Ultrasonic bubble repair

Abstract

A system for measuring a sample contained in a container, comprising: a transducer assembly configured to receive a plurality of electronic transmit signals and to transmit a corresponding plurality of transmit acoustic signals toward the container and the sample, and configured to receive a reflected acoustic signal from the container or the sample and to generate a corresponding plurality of electronic receive signals; a transmit signal circuit configured to generate and transmit an electronic transmit signal to the transducer assembly; a receive signal circuit configured to receive an electronic receive signal from the transducer assembly; and a processor configured to control the transmit signal circuit and receive information corresponding to the electronic receive signal from the receive signal circuit, where the plurality of transmit acoustic signals includes a sequence of a first ping, a first bubble destruction signal, and a second ping, where the first ping and the second ping are coupled to each other. The plurality of reflected acoustic signals includes a first TB reflection corresponding to the first ping and a second TB reflection corresponding to the second ping, and wherein the system is configured to emit a second bubble destruction signal based on a comparison of a characteristic of the first TB reflection and a characteristic of the second TB reflection.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 414,322, filed on October 7, 2022, the entire contents of which are incorporated herein by reference. Background Art

[0003] Acoustic droplet ejection (ADE) is a technique that uses acoustic energy to move liquids without any physical contact. Some examples of ADE technology are disclosed in U.S. Patent No. 10,156,499, the entire contents of which are incorporated herein by reference. Acoustic energy (e.g., in the form of ultrasonic pulses) is emitted from a transducer toward a certain amount of liquid (hereinafter referred to as a "sample"). In some examples, the beam converges on or near the upper surface of the sample, and the acoustic energy is transferred to a portion of the sample, thereby moving the portion upward away from the rest of the sample (e.g., as a droplet). The sample can be contained in a well (also referred to herein as a container) of a plate (e.g., a 96-well microplate or a 384-well microplate). Summary of the invention

[0004] According to an embodiment, a system for measuring a sample contained in a container, wherein the container includes a bottom surface having a bottom bottom interface (BB) and a bottom top interface (TB), the system including: a transducer assembly configured to receive a plurality of electronic transmission signals and transmit a corresponding plurality of transmission acoustic signals toward the container and the sample, and configured to receive reflected acoustic signals from the container or the sample and generate a corresponding plurality of electronic reception signals; a transmission signal circuit configured to generate the electronic transmission signal and transmit it to the transducer assembly; a reception signal circuit configured to receive the electronic reception signal from the transducer assembly; and a processor configured to control the transmission signal circuit and receive information corresponding to the electronic reception signal from the reception signal circuit, wherein the plurality of transmission acoustic signals include a sequence of a first ping, a first bubble destruction signal, and a second ping, wherein the plurality of reflection acoustic signals include a first TB reflection corresponding to the first ping and a second TB reflection corresponding to the second ping, and wherein the system is configured to transmit the second bubble destruction signal based on a comparison of characteristics of the first TB reflection with characteristics of the second TB reflection. The characteristic of the first TB reflection may include a first peak amplitude, the characteristic of the second TB reflection includes a second peak amplitude, and wherein the system may also be configured to: if the first peak amplitude is greater than the second peak amplitude, then transmit a second bubble destruction signal. 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 destruction signal may be greater than the total energy of the first ping and greater than the total energy of the second ping. The transducer assembly may be configured to focus the acoustic energy at a first height when transmitting the first ping, the first bubble destruction signal, and the second ping, and wherein the transducer assembly may be configured to focus the acoustic energy at a second height higher than the first height when transmitting the second bubble destruction signal. The first height may be predetermined relative to the TB, and wherein 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 not cause the droplet to be completely ejected from the sample. The second bubble destruction signal may include a pre-adjustment signal selected to destroy the bubble and a subsequent adjustment signal selected to destroy the bubble. The pre-adjustment signal may be a bubble destruction signal. The transducer assembly may be configured to emit a second bubble destruction signal while focusing the acoustic energy substantially at the surface of the sample.The container may be included in a plate including a plurality of containers, the plurality of containers including a corresponding plurality of samples, wherein each of the plurality of containers may include a respective BB and TB, wherein the system may further include at least one motor configured to move at least one of the plate or the transducer assembly such that the transducer assembly is positioned below each of the plurality of containers, wherein the system may further be configured to cause the transducer assembly to transmit a sequence of a first ping, a first bubble busting signal, and a second ping for each of the plurality of containers, and wherein the system may further be configured to cause the transducer assembly to transmit a second bubble busting signal for a given container of the plurality of containers based on a comparison of a characteristic of a first TB reflection for the given container with a characteristic of a second TB reflection for the given container. The system may further be configured to transmit a second bubble busting signal for a given container of the plurality of containers after the first ping, the first bubble busting signal, and the second ping have been transmitted for each of the plurality of containers.

[0005] According to an embodiment, a method for measuring a sample contained in a container is provided, wherein the container includes a bottom surface having a bottom bottom interface (BB) and a bottom top interface (TB), the method comprising: receiving a plurality of electronic transmission signals at a transceiver assembly; transmitting a plurality of transmission acoustic signals toward the container and the sample by the transceiver assembly, wherein the plurality of transmission acoustic signals correspond to a plurality of electronic transmission signals; receiving a plurality of reflected acoustic signals from the container or the sample at the transceiver assembly; generating a plurality of electronic reception signals corresponding to the plurality of reflected acoustic signals by the transceiver assembly; generating a plurality of electronic transmission signals by a transmission signal circuit and transmitting the plurality of electronic transmission signals to the transceiver assembly; The method further comprises transmitting to a transducer assembly; receiving a plurality of electronic receive signals from the transducer assembly at a receive signal circuit; and controlling a transmit signal circuit with a processor; receiving information corresponding to the electronic receive signals from the receive signal circuit at the processor, wherein the plurality of transmit acoustic signals comprises a sequence of a first ping, a first bubble busting signal, and a second ping, wherein 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 method further comprises: transmitting a second bubble busting signal by the transducer assembly 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 comprises a first peak amplitude, the characteristic of the second TB reflection comprises a second peak amplitude, and the method further comprises: transmitting a second bubble busting signal by the transducer assembly if the first peak amplitude is greater than 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 busting signal may be greater than the total energy of the first ping and greater than the total energy of the second ping. The method may also include: when transmitting the first ping, the first bubble destruction signal, and the second ping, focusing the acoustic energy at a first height using the transceiver assembly; and when transmitting the second bubble destruction signal, focusing the acoustic energy at a second height higher than the first height using the transceiver assembly. The first height can be predetermined relative to the TB, and wherein the second height can be substantially at the surface of the sample. The first height can be between + / -6mm 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 the droplet from being completely ejected from the sample. The second bubble destruction signal may include a pre-adjustment signal selected to destroy the bubble and a subsequent adjustment signal selected to destroy the bubble. The pre-adjustment signal may be a bubble destruction signal. The method may also include: transmitting the second bubble destruction signal by the transceiver assembly when the acoustic energy is substantially focused at the surface of the sample.The container may be included in a plate including a plurality of containers, the plurality of containers including a corresponding plurality of samples, wherein each of the plurality of containers includes 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 located below each of the plurality of containers; transmitting, by the transducer assembly, a sequence of a first ping, a first bubble destruction signal, and a second ping for each of the plurality of containers; and transmitting, by the transducer assembly, a second bubble destruction signal for a given container of the plurality of containers based on a comparison of a characteristic of a first TB reflection for the given container with a characteristic of a second TB reflection for the given container. After the first ping, the first bubble destruction signal, and the second ping have been transmitted for each of the plurality of containers, the transducer assembly may further transmit a second bubble destruction signal for a given container of the plurality of containers. A non-transitory computer-readable medium containing instructions that, when executed by a processor, may perform any 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 with a transducer assembly, wherein the container includes a bottom surface having a bottom bottom interface (BB) and a bottom top interface (TB), wherein bubbles in the sample surround the TB, the system including: a transducer assembly configured to align a focus of an acoustic energy beam, wherein the height of the focus is substantially at the upper surface of the sample, wherein the transducer assembly is further configured to emit a bubble destruction signal when the height of the focus is substantially at the upper surface of the sample, and wherein the bubble destruction signal includes a peak amplitude selected to prevent a droplet from being completely ejected from the sample. The system can be configured to perform at least one measurement to determine the peak amplitude of the bubble destruction signal. The peak amplitude can include a substantially maximum amplitude before a droplet will be completely ejected from the sample. The bubble destruction signal can also include: a maximum amplitude determination signal selected to determine a maximum amplitude of the acoustic signal by processing reflections of the maximum amplitude determination signal from the 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; and a bubble mitigation signal including a peak amplitude determined based on 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 after the first disturbance signal, wherein the first measurement signal is selected to be reflected by the upper surface of the sample, whereby the reflection of the first measurement signal is processed to determine the zero velocity droplet; a second disturbance signal after the first measurement signal, wherein the second disturbance signal is selected to disturb the upper surface of the sample, wherein the peak amplitude of the second disturbance signal is greater than the peak amplitude of the first disturbance signal; and a second measurement signal after the second disturbance signal, wherein the second measurement signal is selected to be reflected by the upper surface of the sample, whereby the reflection of the second measurement signal is processed to determine the zero velocity droplet. The system may also be configured to transmit a sequence of additional disturbance signals and additional measurement signals, wherein the additional disturbance signals have increasing peak amplitudes. The bubble mitigation signal may include a plurality of signals, each having a peak amplitude determined according to the maximum amplitude. Each of the plurality of signals in the bubble mitigation signal may have a duration of at least 8us.

