiple vessels in a vessel plate and a flow diagram illustrating the sequence for performing ADE on each vessel in turn.

[0016] [Figure 6] FIG. 6 shows a container for holding a sample.

[0017] [Figure 7] FIG. 7 shows the reflected signal and corresponding envelope, which is received at the transceiver assembly in response to the emitted signal.

[0018] [Figure 8] FIG. 8 shows an image from a scanning electron microscope of the interior surface at the bottom top interface (TB) of an exemplary container.

[0019] [Figure 9A] Figure 9A shows an image from a scanning electron microscope of a TB in a container holding a sample, unaffected by air bubbles.

[0020] [Figure 9B] Figure 9B shows an image from a scanning electron microscope of a TB in a container holding a sample, affected by an air bubble.

[0021] [Figure 10] FIG. 10 illustrates a sequence for bubble remediation, according to one embodiment.

[0022] [Figure 11] FIG. 11 shows a top view of a reservoir plate with one reservoir having a sample with an air bubble.

[0023] [Figure 12] FIG. 12 shows a flowchart for a method for removing air bubbles, according to an embodiment.

[0024] [Figure 13]FIG. 13 shows a flowchart for a method for removing air bubbles, according to an embodiment.

[0025] [Figure 14] FIG. 14 illustrates the stabilization of reflection from a TB in a container holding a sample as affected by an air bubble.

[0026] [Figure 15] FIG. 15 illustrates the stabilization of a zero velocity droplet (ZVD) in a container holding a sample, as affected by an air bubble.

[0027] [Figure 16] 16A, 16B, and 16C show illustrative examples of ping reflections when different amounts of air bubbles are present, according to an embodiment.

[0028] [Figure 17] 17A, 17B, 17C, and 17D show illustrative examples of ping reflections after different bubble breaking signals are emitted, according to an embodiment.

[0029] [Figure 18] FIG. 18 shows four microscope images of cells with attached air bubbles after a bubble destruction signal is issued, according to an embodiment.

[0030] [Figure 19] FIG. 19 shows a flowchart for a method of removing air bubbles, according to an embodiment.

[0031] [Figure 20] FIG. 20 shows signal graphs illustrating the reduction of air bubbles in different samples according to an embodiment.

[0032] [Figure 21] FIG. 21 shows a graph illustrating the reduction of air bubbles in different samples according to an embodiment.

[0033] [Figure 22] FIG. 22 shows a graph, according to an embodiment, where the x-axis shows transfection efficiency as a percentage for A549 cells and the y-axis shows bubble removal as a percentage.

[0034] [Figure 23] FIG. 23 shows a graph, according to an embodiment, where the x-axis shows transfection efficiency as a percentage for HEK-293 cells and the y-axis shows bubble removal as a percentage.

[0035] The foregoing description, as well as the following detailed description, of certain techniques of the present application will be better understood when read in conjunction with the accompanying drawings. For illustrative purposes, certain techniques are shown in the drawings. It should be understood, however, that the claims are not limited to the arrangements and instrumentation shown in the accompanying drawings. Furthermore, the features shown in the drawings are one of many ornamental features that may be employed to achieve the stated functionality of the present system. DETAILED DESCRIPTION OF THE INVENTION

[0036] Detailed Description FIG. 1 depicts an exemplary representation of an ADE system 100, including a cross-sectional view of a reservoir plate 120 (e.g., a microplate) including multiple reservoirs 122 (e.g., wells of a microplate) that hold multiple individual samples 101; a receiver plate 130 including multiple receiving wells that receive ejected liquid 102 from the samples 101; and a block diagram of electronics 140. ADE system 100 further includes a transducer assembly 110, a coupling fluid 160, an X / Y / Z motor 150 (or a motor in less than all three dimensions, e.g., a non-vertical (Z) dimension motor), and / or a temperature sensor (not shown). FIG. 2 further illustrates transducer assembly 110, including a transducer 112 and an acoustic lens 113. ADE system 100 can determine properties of the reservoirs 122 and / or samples 101 and eject liquid. A given sample 101 is a liquid of interest held in a corresponding reservoir 122. Although this disclosure focuses on vessels 122 that are wells of a microplate, the techniques described herein can be used with other vessels 122, such as tubes, flasks, and beakers, and any samples 101 contained therein.

[0037] To eject the ejected liquid 102 from the sample 101, the transducer 112 generates acoustic energy (e.g., ultrasonic energy), which is focused by the acoustic lens 113 into a beam 170. While the beam 170 is shown in two dimensions in the figures, the beam 170 is actually three-dimensional. Furthermore, although the beam 170 is shown as an equilateral triangle, the beam 170 can have a different shape. By adapting the configuration of the transducer assembly 110, it may be possible to vary the height of the focal point of the acoustic energy beam 170. For example, the focal length of the beam 170 may be changeable by adapting the transducer assembly 110. Such a transducer assembly 110 is described in U.S. Application No. 16 / 369,780 (U.S. Publication No. 2019 / 0302063), which is incorporated herein by reference in its entirety. It may also be possible to vary the height of the focal spot by moving the transducer assembly 110 along the z-axis (ie, the vertical dimension between the container 122 and the transducer assembly 110).

[0038] 1, beam 170 is focused onto the upper surface of sample 101, which may be at the interface between sample 101 and the air above sample 101. First, beam 170 passes through binding liquid 160, bottom wall 123 of container 122, then through the depth of sample 101 to reach surface 103 of sample 101.

[0039] Electronic circuitry 140 includes processor 143, motor controller 142, signal transmission circuitry 144, signal reception circuitry 145, and temperature sensor circuitry 141. While shown as separate components for purposes of illustration, portions of electronics 140 may be combined or integrated. Furthermore, some components shown may include multiple different subcomponents that are not specifically shown. For example, processor 143 may include multiple processors (e.g., multiple processors distributed at different locations).

[0040] The processor 143 causes the signal transmission circuitry 144 to generate or control an analog electrical signal (an electronic transmission signal, such as a radio frequency (RF) signal) that is communicated to the transducer 112. The transducer 112 then vibrates in response to the analog signal (amplitude and frequency) such that a corresponding acoustic signal is emitted. The transducer assembly 110 may also receive and vibrate in sync with an acoustic signal (e.g., an acoustic signal reflected from the container 120 or sample 101 in response to the emitted acoustic signal). This may generate an analog electrical signal (an electronic receive signal), which is then communicated to the signal reception circuitry 145. The processor 143 may receive information from the signal reception circuitry 145 in the form of an electronic receive signal, corresponding to the received acoustic signal. The information in the electronic receive signal will be analyzed by the processor 143.

[0041] The processor 143 can also communicate with a motor controller 142 to control the location of the transducer assembly 110. The motor controller 142 controls one or more of the X / Y / Z motors 150 (again, not all of the X, Y, and Z motors are required) to move the transducer assembly 110 relative to the container plate 120. As shown, the X / Y / Z motors 150 are coupled (directly or indirectly) to the transducer assembly 110, although these or other motors may be coupled (directly or indirectly) to the container plate 120 and / or the receiving plate 130 to control the relative movement between the transducer assembly 110, the container plate 120, and / or the receiving plate 130. The processor 143 can control one or more motors 150 to position the transducer assembly 110 directly beneath a given container 122 in the container plate 120 (e.g., the transducer assembly 110 is centered relative to the center of the given container 122) and then move the transducer assembly 110 to directly beneath another given container 122 in the container plate 120. Alternatively, or in addition, the container plate and / or receiving plate 130 may be translated in one or more of the X, Y, and Z dimensions using motors or other translation devices.

[0042] In some embodiments, the ADE system 100 may include a temperature sensor (not shown) that may be located in the coupling liquid 160 in the area between the vessel plate 120 and the receiving plate 130 or elsewhere. The temperature sensor circuitry 141 receives signals (e.g., electrical or wireless) from the temperature sensor and communicates them with the processor 143 so that temperatures (e.g., of the coupling liquid 160, vessels 122, samples 101, air temperature) may be measured.

[0043] In some embodiments, the transducer assembly 110 can have a cylindrical shape. In some examples, instead of using a single transducer 112 for both transmitting and receiving acoustic signals, the transducer assembly 110 can include separate transmitter and receiver transducers, as disclosed, for example, in U.S. Pat. No. 10,787,670, which is incorporated herein by reference in its entirety. According to one technique, the receiving transducer can substantially surround the transmitting transducer and the acoustic lens.

[0044] FIG. 3 shows a representation of the movement of the transducer assembly 110 relative to the reservoir plate 120 when performing ADE on multiple samples 101. The transducer assembly 110 is moved along the x-axis from reservoir to reservoir 122. The transducer assembly 110 can also be moved along the y-axis to additional reservoirs 122 (not shown), as further described with respect to FIG. 5. For each reservoir 122, the transducer assembly 110 can be substantially centered beneath the reservoir 122. The transducer assembly 110 can move vertically along the z-axis to emit and receive acoustic signals at different z-positions beneath the reservoir 122. The transducer assembly 110 can be positioned along the z-axis to focus a beam 170 onto the surface 103 of the sample 101 to eject the ejected liquid 102 (ADE). As further described below, the transducer assembly 110 is positioned along the z-axis and focuses the beam 170 at a predetermined height relative to the container plate 120, allowing for the destruction of bubbles, for example, bubbles located at the interface between the container 122 and the sample 101, without performing ADE.

