Ultrasonic bubble improvement measures

Abstract

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

Description

[Background technology]

[0001] (Cross-reference of related applications) This application claims priority to U.S. Provisional Patent No. 63 / 414,322, filed on 7 October 2022, which is incorporated herein by reference in its entirety.

[0002] Acoustic droplet ejection (ADE) is a technique that uses acoustic energy to move a liquid without any physical contact. Several embodiments of ADE techniques are disclosed in U.S. Patent No. 10,156,499 (Patent Document 1) (which is incorporated herein by reference as a whole). Acoustic energy (e.g., in the form of ultrasonic pulses) is emitted from a transducer toward a certain volume of liquid (hereinafter, the "sample"). In some embodiments, the beam is focused on or near the upper surface of the sample, and the acoustic energy is transmitted to a portion of the sample, thereby moving this portion away upward from the rest of the sample (e.g., as a droplet). The sample may be contained within wells (also referred herein to as containers) of a plate (e.g., a 96- or 384-well microplate). [Prior art documents] [Patent Documents]

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

[0004] According to the embodiment, a system for measuring a sample contained in a container, wherein the container includes a bottom surface having a bottom interface (BB) and a bottom upper interface (TB), the system comprises a transducer assembly configured to receive a plurality of electron transmission signals and emit corresponding plurality of transmitted acoustic signals toward the container and sample, and to receive reflected acoustic signals from the container or sample and generate corresponding plurality of electron reception signals, and a signal transmission network configured to generate electron transmission signals and communicate with the transducer assembly, and electron reception The system comprises a signal receiving network configured to receive signals from a transducer assembly, and a processor configured to control a signal transmission network and receive information corresponding to electronically received signals from the signal receiving network, wherein multiple transmitted acoustic signals comprise a sequence of a first ping, a first bubble-breaking signal, and a second ping, and multiple reflected acoustic signals comprise a first TB reflection corresponding to the first ping and a second TB reflection corresponding to the second ping, and the system is configured to emit a second bubble-breaking signal based on a comparison of the characteristics of the first TB reflection and the characteristics of the second TB reflection. The characteristics of the first TB reflection may comprise a first peak amplitude, and the characteristics of the second TB reflection may comprise a second peak amplitude, and the system may further be configured to emit a second bubble-breaking signal if the first peak amplitude exceeds the second peak amplitude. The peak amplitudes of the first ping and the second ping may be substantially the same. The total energy of the first bubble-breaking signal may exceed the total energy of the first ping and the total energy of the second ping. The transducer assembly may be configured to focus the acoustic energy to a first height when emitting the first ping, the first bubble-breaking signal, and the second ping, and the transducer assembly may be configured to focus the acoustic energy to a second height above the first height when emitting the second bubble-breaking signal. The first height may be predetermined relative to the TB, and the second height may be substantially at the surface of the sample. The first height is between + / - 6 mm relative to the TB.The first bubble-breaking signal may include a variable frequency. The energy of the second bubble-breaking signal may be selected so as not to completely eject the droplet from the sample. The second bubble-breaking signal may include a pre-adjustment signal selected to break the bubble and a subsequent adjustment signal selected to break the bubble. The pre-adjustment signal may be the bubble-breaking signal. The transducer assembly may be configured to emit the second bubble-breaking signal while focusing the acoustic energy substantially onto the surface of the sample. The containers may be contained within a plate comprising a plurality of containers, each containing a corresponding plurality of samples, each of which may include a corresponding BB and TB, and the system may further include at least one motor configured to move the plate or at least one of the transducer assemblies such that a transducer assembly is positioned directly beneath each of the plurality of containers, and the system may further be configured to cause the transducer assembly to emit a sequence of a first ping, a first bubble-breaking signal, and a second ping for each of the plurality of containers, and the system may further be configured to cause the transducer assembly to emit a second bubble-breaking signal for a given plurality of containers based on a comparison of the characteristics of a first TB reflection for a given container and the characteristics of a second TB reflection for a given container, and the system may further be configured to emit a second bubble-breaking signal for a given plurality of containers after the first ping, the first bubble-breaking signal, and the second ping have been emitted for each of the plurality of containers.