[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, wherein the container includes a bottom surface having a bottom bottom interface (BB) and a bottom top interface (TB), wherein bubbles in the sample surround the TB, the method comprising: using the transducer assembly to align a focus of an acoustic energy beam, wherein the height of the focus is substantially at an upper surface of the sample; and using the transducer assembly to transmit a bubble destruction signal when the height of the focus is substantially at the upper surface of the sample, wherein the bubble destruction signal comprises a peak amplitude selected to prevent a droplet from being completely ejected from the sample. The method may also include performing at least one measurement to determine a peak amplitude of the bubble destruction signal. The peak amplitude may include a substantially maximum amplitude before a droplet will be completely ejected from the sample. The bubble destruction signal may also include: a maximum amplitude determination signal selected to determine a maximum amplitude of an acoustic signal by processing reflections of the maximum amplitude determination signal from the 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; and a bubble mitigation signal comprising 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 after the first disturbance signal, wherein the first measurement signal is selected to be reflected by the upper surface of the sample, whereby the reflection of the first measurement signal is processed to determine the zero velocity droplet; a second disturbance signal after the first measurement signal, wherein the second disturbance signal is selected to disturb the upper surface of the sample, wherein the peak amplitude of the second disturbance signal is greater than the peak amplitude of the first disturbance signal; and a second measurement signal after the second disturbance signal, wherein the second measurement signal is selected to be reflected by the upper surface of the sample, whereby the reflection of the second measurement signal is processed to determine the zero velocity droplet. The method may also include a sequence of additional disturbance signals and additional measurement signals, wherein the additional disturbance signals have an increased peak amplitude. The bubble mitigation signal may include a plurality of signals, each having a peak amplitude determined according to the maximum amplitude. Each of the plurality of signals in the bubble mitigation signal may have a duration of at least 8us. A non-transitory computer-readable medium containing instructions, which when executed by a processor may perform any of the foregoing method embodiments.

[0008] According to an embodiment, a system for determining the presence of bubbles in a sample contained in a container in an ultrasonic system is provided, wherein the container includes a bottom surface having a bottom bottom interface (BB) and a bottom top interface (TB), the system including: an ultrasonic transducer assembly configured to emit a first ping having a first energy, and further configured to emit a second ping having a second energy after a bubble destruction signal, and further configured to emit a higher energy signal after the first ping and before the second ping, wherein the higher energy signal has an energy greater than the first energy and the second energy, the ultrasonic transducer assembly further configured to receive a first reflected signal reflected from the TB from the first ping, and further configured to receive a second reflected signal reflected from the TB from the second ping; and a processor configured to infer the presence of bubbles in the sample when the peak amplitude of the first reflected signal is greater than the peak amplitude of the second reflected signal.

[0009] According to an embodiment, a method for determining the presence of bubbles in a sample contained in a container in an ultrasound system is provided, the ultrasound system having an ultrasound transducer assembly and a processor in communication with the ultrasound transducer assembly, wherein the container includes a bottom surface having a bottom bottom interface (BB) and a bottom top interface (TB), the method comprising: transmitting a first ping having a first energy from the ultrasound transducer assembly; transmitting a second ping having a second energy from the ultrasound transducer assembly after a bubble destruction signal; transmitting a higher energy signal from the ultrasound transducer assembly after the first ping and before the second ping, wherein the higher energy signal has an energy greater than the first energy and the second energy; receiving by the ultrasound transducer assembly a first reflected signal reflected from the TB from the first ping; receiving by the ultrasound transducer assembly a second reflected signal reflected from the TB from the second ping; and inferring by the processor the presence of bubbles in the sample when the peak amplitude of the first reflected signal is greater than the peak amplitude of the second reflected signal. A non-transitory computer readable medium may contain instructions that, when executed by the processor, perform any of the foregoing method embodiments.

[0010] According to an embodiment, a method for performing acoustic perforation on a sample contained in a container, the sample including cells in the sample, wherein the container includes a bottom surface having a bottom-end interface (BB) and a bottom-end interface (TB), the method comprising: sending a ping toward the sample by a transducer assembly configured to send and receive acoustic signals; receiving a reflection signal from the ping at the transducer assembly, the reflection signal including reflections from the TB and from the surface of the sample; measuring the energy of the reflection signal with a processor; and estimating the number of bubbles based on the energy of the reflection signal with the processor. The estimated number of bubbles may be based at least in part on the energy of the reflection from the TB in the reflection signal. The estimated number of bubbles may be based at least in part on the energy of the reflection from the surface of the sample in the reflection signal. The estimated number of bubbles may be based at least in part on the energy of the reflection from the surface of the sample in the reflection signal. For a given reflection, the energy of the reflection signal may correspond to the peak amplitude of the reflection signal. The method may also include: sending a bubble destruction signal with the transducer assembly, the bubble destruction signal being configured to destroy at least a portion of the bubbles; sending a second ping with the transducer assembly toward the sample; receiving a second reflection signal with the transducer assembly in response to the second ping, wherein the second reflection signal includes a reflection from the TB and a reflection from the surface of the sample; measuring the energy of the second reflection signal with the processor; and estimating a second number of bubbles with the processor based on the energy of the second reflection signal. The estimated second number of bubbles may be based at least in part on the energy of the reflection from the TB in the second reflection signal. The estimated number of bubbles may be based at least in part on the energy of the reflection from the surface of the sample in the reflection signal. The estimated number of bubbles may be based at least in part on the energy of the reflection from the surface of the sample in the reflection signal. The method may also include sending a second bubble destruction signal based on the second number of bubbles, the second bubble destruction signal being configured to destroy at least a portion of the bubbles. The second bubble destruction signal may be different from the bubble destruction signal. The second bubble destruction signal may be determined by the processor at least in part based on the second number of bubbles. The method may also include performing a transfection on cells in the sample. The method may also include predicting the efficiency of the transfection based at least in part on the number of bubbles. A system may be configured to perform any of the aforementioned method embodiments. A non-transitory computer readable medium containing instructions, which when executed by a processor may perform any of the aforementioned method embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 A representation of an ADE system is shown, including a cross-sectional view of a container plate including a plurality of containers (or wells) holding a corresponding plurality of samples, a receiver board, a transducer assembly, and a block diagram of electronic circuitry.

[0012] Figure 2 A block diagram of a transducer assembly is shown.

[0013] Figure 3 A representation of the movement of the transducer assembly relative to the container plate while performing ADE on multiple samples is shown.

[0014] Figure 4 A top view of a container plate having a plurality of containers is shown.

[0015] Figure 5 A top view of a plurality of containers in a container plate is shown, and a flow chart showing a sequence for successively performing ADE on each container is shown.

[0016] Figure 6 The container holding the sample is shown.

[0017] Figure 7 A reflected signal and corresponding envelope are shown, wherein the reflected signal is received at a transceiver component in response to a transmitted signal.

[0018] Figure 8 A scanning electron microscope image of the inner surface at the bottom-top interface (TB) of an exemplary container is shown.

[0019] Fig. 9A A scanning electron microscope image of a TB in a container holding a sample not affected by bubbles is shown.

[0020] Fig. 9B A scanning electron microscope image of a TB in a container holding a sample affected by gas bubbles is shown.

[0021] Fig.10 A sequence for bubble remediation is shown in accordance with certain embodiments.

[0022] Fig.11 Shown is a top view of a container plate with certain containers having samples with air bubbles.

[0023] Fig.12 A flow chart of a method for removing bubbles according to an embodiment is shown.

[0024] Fig.13 A flow chart of a method for removing bubbles according to an embodiment is shown.

[0025] Fig.14 The stability of reflections from a TB in a container holding a sample affected by air bubbles is shown.

[0026] Fig.15 The stability of a zero velocity droplet (ZVD) in a container holding a sample affected by an air bubble is shown.

[0027] Fig.16A , Fig. 16B and Fig. 16C Illustrative examples of reflections of pings when different numbers of bubbles are present are shown, in accordance with an embodiment.

[0028] Fig.17A , Fig. 17B , Fig. 17C and Fig.17D An illustrative example of the reflection of a ping after different bubble-busting signals have been emitted is shown, in accordance with an embodiment.

[0029] Fig.18 Four microscope images of cells with attached bubbles after a bubble-destroying signal has been emitted, according to an embodiment, are shown.

[0030] Fig.19 A flow chart of a method for removing bubbles according to an embodiment is shown.

[0031] Fig. 20 Graphs of signals indicative of bubble reduction in different samples are shown, according to an embodiment.

[0032] Fig.21 Shown are graphs indicating reduction of bubbles in different samples, according to embodiments.

[0033] Fig. 22 A graph is shown wherein the x-axis shows the transfection efficiency of A549 cells (in percent) and the y-axis shows the bubble clearance rate (in percent) according to an embodiment.

[0034] Fig.23 A graph is shown wherein the x-axis shows the transfection efficiency of HEK-293 cells (in percent) and the y-axis shows the bubble clearance rate (in percent) according to an embodiment.

[0035] The foregoing summary of the invention and the following detailed description of certain techniques of the present application will be better understood when read in conjunction with the accompanying drawings. Certain techniques are shown in the accompanying drawings for illustrative purposes. However, it should be understood that the claims are not limited to the arrangements and means shown in the accompanying drawings. In addition, the appearance shown in the accompanying drawings is one of many decorative appearances that can be adopted to achieve the described functions of the system. DETAILED DESCRIPTION

[0036] Figure 1An exemplary representation of an ADE system 100 is depicted, including a cross-sectional view of a container plate 120 (e.g., a microplate) including a plurality of containers 122 (e.g., wells of a microplate) holding a corresponding plurality of samples 101, a receiver plate 130 including a plurality of receiver wells that receive ejected liquid 102 from the samples 101, and a block diagram of electronics 140. The ADE system 100 also includes a transducer assembly 110, a coupling liquid 160, an X / Y / Z motor 150 (or less than all three dimensions—e.g., no vertical (Z) motor), and / or a temperature sensor (not shown). Figure 2 Also shown is a transducer assembly 110, which includes a transducer 112 and an acoustic lens 113. The ADE system 100 can determine the characteristics of a container 122 and / or a sample 101 and cause the liquid to be ejected. A given sample 101 is a liquid of interest held within a corresponding container 122. Although the present disclosure focuses on containers 122 that are wells of a microplate, the techniques described herein can be used with other containers 122, such as test tubes, flasks, and beakers and any samples 101 contained therein.