[0045] FIG. 4 shows a top view of a vessel plate 120 having a plurality of vessels 122. The vessel plate 120 shown is a 384-well microplate (e.g., a polypropylene microplate designated as 384-PP). FIG. 5 shows a top view of the plurality of vessels 122 and an exemplary pattern (a serpentine pattern) for performing ADE and / or other ultrasonic techniques (such as bubble destruction) on each vessel 122 and the samples 101 therein, as described with respect to FIG. 3. In this example, a motor 150 moves the transducer assembly 110 along the x- and y-axes to position it under the various vessels 122. Any other suitable pattern may be used (e.g., a raster pattern). It may also be possible to move the vessel plate 120 or a combination of the vessel plate 120 and the transducer assembly 110 to achieve similar results.

[0046] 6 shows a container 122 (or well) that holds a sample 101, which has a surface 103 (i.e., an upper or free surface). A bottom wall 123 of the container 122 defines a bottom-top boundary surface (TB) 124 and a bottom-top boundary surface (BB) 125.

[0047] FIG. 7 shows the reflected signal and corresponding envelope, which is received at the transceiver assembly 110 in response to the emitted signal. BB indicates a reflection from BB 125. TB indicates a reflection from TB 124. SR (surface reflection) indicates a reflection from the surface 103 of the sample 101. The overall time for a reflection to appear is referred to as the time of flight, or TOF. For each reflection BB, TB, SR in FIG. 7, there is a different individual TOF. When the distance between the transducer and the BB, TB, or surface 103 is generally known, a specific reflection can be identified within the overall reflected signal based on the TOF.

[0048] FIG. 8 shows an image from a scanning electron microscope of the interior surface of an exemplary container 122, which contains TBs 124. Surface texturing on the order of 5-10 μm can be seen (light-colored, irregularly shaped). It has been observed that such texturing does not appear on all containers 122, suggesting that the plate 120 may be affecting the containers 122. As shown in FIG. 11, affected containers are referred to as affected containers 126. The affected containers 126 (which may be a subset of containers 122) may occur in locations A11, A14, P11, P14, B11, B14, O11, or O14, although other locations are also possible. Such locations may be near or adjacent to the injection molding port through which the container plate 120 is formed. It has been observed that there may be one to six affected containers 126, although it is possible that the plate 120 may have more affected containers 126. Such texturing may be caused by separation of plastic components during molding and / or molding process parameters (e.g., temperature and pressure gradients). However, the techniques described herein are not necessarily specific to the texture shown in Figure 8. Instead, the techniques described herein are applicable to a variety of conditions that may benefit from bubble remediation measures.

[0049] As shown in Figures 9A and 9B, texturing may occur only in the affected container 126. Figure 9A shows a microscopic view of the TB 124 of an unaffected container 122 containing sample 101. Figure 9A shows that there are few, if any, air bubbles present. Figure 9B shows a similar microscopic view of the TB 124 of an affected container 126 containing sample 101. In Figure 9B, it can be seen that air bubbles 104 have formed in the TB 124. As used herein, the affected container 126 is also a type of container 122.

[0050] Air bubbles 104 have been observed in affected containers 126 filled with samples 101, including, for example, Milli-Q® water, 1x phosphate buffered saline (PBS), critical micelle concentration of Triton® X-100 ≦14% in 1x PBS, or 70%-80% dimethyl sulfoxide in water. The air bubbles 104 may have diameters of 1.5-5 μm, although as will be appreciated, the techniques described herein are not limited to specific air bubble 104 sizes.

[0051] Air bubbles 104 in the TB 124 can affect the acoustic ejection process. For example, the air bubbles 104 can increase acoustic reflection from the TB 124. Referring back to FIG. 7 , the acoustic energy beam refracts through the BB 125 and the TB 124 and reaches the surface 103 of the sample 101. Once at the surface 103, a liquid bulge is created, and then the ejected liquid 102 is ejected. However, in an affected container 126, the air bubbles 104 can cause a larger-than-normal reflection from the TB 124. This can be due to a larger acoustic impedance mismatch between the affected container 126 material (e.g., polypropylene) and the gas trapped within the air bubbles 104 compared to the acoustic impedance mismatch between the container 126 material and the sample 101 without the air bubbles 104. In the affected container 126, the bubbles 104 may not cover 100% of the area, and the acoustic energy beam may intersect the TBs 124, but the bubbles 104 may be relatively evenly distributed across the TBs 124. The reflected signal may include reflections from areas of the TBs 124 where no bubbles 104 are present and areas of the TBs 124 where bubbles 104 are present. For reflections from portions of the beam that do not intersect the bubbles 104, the reflected energy may be of a typical amplitude expected from the TBs 124. For reflections from portions of the beam that intersect the bubbles 104, the emitted signal may actually strike two interfaces: first, the TBs 124 / gas interface at the bottom surface of the bubble 104 attached to the TBs 124, and second, the gas / fluid interface at the top of the bubble 104. Reflections from these two interfaces can be nearly simultaneous, given that they are separated in time by approximately tens of nanoseconds. The combined reflection due to the bubble 104 can be substantially greater in energy than the reflection from the TB 124 without any bubble 104.

[0052] It has been observed that the energy reflected from the TB 124 in an affected container 126 containing a sample 101 is three times higher than in an unaffected container 122 containing a sample 101. In some cases, the energy received from the TB 124 at the transducer assembly 110 can be high enough to saturate the signal receiving circuitry 145 (exceeding the acceptable operating range). This can cause problems such as false identification of an empty container 122. When the energy reflected from the TB 124 saturates (or is otherwise high enough to) the signal receiving circuitry 145, this can identify (or assist in identifying) an empty container 122. However, if an air bubble 104 is present, a given container 122 can be erroneously identified as not containing a sample 101. Another problem with affected containers 126 can be incorrect assessment of the sample 101 for calibration purposes, which takes into account the impedance signatures of the container 122 and / or the sample 101.

[0053] An additional potential problem may be that the bubble 104 may attenuate or reduce the signal directed toward the surface 103 of the sample 101 during the bulge imaging process (MIP) and droplet ejection. The reduction may be due to increased reflection, energy dispersion, and / or energy used in bubble destruction. For example, ADE may use MIP to determine the appropriate power amplitude for droplet ejection, while the duration of the ejected acoustic signal may be fixed. The bubble 104 may require the transducer assembly 110 to emit higher energy or power due to power losses in the TB 124 (e.g., energy lost due to reflection, dispersion, and any energy used in bubble 104 removal). As acoustic power continues to be projected onto the TB 124, the bubble 104 tends to burst, and acoustic attenuation begins to decrease. As a result, over time, more acoustic power or energy reaches the surface 103 of the sample 101, thereby reducing the accuracy of ADE due to the time-varying nature of the signal.

[0054] One known technique for addressing the interference of the gas bubble 104 is to reduce the amplitude of the emitted signal (e.g., interrogation ping). However, the reflection from the TB 124 in the affected vessel 126 may remain higher than in the unaffected vessel 126, vessel 122. This may mean that challenges to signature calibration are not addressed. Signature calibration relies on the TB 124 reflection and the BB 125 reflection to determine properties of the bottom wall 123 and sample 101 (e.g., the acoustic impedance of the sample 101) as they relate to ADE.

[0055] As an example, assume a first amplitude for the emitted signal and a peak reflection from BB 125 (1,000 counts), a peak reflection from TB 124 in unaffected container 122 (1,000 counts), and a peak reflection from TB 124 in affected container 126 (3,000 counts) (counts are increments on an analog-to-digital (A / D) converter). As used herein, "counts" refers to the envelope value being fitted. In this example, the A / D converter has a maximum range of 2,048 counts; therefore, a signal of 3,000 counts would saturate the A / D converter. If the amplitude of the emitted signal is reduced to a second value, the peak amplitude of the reflected energy can be halved (500, 500, and 1,500 counts from BB 125, TB 124 in unaffected container 122, and TB 124 in affected container 126, respectively). In this embodiment, the A / D converter may no longer be saturated by the peak amplitude of the reflected signal from the TB 124 in the affected vessel 126. However, the ratio of the peak amplitude reflection from the TB 124 to the peak amplitude reflection from the BB 125 in the affected vessel may still be relatively high (3x), which may result in incorrect identification of fluid properties by signature.

[0056] Additionally, even if the affected container 126 cannot be considered empty and the process proceeds to ADE transfers, these transfers may suffer from incorrect volume and / or poor placement because the instability of the “zero velocity droplet” (ZVD) (e.g., not repeatedly measuring to be substantially constant) is not addressed. MIP can enable calculation of the ZVD (the amplitude of the acoustic signal required to just break off the droplet 102, but without the energy required to separate the droplet 102 from the surface 103 of the sample 101). Thus, the droplet 102 may fall back into the sample 101. Once determined, the ZVD value may be scaled to the amplitude required to completely eject the droplet 102 from the sample 101 and container 122. ZVD instability may be caused by changing attenuation as the bubble 104 collapses. If the bubble 104 is substantially absent from the TB 124, MIP can yield a stable value. Similarly, if a gas bubble 104 is present in TB 124, acting as an attenuator, but the gas bubble 104 is not destroyed, the MIP may determine the ZVD to be a higher value (to compensate for the attenuation), but the system may still be able to consistently (and reproducibly) eject droplets 102. Instability may arise from the fact that the MIP assesses the system with an additional attenuator (in this case, gas bubble 104), but the attenuation of this additional attenuator is changing (gas bubble 104 is destroyed). As a result, the ZVD may become too high, and droplets 102 may be unintentionally ejected.