[0005] According to the embodiment, a method is provided for measuring a sample contained in a container, wherein the container includes a bottom surface having a bottom interface (BB) and a bottom upper interface (TB), and the method comprises the steps of: receiving a plurality of electron transmission signals in a transceiver assembly; transmitting a plurality of transmitted acoustic signals toward the container and sample by the transceiver assembly, wherein the plurality of transmitted acoustic signals correspond to a plurality of electron transmission signals; receiving a plurality of reflected acoustic signals from the container or sample in the transceiver assembly; generating a plurality of electron reception signals corresponding to the plurality of reflected acoustic signals by the transceiver assembly; and generating a plurality of electron transmission signals by a signal transmission network. The method includes the steps of communicating multiple electronic transmission signals to a transducer assembly, receiving multiple electronic reception signals from the transducer assembly in a signal receiving network, controlling the signal transmission network using a processor, and receiving information corresponding to the electronic reception signals from the signal receiving network in the processor, wherein the multiple transmitted acoustic signals comprise a sequence of a first ping, a first bubble-breaking signal, and a second ping, and the multiple reflected acoustic signals comprise a first TB reflection corresponding to the first ping and a second TB reflection corresponding to the second ping, and the method further includes the step of the transducer assembly emitting a second bubble-breaking signal based on a comparison of the characteristics of the first TB reflection and the characteristics of the second TB reflection. The characteristics of the first TB reflection may comprise a first peak amplitude, and the characteristics of the second TB reflection may comprise a second peak amplitude, and the method further includes the step of the transducer assembly emitting a second bubble-breaking signal if the first peak amplitude exceeds the second peak amplitude. The peak amplitudes of the first ping and the second ping may be substantially the same. The total energy of the first bubble-breaking 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 further include the steps of focusing acoustic energy to a first height using a transceiver assembly when emitting a first ping, a first bubble-breaking signal, and a second ping, and focusing acoustic energy to a second height above the first height using a transceiver assembly when emitting a second bubble-breaking signal. The first height may be predetermined relative to the TB, and the second height may be substantially on the surface of the sample. The first height may be between + / - 6 mm relative to the TB. The first bubble-breaking signal may include a variable frequency. The energy of the second bubble-breaking signal may be selected so as not to completely eject the droplet from the sample. The second bubble-breaking signal may include a pre-adjustment signal selected to break the bubble and a subsequent adjustment signal selected to break the bubble. The pre-adjustment signal may be the bubble-breaking signal. The method may further include the step of emitting a second bubble-breaking signal while focusing acoustic energy substantially on the surface of the sample using a transceiver assembly. The containers may be contained within a plate comprising a plurality of containers, each containing a corresponding plurality of samples, wherein each plurality of containers comprises a corresponding BB and TB. The method may further include the steps of moving the plate or at least one of the transducer assemblies using at least one motor so that a transducer assembly is positioned directly beneath each of the plurality of containers; the transducer assembly emitting a sequence of a first ping, a first bubble-breaking signal, and a second ping for each of the plurality of containers; and the transducer assembly emitting a second bubble-breaking signal for a given plurality of containers based on a comparison of the characteristics of a first TB reflection for a given container with the characteristics of a second TB reflection for a given container. The transducer assembly may further emitting a second bubble-breaking signal for a given plurality of containers after the first ping, the first bubble-breaking signal, and the second ping have been emitted for each of the plurality of containers. A non-transient computer-readable medium contains instructions that, when executed by a processor, can carry out any one of the aforementioned method embodiments.

[0006] According to one embodiment, a system 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 interface (BB) and a bottom upper interface (TB), and the bubbles in the sample are centered at the TB, the system includes a transducer assembly configured to align the focus of an acoustic energy beam, the height of which the focus is substantially at the upper surface of the sample, and the transducer assembly is further configured to emit a bubble-breaking signal while the height of which the focus is substantially at the upper surface of the sample, the bubble-breaking signal having a peak amplitude selected to prevent the complete ejection of droplets from the sample. The system may be configured to perform at least one measurement to determine the peak amplitude of the bubble-breaking signal, the peak amplitude may include substantially the maximum amplitude before the droplet would be completely ejected from the sample. The bubble-breaking signal may further include a maximum amplitude determination signal, which is selected to determine the maximum amplitude of the acoustic signal by processing the reflection of the maximum amplitude determination signal from the upper surface of the sample, and a bubble-reducing signal, which includes a peak amplitude determined according to the maximum amplitude, and an acoustic signal having an amplitude greater than the maximum amplitude, which will cause droplets to be ejected from the sample. The maximum amplitude determination signal may include a first disturbance signal selected to disturb the upper surface of the sample, followed by a first measurement signal, the first measurement signal being selected to be reflected by the upper surface of the sample, so that the reflection of the first measurement signal is processed to determine a zero-velocity droplet, followed by a second disturbance signal, the second disturbance signal being selected to disturb the upper surface of the sample, with the peak amplitude of the second disturbance signal exceeding the peak amplitude of the first disturbance signal, followed by a second measurement signal, the second measurement signal being selected to be reflected by the upper surface of the sample, so that the reflection of the second measurement signal is processed to determine a zero-velocity droplet.The system may also be configured to emit sequences of additional disturbance signals and additional measurement signals, the additional disturbance signals having increasing peak amplitudes. The bubble reduction signal may include multiple signals, each having a peak amplitude determined according to the maximum amplitude. Each of the multiple signals in the bubble reduction signal may have a duration of at least 8 μs.