[0037] In order to eject the liquid 102 from the sample 101, the transducer 112 generates acoustic energy (e.g., ultrasonic energy), which is focused into a beam 170 by the acoustic lens 113. In the figure, the beam 170 is shown in two dimensions, but the beam 170 is actually three-dimensional. In addition, although the beam 170 is shown as a perfect triangle, the beam 170 can have different shapes. The height of the focus of the acoustic energy beam 170 can be changed by adjusting the configuration of the transducer assembly 110. For example, the focal length of the beam 170 can be changed by adjusting the transducer assembly 110. Such a transducer assembly 110 is described in U.S. Application No. 16 / 369,780 (U.S. Publication 2019 / 0302063), the entire contents of which are incorporated herein by reference. The height of the focus can also be changed by moving the transducer assembly 110 along the z-axis (i.e., the vertical dimension between the container 122 and the transducer assembly 110).

[0038] like Figure 1 As depicted, beam 170 is focused on the upper surface of sample 101, which may be at the interface between sample 101 and the air above sample 101. Beam 170 first passes through coupling liquid 160, bottom wall 123 of container 122, and then through the depth of sample 101 to reach surface 103 of sample 101.

[0039] The electronic circuit 140 includes a processor 143, a motor controller 142, a transmission signal circuit 144, a reception signal circuit 145, and a temperature sensor circuit 141. Although shown as separate components for the purpose of explanation, parts of the electronic device 140 can be combined or integrated. In addition, some of the components shown may include multiple different sub-components that are not specifically shown. For example, the processor 143 may include multiple processors (e.g., multiple processors distributed at different locations).

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

[0041] The processor 143 may also communicate with the motor controller 142 to control the position 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, but these or other motors may be coupled (directly or indirectly) to the container plate 120 and / or the receiver plate 130 to control relative movement between the transducer assembly 110, the container plate 120, and / or the receiver plate 130. The processor 143 may control one or more of the motors 150 to position the transducer assembly 110 below a given container 122 in the container plate 120 (e.g., to center the transducer assembly 110 relative to the center of the given container 122), and then move the transducer assembly 110 below another given container 122 in the container plate 120. Alternatively or additionally, motors or other translation devices may be used to translate the container plate and / or receiver plate 130 in one or more X, Y, and Z dimensions.

[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 container plate 120 and the receiver plate 130, or in other locations. The temperature sensor circuit 141 receives a signal (e.g., an electrical signal or a wireless signal) from the temperature sensor and communicates with the processor 143 so that the temperature (e.g., the temperature of the coupling liquid 160, the container 122, the sample 101, the air temperature) can 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 to transmit and receive acoustic signals, the transducer assembly 110 can include separate transmitter transducers and receiver transducers, for example, as disclosed in U.S. Pat. No. 10,787,670, the entire contents of which are incorporated herein by reference. According to one technique, the receiving transducer can substantially surround the transmitting transducer and the acoustic lens.

[0044] Figure 3 1 shows a representation of the movement of the transducer assembly 110 relative to the container plate 120 when performing ADE on multiple samples 101. The transducer assembly 110 moves along the x-axis from container to container 122. The transducer assembly 110 can also move along the y-axis to additional containers 122 (not shown), as shown in FIG. Figure 5 As further described below. For each container 122, the transducer assembly 110 can be substantially centered below the container 122. The transducer assembly 110 can be moved vertically along the z-axis to transmit and receive acoustic signals at different z-positions below the container 122. The transducer assembly 110 can be positioned along the z-axis to focus the beam 170 on the surface 103 of the sample 101 so that the ejected liquid 102 is ejected (ADE). As further described below, the transducer assembly 110 can be positioned along the z-axis to focus the beam 170 at a predetermined height relative to the container plate 120 to destroy bubbles without performing ADE, for example, to destroy bubbles located at the interface between the container 122 and the sample 101.

[0045] Figure 4 A top view of a container plate 120 is shown having a plurality of containers 122. The container plate 120 shown is a 384-well microplate (eg, a polypropylene microplate, designated 384-PP). Figure 5 FIG. 1 is a top view of a plurality of containers 122 and an example pattern (a serpentine pattern) for performing ADE and / or other ultrasonic techniques (eg, bubble destruction) on each container 122 and the sample 101 therein, as shown in FIG. Figure 3As described. In this example, the motor 150 moves the transducer assembly 110 along the x-axis and y-axis to position it under various containers 122. Any other suitable pattern (e.g., a grating pattern) may be used. The container plate 120, or a combination of the container plate 120 and the transducer assembly 110 may also be moved to achieve similar results.

[0046] Figure 6 A container 122 (or well) holding a sample 101 is shown, the container 122 having a surface 103 (ie, an upper surface or free surface). A bottom wall 123 of the container 122 defines a bottom top interface (TB) 124 and a bottom bottom interface (BB) 125 .

[0047] Figure 7 The reflected signal and corresponding envelope are shown, where the reflected signal is received at the transceiver assembly 110 in response to the transmitted signal. BB indicates reflection from BB 125. TB indicates reflection from TB 124. SR (surface reflection) indicates reflection from the surface 103 of the sample 101. The total time that the reflection occurs is called the time of flight or ToF. Figure 7 For each reflection BB, TB, SR in the image, there is a different corresponding ToF. When the distance between the transducer and the BB, TB or surface 103 is generally known, a specific reflection can be identified in the total reflection signal based on the ToF.

[0048] Figure 8 A scanning electron microscope image of the inner surface of an exemplary container 122 is shown, wherein the inner surface includes TB 124. Surface texturing (lighter color, irregular shapes) on the order of about 5 μm to 10 μm can be seen. Such texturing may not be present in all containers 122, but it has been observed that the plate 120 may have affected the container 122. Fig.11 As shown, the affected containers are referred to as affected containers 126, and the affected containers 126 (which may be a subset of containers 122) may appear at the A11, A14, P11, P14, B11, B14, O11, or O14 positions, although other positions are possible. When the container plate 120 is formed, such a location may be close to or adjacent to the injection molding port. It has been observed that there may be one to six affected containers 126, although 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 Figure 8 Texture shown. Rather, the techniques described herein are applicable to a variety of situations that may benefit from bubble repair.

[0049] like Fig. 9A and Fig. 9BAs shown, texturing may result in only affected container 126 . Fig. 9A A microscope view of a TB 124 including an unaffected container 122 of the sample 101 is shown. Fig. 9A Few, if any, bubbles are shown to be present. Fig. 9B A similar microscope view of TB 124 of an affected container 126 including sample 101 is shown. Fig. 9B 1, it can be seen that the bubble 104 is formed at the TB 124. As used herein, the affected container 126 is also a container 122.

[0050] For example, bubbles 104 have been observed in affected containers 126 filled with samples 101, including Milli- Water, 1X phosphate buffered saline (PBS), Triton X-100 at <= 14% critical micelle concentration in 1X PBS, or 70% to 80% dimethyl sulfoxide in water. The bubbles 104 may have a diameter between 1.5 μm and 5 μm, but as will be appreciated, the techniques described herein are not limited to a particular bubble 104 size.

[0051] The air bubble 104 at the TB 124 may affect the acoustic ejection process. For example, the air bubble 104 may increase the acoustic reflection from the TB 124. Return to Reference Figure 7, the acoustic energy beam is refracted through the BB 125 and the TB 124 and reaches the surface 103 of the sample 101. Once reaching the surface 103, a liquid mound is generated, and then the ejected liquid 102 is ejected. However, in the affected container 126, the bubble 104 may cause a larger than normal reflection from the TB 124. This may be due to the larger acoustic impedance mismatch between the affected container 126 material (e.g., polypropylene) and the gas trapped in the bubble 104 than the acoustic impedance mismatch between the container 126 material and the sample 101 without the bubble 104. In the affected container 126, the bubble 104 may not cover 100% of the area where the acoustic energy beam intersects the TB 124, but the bubble 104 may be relatively evenly dispersed across the TB 124. The reflection signal may include reflections from areas of the TB 124 where the bubble 104 is not present, and reflections from areas of the TB 124 where the bubble 104 is present. For reflections from the portion of the beam that does not intersect the bubble 104, the reflected energy may have a typical amplitude expected from the TB 124. For reflections from the portion of the beam that intersects the bubble 104, the transmit signal may actually hit two interfaces: first, the TB 124 to gas interface at the lower surface of the bubble 104 adhered to the TB 124; and second, the gas / fluid interface at the top of the bubble 104. Given that the reflections from these two interfaces are separated in time by an order of tens of nanoseconds, the reflections from these two interfaces may occur nearly simultaneously. The combined reflection caused by the bubble 104 may 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 including a sample 101 is three times higher than that of an unaffected container 122 including a sample 101. In some cases, the energy received from the TB 124 at the transducer assembly 110 may be so high that it saturates the receive signal circuit 145 (exceeds the acceptable operating range). This may lead to problems, such as incorrectly identifying an empty container 122. When the reflected energy from the TB 124 saturates the receive signal circuit 145 (or is otherwise high enough), this may identify (or help identify) an empty container 122. However, if an air bubble 104 is present, a given container 122 may be incorrectly identified as not containing a sample 101. Another problem with an affected container 126 may be that the evaluation of the sample 101 is incorrect for calibration that takes into account the impedance signature of the container 122 and / or the sample 101.