[0057] FIG. 10 illustrates a sequence 1000 for bubble 104 remediation, according to one embodiment. In sequence 1000, transducer assembly 110 may emit acoustic energy beam 170 having a focal length (e.g., between 6 mm and 51 mm, such as 25 mm). Transducer assembly 110 may define a constant focal length (i.e., the focal length cannot be changed), or the focal length may be adjustable. In steps 1010, 1020, and 1030, the focal length of acoustic energy beam 170 extends to a common focus at a predetermined height, such as a predetermined height above and / or relative to TB 124. Such a predetermined height may be between −4 mm and +4 mm below or above TB 124, such as +2.5 mm above TB 124. It may be possible to vary the focal height (or otherwise change the focal length) between steps 1010, 1020, and 1030. It may be beneficial to adapt the position and / or shape of the acoustic energy beam 170 (either by z-axis movement of the transducer assembly 110 or by varying the focal length with the transducer assembly 110). For example, it may be beneficial to match or correlate the cross-section of the acoustic energy beam 170 with the surface area of ​​the TB 124. In steps 1040, 1050, the focal point of the acoustic energy beam 170 is shifted to the surface 103 of the sample 101. Alternatively, the focal point may be between 4 mm above the surface 103 and 16 mm below the surface 103.

[0058] In step 1010, the transducer assembly 110 emits a first ping within the acoustic energy beam 170. The ping may be a type of acoustic signal that may have a relatively short duration (e.g., 5 ns to 200 ns, such as 40 ns) and relatively low energy (e.g., 0.1 μJ to 10 μJ, such as 1 μJ). The ping may have a peak power of up to 100 watts. The ping may include acoustic energy transmitted in a broad spectrum (e.g., a 12 MHz center frequency and a 6 MHz bandwidth). To generate the data shown in FIGS. 16, 17, and 20-23, a ping having a single cycle of a 2.25 MHz square wave was used. The TB 124 reflects a portion of the first ping as a first TB reflection (not shown). The transducer assembly 110 receives the first TB reflection, and the information may be processed by the processor 143. The reflection from the first ping may indicate the presence of a bubble. Turning to Figures 16A-16C, illustrative examples of how the presence of air bubbles can be determined by processing reflections from pings are shown. Figure 16A is an illustrative example in which substantially no air bubbles are present. In this case, the TB reflection is relatively low and the SR reflection is relatively high. Figures 16B and 16C are additional illustrative examples in which an increasing number (concentration) of air bubbles are present. As the number of air bubbles increases, the peak amplitude of the TB reflection increases and the peak amplitude of the SR reflection decreases.

[0059] Returning to FIG. 10 , in step 1020, the transducer assembly 110 emits a first bubble destruction signal. Such a signal may be longer in duration than the first ping or second ping (discussed below). The first bubble destruction signal may be referred to as a dimple. The first bubble destruction signal duration may be 8 μs to 500 μs, such as 90 μs. The first bubble destruction signal may have a peak power of 10 W to 50 W, such as 20 W peak power, and a smaller amplitude than the first ping or second ping. The first bubble destruction signal may have a varying frequency, such as a chirp. The chirp may have a programmable varying pattern. The varying frequency may vary from a low frequency of, for example, 0.5 MHz to a high frequency of, for example, 20 MHz, with an exemplary range of 6 MHz to 14 MHz. The first bubble destruction signal has a total energy that may exceed that of the first ping or the second ping (described below). The total energy of the first bubble destruction signal may be between 0.1 mJ and 10 mJ, such as 2 mJ.

[0060] The first bubble destruction signal may be selected to destroy (or pop) at least some of the bubbles 104. FIG. 10 shows such destroyed bubbles as popped bubbles 105, which are solid black circles. The first bubble destruction signal may impart energy to the bubbles 104 in the TB 124. One bubble destruction mechanism may be bubble cavitation. Energy input to a given bubble 104 at an associated frequency results in the expansion of the bubble 104. As the bubble 104 radius increases, surface tension may no longer be sufficient to sustain the bubble 104. The bubble 104 may increase in size until the signal is stopped, at which point the bubble 104 may break up into many substantially smaller bubbles (i.e., bubble destruction). Another bubble destruction mechanism may be through the bubble being pushed upward and removed from the TB 124.

[0061] According to an embodiment, steps 1010 and 1020 may be repeated before step 1030, either in the same manner or with varying parameters as discussed above.

[0062] In step 1030, the transducer assembly 110 emits a second ping within the acoustic energy beam 170. The second ping may be the same or substantially the same as the first ping, e.g., the peak amplitudes of the first and second pings may be substantially the same. Alternatively, the second ping may be different from the first ping. For example, different ping amplitudes may be useful if the calibration covers a wide range of sample 101 types, with some samples 101 causing relatively small TB reflections and some causing relatively large TB reflections. Using different ping amplitudes during this process may provide useful information for other processes while still being compatible with bubble 104 detection.

[0063] The TB 124 reflects a portion of the second ping as a second TB reflection. The transducer assembly 110 receives the second TB reflection, and the information may be processed by the processor 143. Because some bubbles 104 were popped by the first bubble-breaking signal in step 1020, a smaller fraction of the acoustic energy may be reflected from the TB 124 than that associated with the first ping in step 1010.

[0064] The processor 143 compares the first TB reflection (from step 1010) and the second TB reflection (from step 1030) by comparing at least one characteristic for each. Examples of such a characteristic include overall energy or peak amplitude. If the characteristic for each is peak amplitude, such peak amplitude may be for the signal itself or a corresponding envelope for the signal, such as a Hilbert envelope. If at least one characteristic in the second TB reflection differs from what is expected (e.g., different from what would be the case in the absence of an air bubble 104 or other type of interference), the processor 143 infers that an air bubble 104 is present at the TB 124. For example, the first ping and the second ping may be the same. Thus, theoretically, the first TB reflection and the second TB reflection should be the same under ideal circumstances. However, if the second TB reflection is less than the first TB reflection (e.g., has a lower peak amplitude or overall energy), then based on the reflectivity principles described above, it can be inferred that an air bubble 104 is present at the TB 124. In this example, the second TB reflection was expected to be the same as the first TB reflection in the absence of an air bubble (or other interference). The criterion or criteria for inferring the presence of an air bubble 104 may vary from situation to situation, such as the calibration of a given system.

[0065] Examples of such a criterion / criteria for inferring the presence of an air bubble 104 are various ratios such as TB″ / TB′ or (TB″ / BB″) / (TB′ / BB′), where BB′ and TB′ are the reflections from BB 125 and TB 124 from the first ping, and BB″ and TB″ are the reflections from BB 125 and TB 124 from the second ping. If such a ratio is below a given threshold (e.g., 0.6-0.95), the presence of an air bubble 104 can be inferred.

[0066] If, in step 1030, the presence of an air bubble 104 in the TB 124 is suspected, the flowchart 1000 proceeds to step 1040. Otherwise, the sequence 1000 may skip step 1040 and proceed to step 1050. According to an embodiment, steps 1020 and / or 1030 may be repeated before step 1040, either in the same manner or with varying parameters as discussed above.

[0067] In step 1040, a second bubble-breaking signal is emitted by the transducer assembly 110. The second bubble-breaking signal may be selected to break substantially all of the bubbles 104 (or a substantial portion thereof) in the TB 124. Embodiments of the second bubble-breaking signal are described below in the context of Figures 12, 13, and 15.

[0068] According to one embodiment, the second bubble destruction signal includes a preconditioning signal and an adjustment signal. Each may occur using an acoustic energy beam 170 focused on the surface 103 of the sample 101 (e.g., above the focal height in steps 1010, 1020, and 1030). As the transmission described in step 1050 may occur using the acoustic energy beam 170 focused on the same location, any bubbles 104 in the TB 124 within the path of the beam 170 may be destroyed prior to transmission. The preconditioning signal may destroy some, but not all, of the bubbles 104. The preconditioning may reduce the influence of the bubbles 104 so that the rate of change of the influence of the bubbles 104 is not too large during the adjustment signal, causing excessive force (enough to accidentally eject a droplet), before being recalculated in the next iteration. One possible, but not necessarily inevitable, consequence of skipping preconditioning is that during the conditioning process (discussed below), the energy reaching the surface 103 due to "false transfer" may increase as bubbles 104 are destroyed, and the energy may increase to a level where droplets 102 are unintentionally ejected.

[0069] The preconditioning signal may have an amplitude and frequency selected to prevent liquid from being ejected. The amplitude may be fixed. The amplitude may be predetermined, for example, by sweeping a range of frequencies and amplitudes within the test sample for a given calibration. The preconditioning may include a shorter signal (e.g., of a similar duration to the first bubble collapse signal) emitted repeatedly (e.g., 50 times). During this iteration, the reflection of acoustic energy from the TB 124 may decrease until it stabilizes at a substantially constant value (e.g., within an acceptable range). As the bubble 104 continues to collapse at the TB 124, less and less acoustic energy is reflected, for reasons discussed above. The TB reflection may be monitored by the system 100, for example, to determine when such reflection repeats at a substantially constant value (e.g., within an acceptable range). If stabilization of the TB reflection occurs before the maximum number of iterations, the preconditioning signal may be terminated. 14 illustrates the stabilization of the reflection from the TB 124 in an exemplary affected vessel 126 as the iterations continue. The y-axis represents the ratio of the peak amplitude of the reflection from the TB 124 to the peak amplitude of the reflection from the BB 125. The reflection from the BB 125 can be substantially constant throughout the bubble 104 destruction process.

[0070] The conditioning signal may be used to remove substantially any remaining air bubbles 104 in the TB 124. The preconditioning may not use a substantially maximum amplitude of the acoustic energy so that ejection may be avoided. Instead, the preconditioning may use a fixed amplitude, as discussed above. The conditioning, in contrast, may involve a process in which the maximum amplitude is experimentally determined for each container 122, and then the acoustic energy is emitted at substantially the maximum amplitude.

[0071] The influence of the remaining air bubbles 104 may be a factor in the bulge imaging process (MIP). The adjustment signal may stabilize the bulge imaging solution. The adjustment signal may include bulge imaging followed by multiple "false transfers" of droplets (e.g., 20 droplets falsely ejected) without scaling the ZVD to actually eject liquid. The process of bulge imaging followed by multiple false transfers (no droplets ejected) is repeated until the measured ZVD value stabilizes to a substantially constant value (e.g., within an acceptable range). According to one embodiment, for a given iteration, bulge imaging is performed followed by 20 false transfers, although more or fewer false transfers are possible. If the ZVD value stabilizes before the maximum number of iterations (e.g., 20 sequences of (bulge imaging + multiple false transfers)), the adjustment signal may be terminated. The stabilization of ZVD over 20 iterations in an exemplary affected vessel 126 is shown in FIG. 15. The y-axis represents the ZVD value calculated from each MIP measurement. For each iteration, the surface 103 is perturbed with repeated signals of increasing amplitude, and the raised bulge is measured by measuring the width of the returning surface reflection (SR). The width of the SR corresponds to the size of the bulge. Once a predetermined size is reached, the ZVD can be calculated, i.e., the amplitude required to theoretically detach a droplet so that it is not ejected and falls back into the sample 101. For ADE, the amplitude of the acoustic signal is increased to a value above the ZVD so that a droplet would be ejected. In contrast, pseudotransfer is performed using an amplitude that is the experimentally determined ZVD so that a droplet cannot be ejected.

[0072] As part of the bulge imaging process (MIP), a maximum amplitude above which a droplet will be ejected may be determined using a maximum amplitude determination signal. The maximum amplitude determination signal may include a first disturbance signal, a subsequent first measurement signal, a subsequent second disturbance signal, and a subsequent measurement signal. The first disturbance signal may be selected to disturb the upper surface of the sample but not eject liquid. The peak amplitude and duration of the first disturbance signal may be selected according to an experimentally determined range for a representative fluid. The selected peak amplitude may be slightly below the experimentally determined range. The subsequent first measurement signal may be selected to be reflected by the upper surface of the sample, whereby the reflection is processed by the processor 143 to quantify the effect of the disturbance, for example, the size of the bulge created by the first disturbance signal. The ZVD may be determined based on the size of the bulge compared to the amplitude of the first disturbance signal. The second disturbance signal has a peak amplitude greater than the peak amplitude of the first disturbance signal. The peak amplitude may be increased by a value in the range of 0.02 to 1 dB, such as 0.15 dB. Like the first measurement signal, the second measurement signal may be selected to be reflective so that the processor 143 can quantify the effect of the disturbance, for example, the size of the bulge created by the second disturbance signal. Based on the size of the bulge compared to the amplitude of the second disturbance signal, the ZVD may be determined. Based on a measurement of the bulge size versus the disturbance signal amplitude, the ZVD can be determined. The disturbance and measurement signals may be repeated, for example, with gradually increasing peak amplitudes of the disturbance signal. There may be a total of 1 to 50 pairs of sets (e.g., 10 pairs) of disturbance and measurement signals. It may also be possible to reduce the peak amplitude if excessive force is detected (e.g., if a droplet may begin to detach from the surface 103).

[0073] During false transfer, a bubble mitigation signal is employed with a peak amplitude determined according to a previously identified maximum amplitude. For example, the peak amplitude may be equal to or less than the maximum amplitude (e.g., 0 dB to 10 dB less, such as 0.5 dB less than the maximum amplitude). The bubble destruction signal may include multiple such signals (e.g., two signals), each having a peak amplitude determined according to a previously identified maximum amplitude. The duration of such signals may be 8 μs to 500 μs, such as 150 μs.

[0074] The bubble mitigation signal may have a peak amplitude greater than the maximum amplitude. Depending on the application, it may be acceptable to eject a droplet as long as it does not have enough energy to hit the target or interfere with the assay. For example, the amplitude may be zero to 0.5 dB above the ZVD.

[0075] Another embodiment of the adjustment signal is illustrated in FIG. 13 in flowchart 1300. In step 1310, bulging imaging is performed. In step 1320, multiple false transfers are performed. In steps 1330, 1340, and 1350, bulging imaging and multiple false transfers (e.g., 20 false transfers) are repeatedly performed until the ZVD measurement stabilizes (e.g., within an acceptable tolerance). Steps 1330, 1340, and 1350 may each be repeated at least a predetermined number of times (e.g., three times) until the ZVD measurement stabilizes thereover. Alternatively, or in addition, steps 1330, 1340, and 1350 are repeated a predetermined maximum number of times (e.g., 10 times), regardless of whether the ZVD measurement stabilizes the predetermined number of times. In step 1360, ADE is performed based on the determined ZVD.

[0076] 10, in step 1050, the transducer assembly 110 emits a signal to eject a droplet 102 from the sample 101 in accordance with ADE principles. Some examples of ADE are disclosed in U.S. Publication No. 2021 / 0394171 or PCT Publication No. WO2020 / 092407 (both of which are incorporated herein by reference in their entireties).

[0077] According to an embodiment, a third ping may be emitted before step 1050. The third ping may be similar to the first and / or second ping. The signal reflected from the third ping may be processed similarly to the first and / or second ping in steps 1010, 1030 so that it may be inferred whether the bubble has been substantially destroyed. If the bubble has not been substantially destroyed, steps 1020 and / or 1040 may be performed again, either in the same manner or with varying parameters as described above. Subsequent ping steps and bubble destruction steps may be performed any number of times as desired or according to design.

[0078] FIG. 12 shows a flowchart for a method 1200 for removing air bubbles 104, according to an embodiment. In step 1210, each container 122 is surveyed for air bubbles 104. For each container 112, a transducer assembly 110 may be positioned directly below the given container 112. As another option, containers 122 may not be surveyed, but containers 122 at risk of being affected containers 126 (e.g., A11, A14, B11, B14, O11, O14, P11, or P14, as shown in FIG. 11 ) may be designated. Step 1210 may correspond to steps 1010 and / or 1030, described in the context of FIG. 10 . As described in the context of FIG. 10 , the processor 143 may be able to determine the containers 122 having samples 101 with air bubbles 104 (i.e., affected containers 126) by processing the information gathered in steps 1010 and 1030.

[0079] Preconditioning 1220 and conditioning 1230 may be performed for all affected containers 126 (and / or potentially affected containers) identified in step 1210. It may be possible to perform only preconditioning 1220, instead of both preconditioning 1220 and conditioning 1230, for affected containers 126 or each potentially affected container. It may also be possible to perform preconditioning 1220 and / or conditioning 1230 on one or more containers 122 that are not affected containers 126. After bubble removal in steps 1220 and / or 1230, the process proceeds to step 1240, where transfer (ADE) is performed for each container 122. It may also be possible to perform steps 1210, 1220, and / or 1230 for a plate 120 prior to performing step 1240. Optionally, it may be possible to perform steps 1210, 1220, 1230, and / or 1240 on a given container before proceeding. Step 1240 may correspond to step 1050 .

[0080] FIG. 19 shows a flowchart 1900 for a method of destroying bubbles, according to an embodiment. The method may be implemented by components (e.g., processor 143 and transducer assembly 110) described in the context of FIGS. 1 and 2. The method may be similar to those disclosed in FIGS. 10, 12, and / or 13. It may not be necessary to perform all steps. For example, steps 1910 and / or 1950 may be bypassed and / or omitted. Additional steps may be performed partially or completely in parallel. For example, steps 1910 and 1920 may be performed in parallel. The method corresponding to flowchart 1900 may be used for bubble destruction techniques in which no ADE is present, such as the transfection embodiment described below. For example, preconditioning 1220 and / or conditioning 1230 as described in the context of FIG. 12 may not be used.