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

[0054] One known technique for resolving bubble 104 interference is to reduce the amplitude of the transmitted signal (e.g., survey ping). However, the reflection from TB 124 in the affected container 126 may still be higher than the reflection from TB 124 in the container 122 that is not the affected container 126. This may mean that the challenge of feature calibration is not resolved. Feature calibration can rely on TB 124 reflection and BB 125 reflection to determine the properties of the bottom wall 123 and sample 101 related to ADE (e.g., the acoustic impedance of the sample 101).

[0055] As an example, assume a first amplitude of the transmitted signal, and a peak reflection from BB 125 (1000 counts), a peak reflection from TB 124 in the unaffected container 122 (1000 counts), and a peak reflection from TB 124 in the affected container 126 (3000 counts), where counts are increments on an analog-to-digital (A / D) converter. Herein, "counts" refers to the fitted envelope value. In this example, the A / D converter has a maximum range of 2048 counts, so a signal of 3000 counts will saturate the A / D converter. If the amplitude of the transmitted signal is reduced to a second value, the peak amplitude of the reflected energy may be reduced by half (500, 500, and 1500 counts from BB 125, TB 124 in the unaffected container 122, and TB 124 in the affected container 126, respectively). In this example, the A / D converter may no longer be saturated by the peak amplitude of the reflected signal from TB 124 in the affected container 126. However, the ratio of the peak amplitude reflection from TB 124 to BB 125 in the affected container is still relatively high (3x) and may result in incorrect identification of the fluid properties by the signature.

[0056] Additionally, even though the affected container 126 may not be considered empty and the process does proceed to ADE transfers, these transfers may also suffer from incorrect volumes and / or poor placement because the instability of the "zero velocity droplet" (ZVD) is not accounted for (e.g., not repeatedly measured as substantially constant). The MIP may allow the calculation of the ZVD (the amplitude of the acoustic signal required to just break off the drop 102, but without the energy required to separate the drop 102 from the surface 103 of the sample 101). Therefore, the drop 102 may fall back into the sample 101. Once determined, the ZVD value may be scaled to the amplitude required to fully eject the drop 102 from the sample 101 and container 122. The instability of the ZVD may be caused by the changing attenuation when the bubble 104 is destroyed. If there is substantially no bubble 104 at the TB 124, the MIP may produce a stable value. Likewise, if there is a bubble 104 at TB 124 acting as an attenuator, but the 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 following situation: the MIP is evaluating a system with an additional attenuator (in this case, the bubble 104), but the attenuation level of the additional attenuator is changing (the bubble 104 is being destroyed). Therefore, the ZVD may be too high, and droplets 102 may be ejected unintentionally.

[0057] Fig.10 A sequence 1000 of repairing a bubble 104 according to certain embodiments is shown. In sequence 1000, the transducer assembly 110 may emit an acoustic energy beam 170 having a focal length (e.g., 6 mm to 51 mm, such as 25 mm). The 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 the acoustic energy beam 170 extends to the same focal point at a predetermined height (e.g., a height above TB 124 and / or a height predetermined relative to TB 124). Such a predetermined height may be -4 mm to +4 mm below or above TB 124, such as +2.5 mm above TB 124. The height of the focal point may be changed (or the focal length may be otherwise changed) between steps 1010, 1020, 1030. It may be beneficial to adjust the position and / or shape of the acoustic energy beam 170 (via z-axis movement of the transducer assembly 110 or changing 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 moved 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] At step 1010, the transducer assembly 110 emits a first ping with an acoustic energy beam 170. A ping can be an acoustic signal having a relatively short duration (e.g., 5 ns to 200 ns, such as 40 ns) and a relatively low energy (e.g., 0.1 μJ to 10 μJ, such as 1 μJ). A ping can have a peak power of up to 100 watts. A ping can include acoustic energy transmitted with a wide frequency spectrum (e.g., a center frequency of 12 MHz and a bandwidth of 6 MHz). To generate the acoustic energy beams of FIGS. 16, 17, and 18, the ping can be transmitted with a wide frequency spectrum (e.g., a center frequency of 12 MHz and a bandwidth of 6 MHz). Figure 20 to Figure 23 The data shown uses a ping with a single cycle of a 2.25 MHz square wave. TB 124 reflects a portion of the first ping as a first TB reflection (not shown). Transducer assembly 110 receives the first TB reflection and processor 143 can process the information. The reflection from the first ping can indicate the presence of an air bubble. FIG. 16A to FIG. 16C , shows an illustrative example of how to determine the presence of bubbles by processing reflections from a ping. Fig.16A is an illustrative example in which there are substantially no bubbles. In this case, the TB reflection is relatively low, while the SR reflection is relatively high. Fig. 16B and Fig. 16C is an additional illustrative example of the presence of an increasing number (concentration) of bubbles. As the number of bubbles increases, the peak amplitude of the TB reflection increases, while the peak amplitude of the SR reflection decreases.

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

[0060] The first bubble destruction signal may be selected to destroy (or burst) at least some of the bubbles 104 . Fig.10Such a destroyed bubble is designated as a solid black circle, i.e., a burst bubble 105. The first bubble destruction signal may impart energy to the bubble 104 at the TB 124. One bubble destruction mechanism may be bubble cavitation. Energy input to a given bubble 104 at a relevant frequency may cause the bubble 194 to expand. As the radius of the bubble 104 increases, the surface tension may no longer be sufficient to maintain the bubble 104. The size of the bubble 104 may increase until the signal ceases, and then the bubble 104 may break into many substantially smaller bubbles (i.e., bubble destruction). Another bubble destruction mechanism may be by pushing the bubble upward and out of the TB 124.

[0061] Depending on the implementation, steps 1010 and 1020 may be repeated prior to step 1030, either identically or with varying parameters, as discussed above.

[0062] At step 1030, the transducer assembly 110 emits a second ping with the acoustic energy beam 170. The second ping may be the same or substantially the same as the first ping - for example, the peak amplitudes of the first ping and the second ping may be substantially the same. Alternatively, the second ping may be different from the first ping. For example, if the calibration covers various types of samples 101 (some samples 101 cause relatively small TB reflections, while some samples cause relatively large TB reflections), different ping amplitudes may be useful. Using different ping amplitudes during the process may provide information useful to other processes while still being compatible with bubble 104 detection.

[0063] TB 124 reflects a portion of the second ping as a second TB reflection. Transducer assembly 110 receives the second TB reflection and processor 143 can process the information. Since the first bubble collapse signal in step 1020 has caused some bubbles 104 to burst, a smaller portion of the acoustic energy can be reflected from TB 124 compared to 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 of them. Examples of such characteristics include total energy or peak amplitude. In the case where the characteristic for each is peak amplitude, such peak amplitude can be the signal itself or a corresponding envelope of the signal, such as a Hilbert envelope. If at least one characteristic in the second TB reflection is different from what is expected (e.g., different from the case where there is no bubble 104 or other type of interference), the processor 143 infers that the bubble 104 exists at the TB 124. For example, the first ping and the second ping can be the same. Therefore, in theory, in an ideal case, the first TB reflection and the second TB reflection should be the same. However, if the second TB reflection is smaller than the first TB reflection (e.g., has a lower peak amplitude or total energy), it can be inferred based on the above-mentioned reflection principle that the bubble 104 exists at the TB 124. In this example, if there is no bubble (or other interference), the second TB reflection is expected to be the same as the first TB reflection. The one / multiple criteria used to infer the presence of the bubble 104 may vary depending on the situation, such as the calibration of a given system.

[0065] Examples of such one / more criteria for inferring the presence of the bubble 104 are various ratios, such as: TB" / TB'; or (TB" / BB") / (TB' / BB'), where BB' and TB' are reflections from BB 125 and TB 124 in the first ping, and where BB" and TB" are reflections from BB 125 and TB 124 in the second ping. If such a ratio is below a given threshold (e.g., 0.6 to 0.95), the presence of the bubble 104 can be inferred.

[0066] At step 1030, if it is inferred that a bubble 104 is present at the TB 124, then the flowchart 1000 proceeds to step 1040. Otherwise, the sequence 1000 may skip step 1040 and proceed to step 1050. Depending on the implementation, steps 1020 and / or 1030 may be repeated prior to step 1040, either identically or with varying parameters, as described above.

[0067] At step 1040, the transducer assembly 110 transmits a second bubble destruction signal. The second bubble destruction signal may be selected to destroy substantially all bubbles 104 (or a majority thereof) at the TB 124. Fig.12 , Fig.13 and Fig.15 The implementation of the second bubble destruction signal is described in the context of.

[0068] According to some embodiments, the second bubble destruction signal includes a pre-conditioning signal and a conditioning signal. Each can occur when the acoustic energy beam 170 is focused on the surface 103 of the sample 101 (e.g., above the focus height in steps 1010, 1020, and 1030). Since the transfer described in step 1050 can occur with the acoustic energy beam 170 focused at the same location, any bubbles 104 at the TB 124 in the path of the beam 170 can be destroyed before the transfer. The pre-conditioning signal can destroy some but not all bubbles 104. Pre-conditioning can reduce the impact of bubbles 104 so that during the conditioning signal, the rate of change of the bubble 104 impact is not so large that the power is too high before recalculation in the next iteration (causing droplets to be accidentally ejected). A possible but not necessary result of skipping pre-conditioning is that during the conditioning process (discussed below), the energy for "fake transfers" reaching the surface 103 may increase as the bubbles 104 are destroyed, and the energy may increase to a level where the droplets 102 are accidentally ejected.