[0081] In step 1910, characteristics of the sample may be identified. Such characteristics may include the presence and / or volume of liquid in the sample. Such identification may be determined by implementing techniques such as identifying reflections from physical sample features, including BB, TB, and SR, collected in a pulse-echo ultrasound signal. For example, the volume of the sample may be determined by measuring the time of the SR reflection and knowing the dimensions of the container (which may be standard for a given type of plate).

[0082] In step 1920, the number of bubbles is assessed by inference or estimation. Step 1920 may be similar to steps 1010 or 1030 described in conjunction with FIG. 10 . A ping may be transmitted from the transducer assembly 110, and the reflected signal may be assessed. The number of bubbles may be estimated based on the amount of reflected energy from the TB and / or SR. The degree of reflection may be based on the peak amplitude from the TB reflection and / or the peak amplitude from the SR reflection. Examples of how the energy of the TB and SR reflections varies based on the number of bubbles in the sample are discussed below in the context of FIGS. 16A-16D . The reflected energy may be used by the processor 143 as input to an equation, lookup table, or machine learning model to estimate the amount of bubbles based on previous experimental evaluation of the sample and bubble volume. The number of bubbles in the sample may be estimated as an absolute number or concentration.

[0083] In step 1930, a bubble destruction signal may be emitted by transducer assembly 110. Step 1930 may be similar to step 1020 and / or step 1040. In step 1940, the effectiveness of step 1930 in destroying the bubbles is assessed. Step 1940 may be similar to step 1920 or steps 1010 and / or 1030 described in the context of FIG. 10 . The effectiveness of step 1930 may be determined by comparing the estimated number of bubbles to a threshold or some other predetermined assessment method. If a sufficient number of bubbles are destroyed in step 1930, the process may move to the next vessel in step 1960 (e.g., by moving transducer assembly 110 or plate 120). Alternatively, acoustic droplet ejection may be performed before moving to the next vessel in step 1960. Acoustic droplet ejection may be performed after all relevant samples have had bubbles destroyed or before moving to the next vessel. ADE may or may not be performed with certain transfection techniques. ADE may, for example, be performed after the bubbles have substantially collapsed.

[0084] On the other hand, if a sufficient number of bubbles have not been destroyed, flowchart 1900 may proceed to step 1950. Here, processor 143 may determine parameters of a new bubble destruction signal, which will be used when step 1930 is repeated. Alternatively, parameters of a new bubble destruction signal may not be determined, and the same bubble destruction signal used in the previous iteration of 1930 may be used again. Steps 1930, 1940, and 1950 (optionally) may be repeated until there is a sufficient rate of removal of bubbles from the sample.

[0085] The embodiments described in conjunction with Figure 19 (as well as Figures 10 and 12) may be used to automatically identify when a given sample has a different (higher or lower) concentration of bubbles than expected. Such identification may occur in steps 1920 and / or 1940. Subsequent bubble destruction signals may be adjusted to compensate for any such variations.

[0086] Figures 16A-16C illustrate examples of ping reflections when different amounts of bubbles are present. Pings are emitted toward the sample as described in the context of Figures 10 and 19. In Figure 16A, the sample has a lower number of bubbles, and the amount of bubbles gradually increases in Samples 16B and 16C. The y-axis is shown in counts (counts are increments on an analog-to-digital (A / D) converter), but is essentially arbitrary and intended to provide a way to objectively compare each of Figures 16A-16C with each other. The x-axis is time. As can be seen in each figure, the peak amplitude of the BB reflection is the same. However, the peak amplitude of the TB reflection increases as the number of bubbles increases in the sample. With regard to transfection (discussed below), many bubbles may be attached to cells, which are heavier than the bulk liquid in the sample and therefore tend to settle down to or toward the TB. The bubbles form gas pockets, and where the gas pockets are located, more acoustic energy will be reflected. Furthermore, as the number of bubbles increases, the peak amplitude of the surface reflection (SR) decreases. This is in part because more energy is reflected at the TB and never reaches the surface of the sample. The reduction in the peak amplitude of the surface reflection may also be caused by bubbles suspended in the bulk liquid of the sample and possibly by the relatively chaotic or irregular surface geometry resulting from bubbles floating on the surface.

[0087] By assessing the energy reflected from the TB and / or the surface of the sample, it may be possible to infer the amount of bubbles in the sample, as discussed above. For example, it may be possible to estimate the amount of bubbles using a lookup table based on experimental data. As another example, a machine learning model may be used. Training such a model may involve using training data including the transmitted signal waveform, the voltage supplied to the transducer assembly, the reflected signal peak amplitude (e.g., from the surface and TB of the sample), the reflected signal waveform, the sample bulk liquid composition, the number of cells in a given sample, the type of cells in the sample, the volume of the sample, and / or the dimensions of the container.

[0088] Additionally, bubbles are known to oscillate at specific resonant frequencies related to the size and composition of individual bubbles. Such compositions may include the type of gas inside the bubble and the material (e.g., lipids, proteins, synthetic polymers, or the like) within the bubble shell. Both the density and compressibility of the gas and the elasticity and surface energy of the shell can play a role in modifying the bubble's resonant frequency. This resonant frequency can affect how the bubble absorbs or reflects the ultrasound signal. Frequency analysis of the reflected waveform can be processed to identify shifts in the reflected and / or absorbed frequency spectrum, which can further infer characteristics of the bubbles in the sample and thereby provide information about the size distribution of bubbles within a specific container (e.g., different sizes of bubbles and the number of each size, or a size-to-volume curve). Such information can also be processed by conventional algorithms, lookup tables, or used to train machine learning models. Such trained models can be used to determine the size and composition of bubbles using subsequent waveforms or features extracted therefrom.

[0089] 17A-17D show illustrative examples of signals reflected from pings after different bubble-breaking signals are emitted. These figures are similar to FIGS. 16A-16D in that they show signals reflected from pings. However, FIGS. 17A-17D illustrate the nature of the reflected pings based on the number of bubbles destroyed in the previous step. In each example, the same number of bubbles was originally present in the sample before the bubble-breaking signal was transmitted by the transducer assembly 110. FIG. 17A shows the baseline condition of the signal reflected from the vessel containing the sample when no bubble-breaking signal was transmitted. In FIG. 17B, the transducer assembly 110 was provided with an RF electrical signal having a peak voltage of 12.5% ​​of the maximum that can be generated by the signal transmission circuitry 144 (see FIG. 1). The reflections from the pings indicate that some of the bubbles in the sample have been destroyed, since the peak amplitude of the TB reflection has decreased and the peak amplitude of the SR reflection has increased. In FIG. 17C, the transducer assembly 110 was provided with an RF electrical signal by the signal transmission circuitry 144 having a peak voltage of 25% of the maximum value. As can be seen, the peak amplitude of the TB reflection is lower than that in FIGS. 17A and 17B. Furthermore, the peak amplitude of the SR reflection is higher than that in FIGS. 17A and 17B. This indicates that more bubbles were destroyed by the bubble-destroying signal generated by the RF electrical signal having a peak voltage of 25% of the maximum value than when using RF electrical signals having peak voltages of 0% or 12.5% ​​of the maximum value. In FIG. 17D, the transducer assembly 110 was provided with an RF electrical signal by the signal transmission circuitry 144 having a peak voltage of 50% of the maximum value. As can be seen, the peak amplitude of the TB reflection is lower than that in FIGS. 17A, 17B, and 17C. Furthermore, the peak amplitude of the SR reflection is higher than that in FIGS. 17A, 17B, and 17C. This indicates that more bubbles were destroyed by the bubble destruction signal generated by the RF electrical signal having a peak voltage of 50% of the maximum value than when using RF electrical signals having 0%, 12.5%, or 25% of the maximum value.

[0090] According to embodiments, the bubble remediation process described herein can be used in conjunction with transfection procedures. Transfection refers to the process of intentionally introducing genetic material, including (but not limited to) messenger RNA (mRNA), short interfering RNA (siRNA), or plasmid DNA (pDNA), into eukaryotic cells. One method of performing transfection involves sonoporation. Sonoporation refers to the use of sound in the ultrasonic range to increase the permeability of cell plasma membranes. Increasing cell plasma membrane permeability allows for the uptake of large molecules, such as DNA, into cells, as desired during transfection. Sonoporation employs acoustic cavitation of microbubbles to improve the delivery of these large molecules. The bubbles used for transfection may have a diameter of, for example, 5 μm.

[0091] Information generated by assessing and / or controlling the amount (number and / or concentration) of bubbles in a sample can have certain advantages that can be used for the transfection process. For example, this can be useful for quality control. One advantage is verifying sufficient bubble attachment to cells prior to sonoporation, potentially using, for example, SR and / or TB reflectance information to distinguish between free and bound bubbles and assess (e.g., quantify) the binding efficiency in a given sample. A further advantage is verifying the appropriate rate of bubble removal. This can help identify any issues with the transfection system's software and / or hardware and / or reduce the occurrence of unexplained and / or poor results. A further advantage allows for verification that the appropriate sample (including cells, liquid, and bubbles) is in the expected container. This can flag and / or allow for correction of pipetting errors that would otherwise result in experimental failure.