[0069] The pre-adjustment signal may have an amplitude and frequency selected to prevent liquid from being ejected. The amplitude may be fixed. For example, the amplitude may be predetermined by scanning a series of frequencies and amplitudes within a test sample for a given calibration. The pre-adjustment may include a shorter signal (e.g., a duration similar to the first bubble destruction signal) that is repeatedly transmitted (e.g., 50 times). During this repetitive process, the acoustic energy reflection from TB 124 may decrease until it stabilizes to a substantially constant value (e.g., within an acceptable range). For the reasons discussed above, as the bubble 104 continues to be destroyed at TB 124, less and less acoustic energy is reflected. For example, the system 100 may monitor the TB reflection to determine when such a reflection is repeatedly at a substantially constant value (e.g., within an acceptable range). If the stabilization of the TB reflection occurs earlier than the maximum number of iterations, the pre-adjustment signal may terminate. Fig.14 The stabilization of the reflection from TB 124 in an example affected container 126 is shown as iterations continue. The y-axis represents the ratio of the peak amplitude of the reflection from TB 124 to the peak amplitude of the reflection from BB 125. The reflection from BB 125 may be substantially constant throughout the bubble 104 destruction process.

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

[0071] The effect of the remaining bubbles 104 can be a factor in the ridge imaging process (MIP). The adjustment signal can stabilize the ridge imaging scheme. The adjustment signal can include ridge imaging followed by multiple droplet "pseudo transfers" (e.g., 20 droplets are pseudo-ejected), but without increasing the ZVD proportionally to actually eject liquid. The process of ridge imaging followed by multiple pseudo transfers (without ejecting droplets) is repeated until the measured ZVD value stabilizes to a substantially constant value (e.g., within an acceptable range). According to certain embodiments, for a given iteration, ridge imaging is performed, followed by 20 pseudo transfers, although more or fewer pseudo transfers are possible. If the ZVD value stabilizes before a maximum number of iterations (e.g., 20 sequences of (ridge imaging + multiple pseudo transfers)), the adjustment signal can terminate. Fig.15 The stabilization of the ZVD over 20 iterations in an exemplary affected container 126 is shown. The y-axis represents the ZVD value calculated from each MIP measurement. For each iteration, the surface 103 is disturbed with a repetitive signal of increasing amplitude, and the raised bump is measured by measuring the width of the returning surface reflection (SR). The width of the SR corresponds to the size of the bump. When a predetermined size is reached, the ZVD can be calculated - that is, the amplitude theoretically required to break off the following droplet: the droplet is not ejected but falls back into the sample 101. For ADE, the amplitude of the acoustic signal is increased to a value greater than the ZVD, so that the droplet will be ejected. In contrast, a false transfer is performed where the amplitude is the empirically determined ZVD, making it impossible for the droplet to be ejected.

[0072] As part of a ridge imaging process (MIP), a maximum amplitude determination signal can be used to determine the maximum amplitude above which a droplet will be ejected. The maximum amplitude determination signal can include a first disturbance signal, a subsequent first measurement signal, a subsequent second disturbance signal, and a subsequent measurement signal. The first disturbance signal can be selected to disturb the upper surface of the sample, but without ejecting liquid. The peak amplitude and duration of the first disturbance signal can be selected according to a range determined experimentally for a representative fluid. The selected peak amplitude can be slightly smaller than the range determined experimentally. The subsequent first measurement signal can be selected to be reflected by the upper surface of the sample, whereby the processor 143 processes the reflection to quantify the effect of the disturbance, for example, the size of the ridge generated by the first disturbance signal. Based on the size of the ridge compared to the amplitude of the first disturbance signal, the ZVD can be determined. The peak amplitude of the second disturbance signal is greater than the peak amplitude of the first disturbance signal. The peak amplitude can be increased by a value in the range of 0.02dB to 1dB, for example 0.15dB. As with the first measurement signal, the second measurement signal can be selected to be reflected so that the processor 143 can quantify the effect of the disturbance - for example, the size of the ridge produced by the second disturbance signal. Based on the size of the ridge compared to the amplitude of the second disturbance signal, the ZVD can be determined. Based on a measurement of the size of the ridge relative to the amplitude of the disturbance signal, the ZVD can be determined. The disturbance and measurement signals can be repeated, for example, where the peak amplitude of the disturbance signal is continuously increased. There can be a total of 1 to 50 paired disturbance and measurement signal groups (for example, 10 paired groups). The peak amplitude can also be reduced if the power is detected to be too high (for example, if droplets can begin to break off from the surface 103).

[0073] During the pseudo transfer, a bubble mitigation signal having a peak amplitude determined according to a previously determined maximum amplitude is employed. For example, the peak amplitude may be equal to or less than the maximum amplitude (e.g., between 0 dB and 10 dB less than the maximum amplitude, such as 0.5 dB less). The bubble destruction signal may include a plurality of such signals (e.g., two signals), each having a peak amplitude determined according to a previously determined maximum amplitude. The duration of such a signal may be between 8 μs and 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, ejecting droplets may also be acceptable as long as there is not enough energy to hit the target or interfere with the assay. For example, the amplitude may be between zero and 0.5 dB above the ZVD.

[0075] Fig.13Another embodiment of adjusting the signal is shown in flowchart 1300. At step 1310, uplift imaging is performed. At step 1320, a plurality of pseudo transfers are performed. At steps 1330, 1340, and 1350, uplift imaging and a plurality of pseudo transfers (e.g., 20 pseudo transfers) are repeatedly performed until the ZVD measurement is stable (e.g., within acceptable tolerances). Steps 1330, 1340, 1350 may each be repeated at least a predetermined number of times (e.g., three times) during which the ZVD measurement has stabilized. Alternatively or in addition, regardless of whether the ZVD measurement has stabilized a predetermined number of times, steps 1330, 1340, 1350 are repeated a predetermined maximum number of times (e.g., 10 times). At step 1360, ADE is performed based on the determined ZVD.

[0076] Back to Fig.10 At step 1050, according to the ADE principle, the transducer assembly 110 emits a signal to cause the droplet 102 to be ejected from the sample 101. Some examples of ADE are disclosed in US Publication No. 2021 / 0394171 or PCT Publication No. WO2020 / 092407, both of which are incorporated herein by reference in their entirety.

[0077] Depending on the implementation, a third ping may be emitted prior to step 1050. The third ping may be similar to the first ping and / or the second ping. The reflected signal from the third ping may be processed in a similar manner as the first ping and / or the 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 identically or with varying parameters, as described above. Subsequent ping steps and bubble destruction steps may be performed any number of times as desired or as designed.

[0078] Fig.12 104 according to an embodiment. At step 1210, each container 122 is surveyed for bubbles 104. For each container 112, the transducer assembly 110 may be located below the given container 112. Alternatively, rather than surveying the containers 122, containers 122 that are at risk of becoming affected containers 126 may be designated (e.g., Fig.11 As shown, A11, A14, B11, B14, O11, O14, P11 or P14). Step 1210 may correspond to Fig.10 Steps 1010 and / or 1030 are described in the context of Fig.10As described in the context of , the processor 143 may be able to determine which containers 122 have samples 101 with bubbles 104 (ie, affected containers 126 ) by processing the information collected at steps 1010 and 1030 .

[0079] For each affected container 126 (and / or potentially affected container) identified in step 1210, preconditioning 1220 and conditioning 1230 may be performed. For each affected container 126 or potentially affected container, only preconditioning 1220 may be performed, rather than both preconditioning 1220 and conditioning 1230. Preconditioning 1220 and / or conditioning 1230 may be performed on one or more containers 122 that are not affected containers 126. After removing bubbles at steps 1220 and / or 1230, the process proceeds to step 1240, where a transfer (ADE) is performed for each container 122. Steps 1210, 1220, and / or 1230 may be performed for the plate 120 before performing step 1240. Alternatively, steps 1210, 1220, 1230, and / or 1240 may be performed for a given container before being sorted to the next. Step 1240 may correspond to step 1050.

[0080] Fig.19 A flowchart 1900 of a method of destroying bubbles according to an embodiment is shown. The method may be performed by Figure 1 and Figure 2 The method may be performed by components described in the context of FIG. 1 (e.g., processor 143 and transducer assembly 110). Fig.10 , Fig.12 and / or Fig.13 1900. Not all steps need to be performed. For example, steps 1910 and / or 1950 may be bypassed and / or omitted. Other steps may be performed partially or completely simultaneously. For example, steps 1910 and 1920 may be performed simultaneously. The method corresponding to flowchart 1900 may be used for bubble destruction techniques without ADE, such as the transfection embodiments described below. For example, it may not be used. Fig.12 The pre-conditioning 1220 and / or conditioning 1230 described in the context of.

[0081] At 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 based on physical sample features (including BB, TB, and SR) in the collected pulse-echo ultrasonic 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] At step 1920, the number of bubbles is evaluated by inference or estimation. Step 1920 may be combined with Fig.10 Steps 1010 or 1030 described are similar. A ping may be sent from the transducer assembly 110 and the reflected signal may be evaluated. Based on the amount of reflected energy from the TB and / or SR, the number of bubbles may be estimated. The degree of reflection may be based on the peak amplitude of the reflection from the TB and / or the peak amplitude of the reflection from the SR. FIG. 16A to FIG. 1 An example of how the energy reflected by the TB and SR varies based on the number of bubbles in a sample is discussed in the context of FIG6D. The processor 143 can use the reflected energy as an input to an equation, a lookup table, or a machine learning model to estimate the number of bubbles based on previous empirical evaluations of samples and bubble numbers. The number of bubbles in a sample can be estimated as an absolute number or a concentration.

[0083] At step 1930, transducer assembly 110 may transmit a bubble destruction signal. Step 1930 may be similar to step 1020 and / or step 1040. At step 1940, the effectiveness of step 1930 in destroying the bubble is evaluated. Step 1940 may be similar to step 1920 or step 1040. Fig.10 The effectiveness of step 1930 may be determined by comparing the estimated number of bubbles to a threshold value or some other predetermined evaluation method. If a sufficient number of bubbles are destroyed at step 1930, the process may move to the next container at step 1960 (e.g., by moving the transducer assembly 110 or the plate 120). Alternatively, acoustic droplet ejection may be performed before moving to the next container at step 1960. Acoustic droplet ejection may be performed after all bubbles of the relevant sample have been destroyed, or before moving to the next container. ADE may or may not be performed with certain transfection techniques. ADE may be performed, for example, after the bubbles are substantially destroyed.