[0092] The techniques used herein may be used for real-time and / or closed-loop processes to improve transfection results. This may allow for dynamic adjustment of the number, amplitude, and / or duration of bubble destruction signals to suit individual samples or containers. This may provide additional flexibility when evaluating unknown samples, and may provide improved process reliability by allowing for dynamic adjustment of the number, amplitude, and / or duration of bubble destruction signals to compensate for variations in bubble density resulting from sample preparation for sonoporation.

[0093] As shown below, data collected during acoustic transfection shows a correlation between the TB reflection signal, the initial bubble density, the amplitude of the bubble destruction signal, and the number of bubble destruction signals applied.

[0094] Furthermore, when ADE is used to eject droplets from a sample, as described herein, it may be useful to substantially eliminate air bubbles after transfection.

[0095] There may be different bubble populations that can be detected using the processes described herein. One population is cell-bound microbubbles. As discussed, these may be localized toward the bottom of the vessel (TB), effectively leading to the formation of a gas layer. This gas may cause a larger reflected signal from the TB compared to the reflection from a substantially bubble-free sample (i.e., no gas). Other microbubble populations may include those suspended in the bulk of the fluid or at the fluid surface. Microbubbles in these locations will not affect the signal from the TB, but may still affect the signal from surface reflection (SR) via acoustic scattering.

[0096] Figure 18 shows four images from a microscope of cells in a sample with attached microbubbles used as part of the transfection process. The first image is a baseline image, with no bubble destruction signal being emitted (0% maximum signal). In subsequent images, the number of bubbles on the cells is reduced by increasing amounts according to the increasing energy of the bubble destruction signal (the peak voltage provided by signal transmission circuitry 144 to transducer assembly 110 increases from 12.5% ​​of maximum for a weaker bubble destruction signal to 50% of maximum for a stronger bubble destruction signal). For these particular examples, the bubble destruction signal had a frequency of 2.25 MHz, a duration of 12 μs, and a sinusoidal shape.

[0097] Figures 20-23 show the effect and effectiveness of certain techniques disclosed herein on a sample containing gas bubbles and mammalian cells. The sample being assessed was a suspension of mammalian cells at various densities with microbubbles attached. Ultrasonic pings were transmitted to the sample by the transducer assembly, and reflections therefrom were received by the transducer assembly. Pings were transmitted, and corresponding reflections were assessed before and after transmission of the bubble destruction signal. The reflected data was interpreted using a custom MATLAB script to read all of the stored data and then determine the peak amplitudes of reflections from the vessel bottom beam, vessel top beam, and sample surface.

[0098] A parameter was used to calculate the bubble removal rate based on the peak amplitude of the TB reflection. This parameter is based on the decrease in TB reflection amplitude after application of a bubble breaking signal to the sample. Removal Rate = (TB initial -TB current ) / (TB initial -TB control ) wherein TB initial is the TB reflection amplitude in the container before the bubble destruction signal is transmitted, and TB current is the real-time TB reflection amplitude, and TB controlis the TB reflection amplitude of a comparable vessel without any microbubbles.

[0099] In addition to the differences observed in the TB reflection signal, there are also differences, likely induced by air bubbles, in the signal reflected by the surface of a given sample. SR reflections could also potentially be assessed, as explained above, but were not considered for the generation of the data depicted in Figures 20-23.

[0100] FIG. 20 shows signal graphs illustrating the reduction of bubbles in different samples according to an embodiment. For three types of samples (low cell density, medium cell density, and high cell density), the peak amplitude of the TB reflection in response to the same ping was measured in counts (counts are increments on an analog-to-digital (A / D) converter), although other measurement metrics could also be considered. The number of cells in a given sample corresponds to the number of bubbles in a given sample, and it is assumed that approximately the same number of bubbles bind to a given cell. A first bubble destruction signal was transmitted, and the peak TB reflection amplitude from the ping was then measured. The magnitude of the first bubble destruction signal is indicated by the voltage supplied to the transducer assembly on the x-axis. The peak TB reflection amplitude is plotted against the magnitude of the first bubble destruction signal in the dashed curve. Additionally, a second bubble destruction signal was transmitted, and the peak TB reflection amplitude from the same ping was then measured. The magnitude of the second bubble destruction signal is represented by the voltage supplied to the transducer assembly on the x-axis. The peak TB reflection amplitude is plotted against the magnitude of the second bubble destruction signal in the solid curve. As can be seen, a larger amplitude bubble destruction signal results in more bubbles being destroyed.

[0101] FIG. 21 shows a graph illustrating the reduction of air bubbles in different samples according to an embodiment. FIG. 21 is similar to FIG. 20, but FIG. 21 shows the percentage of bubble removal. The parameters for the removal rate as shown in FIG. 21 are explained above, i.e., removal rate = (TB initial -TB current) / (TB initial -TB contorl )

[0102] FIG. 22 shows a graph, according to an embodiment, where the x-axis shows transfection efficiency as a percentage and the y-axis shows air bubble removal rate as a percentage. Samples with A549 cells were assessed. The removal rate was measured as described with respect to FIG. 21. The transfection efficiency for the samples was measured using flow cytometry. FIG. 22 shows the correlation between the air bubble removal rate measured during transfection and the percentage of cells successfully transfected with green fluorescent protein (GFP)-encoding mRNA. Transfection efficiency was assessed using flow cytometry to measure the expression rate of GFP in individual cells. A similar correlation was observed with HEK-293 cells, as shown in FIG. 23. These correlations indicate that bubble assessment before and after the disruption technique can be used as a predictor of transfection success for one or more cell types, thereby enabling real-time process improvement through dynamic adjustment of the number, amplitude, and / or duration of bubble disruption signals to correct for insufficient removal rates of microbubbles.

[0103] Many of the embodiments described herein may be implemented on or in conjunction with a computer storage product involving a non-transitory computer-readable medium (which may also be referred to as a non-transitory processor-readable medium) having instructions or computer code thereon for performing various computer-implemented operations, as will be understood. These embodiments may include those involving a processor 143 (or relevant portions of such an embodiment). The medium may include one or more distinct media. The code may execute on one or more processors, such as processor 143 (which may itself include multiple processors). The computer-readable medium (or processor-readable medium) is non-transitory in the sense that it does not itself include a transient, propagating signal (e.g., a propagating electromagnetic wave that carries information over a transmission medium such as space or cable). The medium and computer code (which may also be referred to as code) may be designed and constructed for a specific purpose or purposes. Examples of non-transitory computer-readable media include, but are not limited to, magnetic storage media such as hard disks, floppy disks, and magnetic tape, optical storage media such as compact discs / digital video discs (CD / DVDs), compact disc-read only memories (CD-ROMs), and holographic devices, magneto-optical storage media such as optical discs, carrier wave signal processing modules, and hardware devices specially configured to store and execute program code such as application specific integrated circuits (ASICs), programmable logic devices (PLDs), read-only memories (ROMs), and random access memory (RAM) devices, etc. Other embodiments described herein relate to computer program products, which may include, for example, the instructions and / or computer code discussed herein.

[0104] Some embodiments and / or methods described herein can be implemented by software (executing on hardware), hardware (e.g., processor 143), or a combination thereof. Hardware modules may include, for example, general-purpose processors, field programmable gate arrays (FPGAs), and / or application-specific integrated circuits (ASICs). Software modules (executing on hardware) may be implemented in C, C++, Java, Ruby, Visual Basic, TM , and / or other object-oriented, procedural, or other programming languages ​​and development tools. Examples of computer code include, but are not limited to, microcode or microinstructions, machine instructions such as those produced by a compiler, code used to produce web services, and files containing high-level instructions executed by a computer using an interpreter. For example, embodiments may be implemented using imperative programming languages ​​(e.g., C, Fortran, etc.), functional programming languages ​​(Haskell, Erlang, etc.), logic programming languages ​​(e.g., Prolog), object-oriented programming languages ​​(e.g., Java, C++, etc.), interpreted languages ​​(JavaScript, Typescript, Perl), or other suitable programming languages ​​and / or development tools. Additional examples of computer code include, but are not limited to, control signals, encrypted code, and compressed code.

[0105] It will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the scope of the novel techniques disclosed herein. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the novel techniques without departing from their scope. Therefore, the novel techniques are not limited to the particular techniques disclosed, but are intended to include all techniques falling within the scope of the appended claims.

Claims

1. 1. A system for analyzing a sample contained in a vessel, the vessel including a bottom surface having a bottom bottom boundary (BB) and a bottom top boundary (TB), the system comprising: a transducer assembly configured to receive a plurality of electronic transmit signals and emit a corresponding plurality of transmitted acoustic signals toward the container and the sample, and configured to receive reflected acoustic signals from at least one of the container or the sample and generate a corresponding plurality of received electronic signals; signal transmission circuitry configured to provide the electronic transmission signal to the transducer assembly; signal receiving circuitry configured to receive the electronic receive signal from the transducer assembly; a processor configured to control the signal transmission circuitry and to receive information corresponding to the received electronic signal from the signal reception circuitry; Equipped with the plurality of transmitted acoustic signals comprising a sequence of a first ping, a first bubble breaking signal, and a second ping; the plurality of reflected acoustic signals comprising a first TB reflection corresponding to the first ping and a second TB reflection corresponding to the second ping; The system is configured to issue a second bubble collapse signal based on a comparison of a characteristic of the first TB reflection and a characteristic of the second TB reflection.