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

[0085] Combination Fig.19 (as well as Fig.10 and Fig.12) The embodiments described above can be used to automatically identify when a given sample has a different bubble concentration than expected (higher or lower). Such identification can occur at steps 1920 and / or 1940. Subsequent bubble destruction signals can be adjusted to compensate for any such changes.

[0086] FIG. 16A to FIG. 16C An example of the reflection of a ping when different numbers of bubbles are present is shown. A ping is fired towards a sample such as Fig.10 and Fig.19 described in the context of Fig.16A In , sample 16B has a smaller number of bubbles, and the amount of bubbles increases gradually in samples 16B and 16C. The y-axis is shown in counts (where counts are increments on an analog-to-digital (A / D) converter), but is arbitrary in nature and is intended to provide an objective way to compare the FIG. 16A to FIG. 16C How each of these is compared to each other. The x-axis is time. It can be seen that in each graph, the peak amplitude of the BB reflection is the same. However, the peak amplitude of the TB reflection increases as the number of bubbles in the sample increases. For transfection (discussed below), many of the bubbles can be cell-bound, and cells tend to be heavier than the bulk liquid in the sample and therefore will sink to or toward the TB. Bubbles form gas pockets, and where gas pockets are located, more acoustic energy will be reflected. In addition, as the number of bubbles increases, the peak amplitude of the surface reflection (SR) decreases. This is partly 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 can also be caused by suspended bubbles in the bulk liquid of the sample and can be caused by the relatively chaotic or irregular surface geometry caused by bubbles floating at the surface.

[0087] By evaluating the reflected energy from the TB and / or sample surface, the amount of bubbles in the sample can be inferred, as discussed above. For example, a lookup table based on empirical data can be used to estimate the number of bubbles. As another example, a machine learning model can be used. The training of such a model involves the use of training data, including the transmitted signal waveform, the voltage supplied to the transducer assembly, the reflected signal peak amplitude (e.g., from the sample surface and TB), the reflected signal waveform, the sample bulk liquid composition, the number of cells in a given sample, the cell type in the sample, the sample volume, and / or the container size.

[0088] In addition, it is known that bubbles oscillate at a specific resonant frequency, which is related to the size and composition of each bubble. Such composition can include the type of gas in the bubble and the material in the bubble shell (e.g., lipids, proteins, synthetic polymers, etc.). The density and compressibility of the gas and the elasticity and surface energy of the shell can all play a role in changing the resonant frequency of the bubble. The resonant frequency may affect how the bubble absorbs or reflects the ultrasonic signal. The frequency analysis of the reflected waveform can be processed to identify the offset in the reflection and / or absorption spectrum, which can further infer the characteristics of the bubble in the sample, thereby providing information about the size distribution of the bubble in a specific container (e.g., the different sizes of the bubble and the number of each size in the different sizes, or the size relative to the number curve). Such information can also be processed by conventional algorithms, lookup tables, or used to train machine learning models. Such a training model can be used to determine the size and composition of the bubble using subsequent waveforms or features extracted therefrom.

[0089] FIG. 17A to FIG. 17D illustrative examples of reflected signals from a ping after different bubble-busting signals have been transmitted. FIG. 16A to FIG. 1 6D are similar in that they show the signal reflected from the ping. However, FIG. 17A to FIG. 17D The properties of the reflected pings are shown based on how many bubbles have been destroyed in the previous step. In each example, the same number of bubbles are initially present in the sample before the transducer assembly 110 sends the bubble destruction signal. Fig.17A 1 shows the baseline condition of the reflected signal from the container containing the sample when no bubble destruction signal is sent. Fig. 17B In the embodiment, the transducer assembly 110 is provided with an RF electrical signal having a transmit signal circuit 144 (see Figure 1 ) generated a peak voltage of 12.5% ​​of the maximum value. As indicated by the reflections from the ping, some bubbles in the sample have been destroyed, as the peak amplitude of the TB reflection has decreased, while the peak amplitude of the SR has increased. Fig. 17C In FIG. 1 , the transducer assembly 110 is provided with an RF electrical signal having a peak voltage of 25% of the maximum value of the transmit signal circuit 144. It can be seen that the peak amplitude of the TB reflection is greater than Fig.17A and Fig. 17B In addition, the peak amplitude of SR reflection is smaller than Fig.17A and Fig. 17B This indicates that the bubble destruction signal generated by the RF electrical signal with a peak voltage of 25% of the maximum value destroys more bubbles than the RF electrical signal with a peak voltage of 0% or 12.5% ​​of the maximum value. Fig.17DIn FIG. 1 , the transducer assembly 110 is provided with an RF electrical signal having a peak voltage of 50% of the maximum value of the transmit signal circuit 144. It can be seen that the peak amplitude of the TB reflection is greater than Fig.17A , Fig. 17B and Fig. 17C In addition, the peak amplitude of SR reflection is smaller than Fig.17A , Fig. 17B and Fig. 17C This indicates that the bubble destruction signal generated by the RF electric signal having a peak voltage of 50% of the maximum value destroys more bubbles than the RF electric signal having a peak voltage of 0%, 12.5% ​​or 25% of the maximum value.

[0090] According to embodiments, the bubble repair process described herein can be used in association with a transfection procedure. 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 way to perform transfection involves sonoporation. Sonoporation refers to the use of sound in the ultrasonic range to increase the permeability of the cell plasma membrane. Increasing the permeability of the cell plasma membrane allows macromolecules (e.g., DNA) to enter the cell, for example, as desired during transfection. Sonoporation uses acoustic cavitation of microbubbles to enhance the delivery of these macromolecules. The bubbles used for transfection can have a diameter of, for example, 5 μm.

[0091] The information generated by evaluating and / or controlling the quantity (number and / or concentration) of bubbles in the sample can have certain advantages that can be used for the transfection process. For example, this can contribute to quality control. One advantage is to verify that enough bubbles are attached to the cells before sonoporation, and to use, for example, SR and / or TB reflection information to potentially distinguish free / bound bubbles to evaluate (e.g., quantify) the binding efficiency in a given sample. Another advantage is to verify the appropriate clearance of bubbles. This can help identify any problems with the software and / or hardware of the transfection system, and / or help reduce the occurrence of unexplained and / or adverse results. Another advantage allows verification that the appropriate sample (including cells, liquids and bubbles) is in the expected container. This can mark and / or be able to correct pipetting errors, which otherwise may cause experimental failure.

[0092] The techniques used herein can be used to improve real-time and / or closed-loop processes for transfection results. This can dynamically adjust the number, amplitude, and / or duration of bubble destruction signals to suit individual samples or containers. This can provide greater flexibility when evaluating unknown samples and can provide improved process reliability by being able to dynamically adjust the number, amplitude, and / or duration of bubble destruction signals to correct for changes in bubble density resulting from preparing samples for sonoporation.

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

[0094] Furthermore, as described herein, if ADE is used to eject droplets from a sample, it can help to substantially eliminate air bubbles after transfection.

[0095] There can be different groups of bubbles that can be detected using the process described herein. One group is cell-bound microbubbles. As discussed, these can be confined to the bottom (TB) of the container, effectively forming a gas layer. Compared with the reflection from a substantially bubble-free sample (i.e., no gas), this gas can cause a larger reflected signal from the TB. Other groups of microbubbles can include microbubbles suspended in the fluid body or at the surface of the fluid. The microbubbles at these locations will not affect the signal from the TB, but can still affect the signal from the surface reflection (SR) via acoustic scattering.

[0096] Fig.18 Four images from a microscope of cells in a sample with attached microbubbles that were used as part of a transfection process are shown. The first image is a baseline image in which no bubble destruction signal is emitted (0% of maximum signal). In subsequent images, the number of bubbles on the cells is reduced by an increasing amount in accordance with the increasing energy of the bubble destruction signal (the peak voltage provided to the transducer assembly 110 by the transmit signal circuit 144 increases from 12.5% ​​of the maximum value for the weaker bubble destruction signal to 50% of the maximum value for the stronger bubble destruction signal). For these particular examples, the bubble destruction signal has a frequency of 2.25 MHz, a duration of 12 μs, and a sinusoidal shape.

[0097] Figure 20 to Figure 23 The effects and effectiveness of certain techniques disclosed herein on samples containing bubbles and mammalian cells are shown. The samples evaluated are suspensions of mammalian cells with varying densities of attached microbubbles. The transducer assembly sends an ultrasonic ping to the sample, and the transducer assembly receives reflections from the sample. Pings are sent before and after sending a bubble destruction signal and the corresponding reflections are evaluated. The reflection data is parsed using a custom MATLAB script that reads all stored data and then determines the peak amplitude of the reflection from the container's BB, the container's TB, and the sample surface.

[0098] Based on the peak amplitude of the TB reflection, a specific parameter is used to calculate the bubble clearance rate. This parameter is based on the reduction in the TB reflection amplitude after the bubble destruction signal is applied to the sample. Clearance = (TB initial -TBcurrent ) / (TB initial -TB control ), where TB initial is the TB reflection amplitude in the container before sending the bubble destruction signal, TB current is the real-time TB reflection amplitude, TB control is the TB reflection amplitude of a comparable container without any microbubbles.

[0099] In addition to the differences observed in the TB reflection signal, there are also differences that may be caused by bubbles in the signal reflected by the surface of a given sample. As previously explained, the SR reflection can also potentially be evaluated, but in Figure 20 to Figure 23 SR reflections were not considered in the data generation depicted in .