2. 2. The system of claim 1, wherein the characteristic of the first TB reflection comprises a first peak amplitude and the characteristic of the second TB reflection comprises a second peak amplitude, and the system is further configured to emit the second bubble collapse signal when the first peak amplitude exceeds the second peak amplitude.

3. The system of claim 1 , wherein the peak amplitude of the first ping and the peak amplitude of the second ping are substantially the same.

4. The system of claim 1 , wherein the overall energy of the first bubble breaking signal exceeds the overall energy of the first ping and exceeds the overall energy of the second ping.

5. 2. The system of claim 1, wherein the transducer assembly is configured to focus acoustic energy at a first height when emitting the first ping, the first bubble breaking signal, and the second ping, and wherein the transducer assembly is configured to focus acoustic energy at a second height above the first height when emitting the second bubble breaking signal.

6. The system of claim 5 , wherein the first height is predetermined relative to the TB and the second height is substantially at the surface of the sample.

7. The system of claim 5 , wherein the first height is between + / −6 mm relative to the TB.

8. The system of claim 1 , wherein the first bubble collapse signal has a variable frequency.

9. The system of claim 1 , wherein the energy of the second bubble disruption signal is selected to prevent droplets from being completely ejected from the sample.

10. The system of claim 1 , wherein the second bubble destruction signal comprises a pre-conditioning signal selected to destroy bubbles and a post-conditioning signal selected to destroy bubbles.

11. The system of claim 10 , wherein the preconditioning signal is a bubble breaking signal.

12. The system of claim 1 , wherein the transducer assembly is configured to emit the second bubble disruption signal while focusing acoustic energy substantially at a surface of the sample.

13. the vessels are contained within a plate comprising a plurality of vessels containing a corresponding plurality of samples; Each of the plurality of containers includes a corresponding BB and TB; the system further comprising at least one motor configured to move at least one of the plate or the transducer assembly such that the transducer assembly is positioned beneath each of the plurality of containers; the system is further configured to cause the transducer assembly to emit a sequence of the first ping, the first bubble breaking signal, and the second ping for each of the plurality of containers; the system is further configured to cause the transducer assembly to emit the second bubble collapse signal for a given one of the plurality of containers based on a comparison of a characteristic of the first TB reflection for the given container and a characteristic of the second TB reflection for the given container. The system of claim 1 .

14. 14. The system of claim 13, wherein the system is further configured to issue the second bubble breaking signal for a given one of the plurality of containers after the first ping, the first bubble breaking signal, and the second ping have been issued for each of the plurality of containers.

15. 1. A method for measuring a sample contained in a vessel, the vessel including a bottom surface having a bottom bottom boundary (BB) and a bottom top boundary (TB), the system comprising: receiving a plurality of electronic transmission signals at a transceiver assembly; transmitting, by the transceiver assembly, a plurality of transmitted acoustic signals toward the container and the sample, the plurality of transmitted acoustic signals corresponding to the plurality of electronic transmit signals; receiving, at the transmitter / receiver assembly, a plurality of reflected acoustic signals from at least one of the container or the sample; generating, by the transceiver assembly, a plurality of received electronic signals corresponding to the plurality of reflected acoustic signals; providing the plurality of electronic transmission signals to the transducer assembly through signal transmission circuitry; receiving, in signal receiving circuitry, the plurality of electronic receive signals from the transducer assembly; controlling the signal transmission network with a processor; receiving, at said processor, information corresponding to said received electronic signal from said signal receiving circuitry; Including, the plurality of transmitted acoustic signals comprising a sequence of a first ping, a first bubble breaking signal, and a second ping; the plurality of reflected acoustic signals comprising a first TB reflection corresponding to the first ping and a second TB reflection corresponding to the second ping; The method further includes generating, by the transducer assembly, a second bubble collapse signal based on a comparison of a characteristic of the first TB reflection and a characteristic of the second TB reflection.

16. 16. The method of claim 15, wherein the characteristic of the first TB reflection comprises a first peak amplitude and the characteristic of the second TB reflection comprises a second peak amplitude, and further comprising emitting the second bubble collapse signal by the transducer assembly when the first peak amplitude exceeds the second peak amplitude.

17. 16. The method of claim 15, wherein the peak amplitude of the first ping and the peak amplitude of the second ping are substantially the same.

18. 16. The method of claim 15, wherein the overall energy of the first bubble collapse signal exceeds the overall energy of the first ping and exceeds the overall energy of the second ping.

19. focusing acoustic energy at a first height using the transceiver assembly when emitting the first ping, the first bubble breaking signal, and the second ping; focusing acoustic energy at a second elevation above the first elevation using the transceiver assembly when issuing the second bubble breaking signal; 16. The method of claim 15, further comprising:

20. 20. The method of claim 19, wherein the first height is predetermined relative to the TB and the second height is substantially at the surface of the sample.

21. 16. The method of claim 15, wherein the first height is between + / - 6 mm relative to the TB.

22. The method of claim 15 , wherein the first bubble collapse signal has a variable frequency.

23. 16. The method of claim 15, wherein the energy of the second bubble disruption signal is selected to prevent a droplet from being completely ejected from the sample.

24. The method of claim 15 , wherein the second bubble destruction signal includes a pre-conditioning signal selected to destroy a bubble and a post-conditioning signal selected to destroy a bubble.

25. 25. The method of claim 24, wherein the second bubble destruction signal comprises a preconditioning signal selected to destroy bubbles.

26. 16. The method of claim 15, further comprising emitting a second bubble disruption signal while focusing acoustic energy substantially at the surface of the sample with the transceiver assembly.

27. the vessel is contained within a plate having a plurality of vessels containing a corresponding plurality of samples, each of the plurality of vessels including a corresponding BB and TB; moving, using at least one motor, at least one of the plate or the transducer assembly so that the transducer assembly is positioned beneath each of the plurality of containers at different times; issuing, with the transducer assembly, a sequence of the first ping, the first bubble breaking signal, and the second ping for each of the plurality of containers; generating, by the transducer assembly, the second bubble collapse signal for a given one of the plurality of containers based on a comparison of a characteristic of the first TB reflection for the given container and a characteristic of the second TB reflection for the given container; 16. The method of claim 15, further comprising:

28. 28. The method of claim 27, further comprising issuing the second bubble breaking signal for a given one of the plurality of containers after the first ping, the first bubble breaking signal, and the second ping have been issued for each of the plurality of containers.

29. 1. A system for reducing air bubbles in a sample contained in a vessel using an ultrasonic system having a transducer assembly, the vessel including a bottom surface having a bottom bottom interface (BB) and a bottom top interface (TB), a portion of the air bubbles in the sample being located substantially at the TB, the system comprising: a transducer assembly configured to align a focal point of an acoustic energy beam, the focal point being at a height substantially at an upper surface of the sample; Equipped with the transducer assembly is further configured to emit a bubble collapse signal while the focal height is substantially at an upper surface of the sample; The system wherein the bubble disruption signal has a peak amplitude selected to prevent complete ejection of a droplet from the sample.

30. 30. The system of claim 29, wherein the system is configured to perform at least one measurement to determine a peak amplitude of the bubble collapse signal.

31. 31. The system of claim 30, wherein the peak amplitude comprises substantially a maximum amplitude before a droplet will be completely ejected from the sample.

32. The bubble collapse signal is generated by the transducer assembly: a maximum amplitude determination signal selected to determine a maximum amplitude of an acoustic signal by processing a reflection of the maximum amplitude determination signal from an upper surface of the sample, wherein an acoustic signal having an amplitude greater than the maximum amplitude will cause a droplet to be ejected from the sample; a bubble mitigation signal having a peak amplitude determined according to the maximum amplitude; 30. The system of claim 29, further comprising transmitting:

33. The maximum amplitude determination signal is a first disturbance signal selected to disturb an upper surface of the sample; a first measurement signal following the first disturbance signal, the first measurement signal being selected to be reflected by an upper surface of the sample, whereby a reflection of the first measurement signal is processed to determine zero velocity droplets; and a second disturbance signal subsequent to the first measurement signal, the second disturbance signal being selected to disturb an upper surface of the sample, the second disturbance signal having a peak amplitude greater than the peak amplitude of the first disturbance signal; a second measurement signal following the second disturbance signal, the second measurement signal being selected to be reflected by an upper surface of the sample, whereby a reflection of the second measurement signal is processed to determine the zero velocity droplet; and 33. The system of claim 32, comprising:

34. 34. The system of claim 33, wherein the transducer assembly is further configured to emit a sequence of additional disturbance signals and additional measurement signals, the additional disturbance signals having increasing peak amplitudes.

35. 30. The system of claim 29, wherein the bubble collapse signal comprises a plurality of signals each having a peak amplitude determined according to the maximum amplitude.

36. 36. The system of claim 35, wherein each of the plurality of signals in the bubble collapse signal has a duration of at least 8 μs.

37. 1. A method for reducing air bubbles in a sample contained in a vessel using an ultrasound system having a transducer assembly, the vessel including a bottom surface having a bottom bottom interface (BB) and a bottom top interface (TB), wherein air bubbles in the sample abut against the TB, the method comprising: using the transducer assembly to focus an acoustic energy beam, the height of the focus being substantially at an upper surface of the sample; using the transducer assembly to emit a bubble collapse signal while the focal height is substantially at the upper surface of the sample; Including, A method wherein the bubble disruption signal has a peak amplitude selected to prevent complete ejection of a droplet from the sample.

38. 38. The method of claim 37, further comprising performing at least one measurement to determine a peak amplitude of the bubble collapse signal.

39. 39. The method of claim 38, wherein the peak amplitude comprises substantially a maximum amplitude before a droplet will be completely ejected from the sample.

40. The bubble collapse signal is a maximum amplitude determination signal selected to determine a maximum amplitude of an acoustic signal by processing a reflection of the maximum amplitude determination signal from an upper surface of the sample, wherein an acoustic signal having an amplitude greater than the maximum amplitude will cause a droplet to be ejected from the sample; a bubble destruction sub-signal having a peak amplitude determined according to the maximum amplitude; 38. The method of claim 37, further comprising:

41. The maximum amplitude determination signal is a first disturbance signal selected to disturb an upper surface of the sample; a first measurement signal following the first disturbance signal, the first measurement signal being selected to be reflected by an upper surface of the sample, whereby a reflection of the first measurement signal is processed to determine zero velocity droplets; and a second disturbance signal subsequent to the first measurement signal, the second disturbance signal being selected to disturb an upper surface of the sample, the second disturbance signal having a peak amplitude greater than the peak amplitude of the first disturbance signal; a second measurement signal following the second disturbance signal, the second measurement signal being selected to be reflected by an upper surface of the sample, whereby a reflection of the second measurement signal is processed to determine the zero velocity droplet; and 41. The method of claim 40, comprising:

42. 42. The method of claim 41, further comprising a sequence of additional disturbance signals and additional measurement signals, the additional disturbance signals having increasing peak amplitudes.

43. 40. The method of claim 39, wherein the bubble collapse signal comprises a plurality of signals each having a peak amplitude determined according to the maximum amplitude.

44. 44. The method of claim 43, wherein each of the plurality of signals in the bubble collapse signal has a duration of at least 8 μs.

45. 1. A system for determining the presence of gas bubbles in a sample contained in a container in an ultrasound system, the container including a bottom surface having a bottom bottom interface (BB) and a bottom top interface (TB), the method comprising: an ultrasonic transducer assembly configured to emit a first ping having a first energy, further configured to emit a second ping having a second energy following the first ping, and further configured to emit a higher energy signal following the first ping and prior to the second ping, the higher energy signal having energy greater than the first energy and the second energy, the ultrasonic transducer assembly further configured to receive a first reflected signal from the first ping that is reflected from the TB, and further configured to receive a second reflected signal from the second ping that is reflected from the TB; a processor configured to infer the presence of an air bubble in the sample when a peak amplitude of the first reflected signal exceeds a peak amplitude of the second reflected signal; A system comprising:

46. 1. A method for determining the presence of gas bubbles in a sample contained in a vessel in an ultrasound system having an ultrasound transducer assembly and a processor in communication with the ultrasound transducer assembly, the vessel including a bottom surface having a bottom boundary surface (BB) and a bottom top boundary surface (TB), the method comprising: emitting a first ping having a first energy from the ultrasonic transducer assembly; emitting a second ping from the ultrasonic transducer assembly following the bubble collapse signal and having a second energy; emitting a higher energy signal from the ultrasonic transducer assembly subsequent to the first ping and prior to the second ping, the higher energy signal having an energy greater than the first energy and the second energy; receiving, by the ultrasonic transducer assembly, a first reflected signal from the first ping that is reflected from the TB; receiving, by the ultrasonic transducer assembly, a second reflected signal from the second ping that is reflected from the TB; inferring, by the processor, the presence of an air bubble in the sample when a peak amplitude of the first reflected signal exceeds a peak amplitude of the second reflected signal; A method comprising:

47. 1. A method for performing sonoporation on a sample, the sample containing cells, the sample being contained in a vessel, the vessel including a bottom surface having a bottom bottom interface (BB) and a bottom top interface (TB), the method comprising: transmitting a ping towards the sample by a transducer assembly configured to transmit and receive acoustic signals; receiving a reflected signal at the transducer assembly, the reflected signal including reflections from the ping, from the TB and from the surface of the sample; measuring the energy of the reflected signal with a processor; using the processor to estimate the volume of the bubbles based on the energy of the reflected signal; A method comprising:

48. 48. The method of claim 47, wherein estimating the volume of the air bubble is based, at least in part, on the energy of reflections from the TB in the reflected signal.

49. 48. The method of claim 47, wherein estimating the volume of the bubbles is based at least in part on the energy of reflection from the surface of the sample in the reflected signal.

50. 49. The method of claim 48, wherein estimating the volume of the bubbles is based, at least in part, on the energy of reflection from the surface of the sample in the reflected signal.

51. 48. The method of claim 47, wherein the energy of the reflected signal corresponds to a peak amplitude of the reflected signal for a given reflection.

52. transmitting, with the transducer assembly, a bubble-breaking signal configured to break at least a portion of the bubbles; transmitting a second ping toward the sample using the transducer assembly; receiving, with the transducer assembly, a second reflected signal in response to the second ping, the second reflected signal including a reflection from the TB and a reflection from a surface of the sample; measuring, with the processor, the energy of the second reflected signal; using the processor to estimate a volume of the second bubble based on the energy of the second reflected signal; and 48. The method of claim 47, further comprising:

53. 53. The method of claim 52, wherein estimating the volume of the second bubble is based, at least in part, on the energy of reflections from the TB in the second reflected signal.

54. 53. The method of claim 52, wherein estimating the volume of the bubbles is based, at least in part, on the energy of reflection from the surface of the sample in the reflected signal.

55. 54. The method of claim 53, wherein estimating the volume of the bubbles is based, at least in part, on the energy of reflection from the surface of the sample in the reflected signal.

56. 55. The method of claim 54, further comprising transmitting a second bubble destruction signal configured to destroy at least a portion of the bubbles based on an amount of the second bubbles.

57. 57. The method of claim 56, wherein the second bubble breaking signal is different from the bubble breaking signal.

58. 57. The method of claim 56, wherein the second bubble collapse signal is determined by the processor based at least in part on the volume of the second bubble.

59. 48. The method of claim 47, further comprising performing transfection on cells in the sample.

60. 60. The method of claim 59, further comprising predicting the efficiency of the transfection based, at least in part, on the amount of the bubbles.

61. 1. A system for performing sonoporation on a sample, the sample containing cells, the sample being contained in a vessel, the vessel including a bottom surface having a bottom bottom interface (BB) and a bottom top interface (TB), the system comprising: a transducer assembly configured to transmit and receive acoustic signals, the transducer assembly further configured to transmit pings towards the sample and receive reflected signals from the pings, including reflections from the TB and from the surface of the sample; a processor configured to measure an energy of the reflected signal and estimate a volume of the air bubble based on the energy of the reflected signal; A system comprising:

62. 62. The system of claim 61, wherein the processor is configured to estimate the volume of the air bubble based at least in part on the energy of reflections from the TB in the reflected signal.

63. 62. The system of claim 61, wherein the processor is configured to estimate the volume of the bubbles based, at least in part, on energy of reflection from the surface of the sample in the reflected signal.

64. 63. The system of claim 62, wherein the processor is configured to estimate the volume of the bubbles based, at least in part, on energy of reflection from the surface of the sample in the reflected signal.

65. 62. The system of claim 61, wherein the energy of the reflected signal corresponds to a peak amplitude of the reflected signal for a given reflection.

66. the transducer assembly is further configured to transmit a bubble-breaking signal configured to break at least a portion of the bubble; transmit a second ping toward the sample; and receive a second reflected signal in response to the second ping, the second reflected signal including a reflection from the TB and a reflection from a surface of the sample; the processor is further configured to measure an energy of the second reflected signal and estimate a volume of the second bubble based on the energy of the second reflected signal.

62. The system of claim 61.

67. 67. The system of claim 66, wherein the processor is configured to estimate a volume of the second bubble based, at least in part, on energy of reflection from the TB in the second reflected signal.

68. 67. The system of claim 66, wherein the processor is configured to estimate the volume of the bubbles based, at least in part, on energy of reflection from the surface of the sample in the reflected signal.

69. 68. The system of claim 67, wherein the processor is configured to estimate the volume of the bubbles based, at least in part, on energy of reflection from the surface of the sample in the reflected signal.

70. 67. The system of claim 66, wherein the transducer assembly is configured to transmit a second bubble destruction signal configured to destroy at least a portion of the bubbles based on an amount of the second bubbles.

71. 71. The system of claim 70, wherein the second bubble breaking signal is different from the bubble breaking signal.

72. 71. The system of claim 70, wherein the second bubble collapse signal is determined by the processor based at least in part on the volume of the second bubble.

73. 62. The system of claim 61, further comprising performing transfection on cells in the sample.

74. 74. The system of claim 73, further comprising predicting an efficiency of the transfection based, at least in part, on the amount of the bubbles.

Images