[0100] Fig. 20 A graph of a signal indicating a reduction in bubbles in different samples according to an embodiment is shown. 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 is measured in counts (where counts are increments on an analog-to-digital (A / D) converter), but other measurement metrics are also possible. The number of cells in a given sample corresponds to the number of bubbles in a given sample, assuming that approximately the same number of bubbles are bound to a given cell. A first bubble destruction signal is sent, and then the peak TB reflection amplitude from the ping is measured. The size of the first bubble destruction signal is indicated by the voltage provided to the transducer assembly on the x-axis. In the dashed curve, the peak TB reflection amplitude is plotted for the size of the first bubble destruction signal. In addition, a second bubble destruction signal is sent, and then the peak TB reflection amplitude from the same ping is measured. The size of the second bubble destruction signal is indicated by the voltage provided to the transducer assembly on the x-axis. In the solid curve, the peak TB reflection amplitude is plotted for the size of the second bubble destruction signal. It can be seen that a larger size bubble destruction signal causes more bubbles to be destroyed.

[0101] Fig.21 Shown are graphs indicating reduction of bubbles in different samples, according to embodiments. Fig.21 and Fig. 20 Similar, except Fig.21 The percentage of bubble removal is shown. Fig.21 The clearance parameters shown are as above - that is, clearance = (TB initial -TB current ) / (TB initial -TB control ).

[0102] Fig. 22A graph is shown according to an embodiment, wherein the x-axis shows transfection efficiency (in percent) and the y-axis shows bubble clearance (in percent). Samples with A549 cells were evaluated. Fig.21 Clearance was measured as described. Transfection efficiency of samples was measured using flow cytometry. Fig. 22 The correlation between the bubble clearance rate measured during transfection and the percentage of cells successfully transfected with mRNA encoding green fluorescent protein (GFP) is shown. The transfection efficiency was assessed using flow cytometry to measure the expression of GFP in individual cells. Fig.23 Similar correlations were observed in the case of HEK-293 cells as shown in Figure 1. 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 improvements by dynamically adjusting the number, amplitude, and / or duration of the bubble disruption signal to correct for inadequate clearance of microbubbles.

[0103] As will be appreciated, many of the embodiments described herein may be implemented on or in conjunction with a computer storage product having a non-transient computer readable medium (also referred to as a non-transient processor readable medium) having instructions or computer codes for performing various computer-implemented operations. These embodiments may include embodiments involving processor 143 (or relevant portions of such embodiments). The medium may include one or more different media. The code may be executed on one or more processors, such as processor 143 (which itself may include multiple processors). The computer readable medium (or processor readable medium) is non-transient in the sense that it does not itself include transient propagation signals (e.g., propagating electromagnetic waves carrying information on a transmission medium such as space or a cable). The medium and computer code (also referred to as code) may be those designed and constructed for a specific purpose. Examples of non-transitory computer-readable media include, but are not limited to: magnetic storage media, such as hard disks, floppy disks, and magnetic tapes; optical storage media, such as compact disks / digital video disks (CD / DVD), compact disk read-only memories (CD-ROMs), and holographic devices; magneto-optical storage media, such as optical disks; carrier signal processing modules; and hardware devices specifically configured to store and execute program codes, such as application specific integrated circuits (ASICs), programmable logic devices (PLDs), read-only memories (ROMs), and random access memory (RAM) devices. Other embodiments described herein relate to computer program products, which may include, for example, instructions and / or computer codes discussed herein.

[0104] Some embodiments and / or methods described herein may be implemented by software (implemented 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 (implemented on hardware) may be expressed in a variety of software languages ​​(e.g., computer code), including C, C++, Java, and the like. TM , 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 generated by a compiler, codes for generating web services, and files containing high-level instructions executed by a computer using an interpreter. For example, the following programming languages ​​can be used to implement implementation: imperative programming languages ​​(e.g., C, Fortran, etc.), functional programming languages ​​(Haskell, Erlang, etc.), logical 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, encryption codes, and compression codes.

[0105] Those skilled in the art will appreciate that various changes may be made and may be replaced with equivalents without departing from the scope of the novel technology disclosed in this application. In addition, many modifications may be made to adapt specific situations or materials to the teachings of the novel technology without departing from the scope of the novel technology disclosed in this application. Therefore, it is intended that the novel technology is not limited to the specific technology disclosed, but will include all technologies falling within the scope of the appended claims.

Claims

1. A system for analyzing a sample contained in a container, wherein: The container comprises a bottom surface having a bottom bottom interface (BB) and a bottom top interface (TB), the system comprising: a transducer assembly configured to receive a plurality of electronic transmit signals and emit a corresponding plurality of transmit 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 electronic receive signals; a transmit signal circuit configured to provide the electronic transmit signal to the transducer assembly; a receive signal circuit configured to receive the electronic receive signal from the transducer assembly; and a processor configured to control the transmit signal circuit and receive information corresponding to the electronic receive signal from the receive signal circuit, wherein the plurality of transmitted acoustic signals comprises a sequence of a first ping, a first bubble destruction signal, and a second ping, wherein the plurality of reflected acoustic signals include a first TB reflection corresponding to the first ping and a second TB reflection corresponding to the second ping, and Wherein the system is configured to transmit a second bubble destruction signal based on a comparison of a characteristic of the first TB reflection and a characteristic of the second TB reflection.

2. The system according to claim 1, wherein: The characteristic of the first TB reflection includes a first peak amplitude, the characteristic of the second TB reflection includes a second peak amplitude, and wherein the system is further configured to transmit the second bubble destruction signal if the first peak amplitude is greater than the second peak amplitude.

3. The system according to 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 according to claim 1, wherein: A total energy of the first bubble-destroying signal is greater than a total energy of the first ping and greater than a total energy of the second ping.

5. The system according to claim 1, wherein: The transducer assembly is configured to focus acoustic energy at a first height when transmitting the first ping, the first bubble-busting signal, and the second ping, and wherein the transducer assembly is configured to focus acoustic energy at a second height higher than the first height when transmitting the second bubble-busting signal.

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

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

8. The system according to claim 1, wherein: The first bubble collapse signal comprises a variable frequency.

9. The system according to claim 1, wherein: The energy of the second bubble-destroying signal is selected so as not to cause a droplet to be completely ejected from the sample.

10. The system according to claim 1, wherein: The second bubble destruction signal includes a pre-adjustment signal selected to destroy the bubble and a subsequent adjustment signal selected to destroy the bubble.

11. The system according to claim 10, wherein: The pre-conditioning signal is a bubble destruction signal.

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

13. The system of claim 1, wherein: The container is included in a plate, the plate comprising a plurality of containers, the plurality of containers containing a corresponding plurality of samples, wherein each of the plurality of containers comprises a corresponding BB and TB, wherein the system further comprises at least one motor configured to move at least one of the plate or the transducer assembly such that the transducer assembly is positioned below each of the plurality of containers, wherein the system is further configured to cause the transducer assembly to transmit a sequence of the first ping, the first bubble-destroying signal, and the second ping for each of the plurality of containers, and Wherein the system is further configured to cause the transducer assembly to transmit the second bubble destruction signal for a given container of the plurality of containers based on a comparison of a characteristic of the first TB reflection for the given container with a characteristic of the second TB reflection for the given container.

14. The system according to claim 13, wherein: The system is further configured to transmit the second bubble busting signal for a given container of the plurality of containers after the first ping, the first bubble busting signal, and the second ping have been transmitted for each of the plurality of containers.

15. A method for measuring a sample contained in a container, wherein: The container comprises a bottom surface having a bottom bottom interface (BB) and a bottom top interface (TB), the system comprising: receiving a plurality of electronic transmission signals at a transceiver assembly; transmitting, by the transceiver assembly, a plurality of transmit acoustic signals toward the container and the sample, wherein the plurality of transmit acoustic signals correspond to the plurality of electronic transmit signals; receiving, at the transceiver 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 electronic receive signals corresponding to the plurality of reflected acoustic signals; providing the plurality of electronic transmit signals to the transducer assembly by a transmit signal circuit; receiving the plurality of electronic receive signals from the transducer assembly at a receive signal circuit; controlling the signal transmission circuit using a processor; and receiving, at the processor, information corresponding to the electronic receive signal from the receive signal circuit, wherein the plurality of transmitted acoustic signals comprises a sequence of a first ping, a first bubble destruction signal, and a second ping, wherein the plurality of reflected acoustic signals include a first TB reflection corresponding to the first ping and a second TB reflection corresponding to the second ping, and The method also includes transmitting, by the transducer assembly, a second bubble destruction signal based on a comparison of a characteristic of the first TB reflection and a characteristic of the second TB reflection.

16. The method according to claim 15, wherein: The characteristic of the first TB reflection includes a first peak amplitude, the characteristic of the second TB reflection includes a second peak amplitude, and the method further includes transmitting, by the transducer assembly, the second bubble collapse signal if the first peak amplitude is greater than the second peak amplitude.

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

18. The method according to claim 15, wherein: A total energy of the first bubble-destroying signal is greater than a total energy of the first ping and greater than a total energy of the second ping.

19. The method according to claim 15, further comprising: focusing acoustic energy at a first height using the transceiver assembly while transmitting the first ping, the first bubble-destroying signal, and the second ping; as well as When transmitting the second bubble-destroying signal, acoustic energy is focused with the transceiver assembly at a second height that is higher than the first height.

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

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

22. The method according to claim 15, wherein: The first bubble collapse signal comprises a variable frequency.

23. The method according to claim 15, wherein: The energy of the second bubble-destroying signal is selected so as not to cause a droplet to be completely ejected from the sample.

24. The method according to claim 15, wherein: The second bubble destruction signal includes a pre-adjustment signal selected to destroy the bubble and a subsequent adjustment signal selected to destroy the bubble.

25. The method according to claim 24, wherein: The second bubble destroying signal comprises a pre-conditioned signal selected to destroy bubbles.

26. The method of claim 15, further comprising: A second bubble destruction signal is transmitted by the transceiver assembly while focusing the acoustic energy substantially at the surface of the sample.

27. The method of claim 15, wherein: The container is included in a plate, the plate comprising a plurality of containers, the plurality of containers containing a corresponding plurality of samples, wherein each of the plurality of containers comprises a respective BB and TB, and the method further comprises: moving at least one of the plate or the transducer assembly using at least one motor so that the transducer assembly is positioned beneath each of the plurality of containers at a different time; transmitting, by the transducer assembly, a sequence of the first ping, the first bubble-destroying signal, and the second ping for each of the plurality of containers; and The second bubble destruction signal is transmitted by the transducer assembly for a given container of the plurality of containers based on a comparison of a characteristic of the first TB reflection for the given container with a characteristic of the second TB reflection for the given container.

28. The method according to claim 27, further comprising: The second bubble busting signal is transmitted for a given container of the plurality of containers after the first ping, the first bubble busting signal, and the second ping have been transmitted for each of the plurality of containers.

29. A system for reducing bubbles in a sample contained in a container using an ultrasonic system having a transducer assembly, wherein: The container comprises a bottom surface having a bottom bottom interface (BB) and a bottom top interface (TB), wherein a portion of bubbles in the sample are substantially located at the TB, the system comprising: a transducer assembly configured to align a focus of an acoustic energy beam, wherein the height of the focus is substantially at an upper surface of the sample, wherein the transducer assembly is further configured to emit a bubble destruction signal when the height of the focal point is substantially at the upper surface of the sample, and Wherein the bubble collapse signal comprises a peak amplitude selected to prevent complete ejection of a droplet from the sample.

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

32. The system of claim 29, wherein: The bubble destruction signal also includes the following: a maximum amplitude determination signal selected to determine a maximum amplitude of an acoustic signal by processing reflections 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; and A bubble mitigation signal includes a peak amplitude determined based on the maximum amplitude.

33. The system of claim 32, wherein: The maximum amplitude determination signal comprises: a first perturbation signal selected to perturb the upper surface of the sample; a first measurement signal subsequent to the first disturbance signal, wherein the first measurement signal is selected to be reflected by an upper surface of the sample, whereby reflections of the first measurement signal are processed to determine a zero velocity droplet; a second disturbance signal subsequent to the first measurement signal, wherein the second disturbance signal is selected to disturb an upper surface of the sample, wherein a peak amplitude of the second disturbance signal is greater than a peak amplitude of the first disturbance signal; and a second measurement signal subsequent to the second disturbance signal, wherein the second measurement signal is selected to be reflected by an upper surface of the sample, whereby reflections of the second measurement signal are processed to determine the zero velocity droplet.

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

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

36. The system of claim 35, wherein: Each of the plurality of the bubble destruction signals has a duration of at least 8 us.

37. A method of reducing bubbles in a sample contained in a container using an ultrasonic system having a transducer assembly, wherein: The container comprises a bottom surface having a bottom-end interface (BB) and a bottom-end interface (TB), wherein bubbles in the sample are adjacent to the TB, the method comprising: aligning a focus of an acoustic energy beam using the transducer assembly, wherein the height of the focus is substantially at an upper surface of the sample; and emitting a bubble destruction signal using the transducer assembly when the height of the focal point is substantially at the upper surface of the sample, Wherein the bubble collapse signal comprises a peak amplitude selected to prevent complete ejection of a droplet from the sample.

38. The method of claim 37, further comprising: At least one measurement is performed to determine a peak amplitude of the bubble collapse signal.

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

40. The method of claim 37, wherein: The bubble destruction signal also includes: a maximum amplitude determination signal selected to determine a maximum amplitude of an acoustic signal by processing reflections 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; and A bubble collapse sub-signal includes a peak amplitude determined based on the maximum amplitude.

41. The method of claim 40, wherein: The maximum amplitude determination signal comprises: a first perturbation signal selected to perturb the upper surface of the sample; a first measurement signal subsequent to the first disturbance signal, wherein the first measurement signal is selected to be reflected by an upper surface of the sample, whereby reflections of the first measurement signal are processed to determine a zero velocity droplet; a second disturbance signal subsequent to the first measurement signal, wherein the second disturbance signal is selected to disturb an upper surface of the sample, wherein a peak amplitude of the second disturbance signal is greater than a peak amplitude of the first disturbance signal; and a second measurement signal subsequent to the second disturbance signal, wherein the second measurement signal is selected to be reflected by an upper surface of the sample, whereby reflections of the second measurement signal are processed to determine the zero velocity droplet.

42. The method of claim 41 further comprising a sequence of additional disturbance signals and additional measurement signals, wherein: The additional disturbance signal has an increased peak amplitude.

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

44. The method of claim 43, wherein: Each of the plurality of the bubble destruction signals has a duration of at least 8 us.

45. A system for determining the presence of bubbles in a sample contained in a container in an ultrasound system, wherein: The container comprises a bottom surface having a bottom bottom interface (BB) and a bottom top interface (TB), the method comprising: an ultrasonic transducer assembly configured to transmit a first ping having a first energy, further configured to transmit a second ping having a second energy after the first ping, and further configured to transmit a higher energy signal after the first ping and before the second ping, wherein the higher energy signal has an energy greater than the first energy and the second energy, wherein the ultrasonic transducer assembly is further configured to receive a first reflected signal from the first ping reflected from the TB, and further configured to receive a second reflected signal from the second ping reflected from the TB; and A processor is configured to infer the presence of bubbles in the sample when the peak amplitude of the first reflected signal is greater than the peak amplitude of the second reflected signal.

46. ​​A method for determining the presence of 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, wherein: The container comprises a bottom surface having a bottom bottom interface (BB) and a bottom top interface (TB), the method comprising: transmitting a first ping having a first energy from the ultrasonic transducer assembly; transmitting a second ping having a second energy from the ultrasonic transducer assembly subsequent to the bubble collapse signal; transmitting a higher energy signal from the ultrasonic transducer assembly after the first ping and before the second ping, wherein the higher energy signal has 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 reflected from the TB; receiving, by the ultrasonic transducer assembly, a second reflected signal from the second ping reflected from the TB; and When the peak amplitude of the first reflection signal is greater than the peak amplitude of the second reflection signal, the processor infers that bubbles exist in the sample.

47. A method for performing sonoporation on a sample contained in a container, the sample comprising cells in the sample, wherein: The container comprises a bottom surface having a bottom bottom interface (BB) and a bottom top interface (TB), the method comprising: sending a ping toward the sample by a transducer assembly configured to send and receive acoustic signals; receiving, at the transducer assembly, a reflected signal from the ping, the reflected signal comprising reflections from the TB and from a surface of the sample; measuring, using a processor, the energy of the reflected signal; and The processor estimates a number of bubbles based on the energy of the reflected signal.

48. The method of claim 47, wherein: The estimated number of bubbles is based at least in part on energy reflected from the TB in the reflection signal.

49. The method of claim 47, wherein: The estimated number of bubbles is based at least in part on energy in the reflection signal that is reflected from a surface of the sample.

50. The method of claim 48, wherein: The estimated number of bubbles is based at least in part on energy in the reflection signal that is reflected from a surface of the sample.

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

52. The method of claim 47, further comprising: transmitting a bubble destruction signal using the transducer assembly, the bubble destruction signal being configured to destroy at least a portion of the bubbles; sending 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, wherein the second reflected signal includes a reflection from the TB and a reflection from a surface of the sample; measuring, using the processor, energy of the second reflected signal; and A second number of bubbles is estimated, using the processor, based on energy of the second reflected signal.

53. The method of claim 52, wherein: The estimated second number of bubbles is based at least in part on energy reflected from the TB in the second reflected signal.

54. The method of claim 52, wherein: The estimated number of bubbles is based at least in part on energy in the reflection signal that is reflected from a surface of the sample.

55. The method of claim 53, wherein: The estimated number of bubbles is based at least in part on energy in the reflection signal that is reflected from a surface of the sample.

56. The method of claim 54, further comprising sending a second bubble destroying signal based on the second number of bubbles, the second bubble destroying signal configured to destroy at least a portion of the bubbles.

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

58. The method of claim 56, wherein: The second bubble destruction signal is determined by the processor based at least in part on the second number of bubbles.

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

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

61. A system for performing sonoporation on a sample contained in a container, the sample comprising cells in the sample, wherein: The container comprises a bottom surface having a bottom bottom interface (BB) and a bottom top interface (TB), the system comprising: a transducer assembly configured to send and receive acoustic signals and further configured to send a ping toward the sample and receive a reflected signal from the ping, the reflected signal including reflections from the TB and from a surface of the sample; and A processor is configured to measure the energy of the reflected signal and estimate the number of bubbles based on the energy of the reflected signal.

62. The system of claim 61, wherein: The processor is configured to estimate the number of bubbles based at least in part on energy in the reflection signal from the TB.

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

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

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

66. The system of claim 61, wherein: The transducer assembly is further configured to send a bubble destruction signal configured to destroy at least a portion of the bubbles, the transducer assembly is further configured to send a second ping toward the sample, and receive a second reflected signal in response to the second ping, wherein the second reflected signal includes a reflection from the TB and a reflection from a surface of the sample; and The processor is further configured to measure an energy of the second reflected signal and estimate a second number of bubbles based on the energy of the second reflected signal.

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

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

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

70. The system of claim 66, wherein: The transducer assembly is configured to transmit a second bubble-destroying signal based on a second number of the bubbles, the second bubble-destroying signal being configured to destroy at least a portion of the bubbles.

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

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

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

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

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