[0007] According to an embodiment, a method is provided for reducing bubbles in a sample contained in a container using an ultrasonic system having a transducer assembly, wherein the container includes a bottom surface having a bottom interface (BB) and a bottom upper interface (TB), and the bubbles in the sample are centered at the TB, and the method includes the steps of: aligning the focus of an acoustic energy beam using the transducer assembly, wherein the height of the focus is substantially at the upper surface of the sample; and emitting a bubble-breaking signal using the transducer assembly while the height of the focus is substantially at the upper surface of the sample, wherein the bubble-breaking signal has a peak amplitude selected to prevent the complete ejection of droplets from the sample. The method may further include the step of performing at least one measurement to determine the peak amplitude of the bubble-breaking signal, wherein the peak amplitude may include substantially the maximum amplitude before the droplet would be completely ejected from the sample. The bubble-breaking signal may further include a maximum amplitude determination signal, which is selected to determine the maximum amplitude of the acoustic signal by processing the reflection of the maximum amplitude determination signal from the upper surface of the sample, and a bubble-reducing signal, which includes a peak amplitude determined according to the maximum amplitude, and an acoustic signal having an amplitude greater than the maximum amplitude, which will cause droplets to be ejected from the sample. The maximum amplitude determination signal may include a first disturbance signal selected to disturb the upper surface of the sample, followed by a first measurement signal, the first measurement signal being selected to be reflected by the upper surface of the sample, so that the reflection of the first measurement signal is processed to determine a zero-velocity droplet, followed by a second disturbance signal, the second disturbance signal being selected to disturb the upper surface of the sample, with the peak amplitude of the second disturbance signal exceeding the peak amplitude of the first disturbance signal, followed by a second measurement signal, the second measurement signal being selected to be reflected by the upper surface of the sample, so that the reflection of the second measurement signal is processed to determine a zero-velocity droplet.The method may further include a sequence of additional disturbance signals and additional measurement signals, the additional disturbance signals having increasing peak amplitudes. The bubble reduction signals 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 reduction signals may have a duration of at least 8 μs. A non-transient computer-readable medium contains instructions that, when executed by a processor, can carry out any one of the aforementioned method embodiments.

[0008] According to one embodiment, a system is provided for determining the presence of bubbles in a sample contained within a container in an ultrasonic system, wherein the container includes a bottom surface having a bottom interface (BB) and a bottom upper interface (TB), and the system comprises an ultrasonic transducer assembly configured to emit a first ping having a first energy, further configured to emit a second ping having a second energy following a bubble-breaking signal, and further configured to emit a higher energy signal following the first ping and preceding the second ping, wherein the higher energy signal has an energy greater than the first and second energies, and 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 exceeds the peak amplitude of the second reflected signal.

[0009] According to the embodiment, a method is provided for determining the presence of bubbles in a sample contained in a container within an ultrasonic system, the system comprising an ultrasonic transducer assembly and a processor communicating with the ultrasonic transducer assembly, wherein the container includes a bottom surface having a bottom interface (BB) and a bottom upper interface (TB), and the method comprises the steps of: emitting a first ping having a first energy from the ultrasonic transducer assembly; emitting a second ping having a second energy following a bubble-breaking signal from the ultrasonic transducer assembly; and emitting a signal from the ultrasonic transducer assembly following the first ping. The method subsequently includes, and prior to, a step of emitting a higher energy signal, wherein the higher energy signal has energy greater than the first and second energies; a step of receiving a first reflected signal reflected from the TB from a first ping by an ultrasonic transducer assembly; a step of receiving a second reflected signal reflected from the TB from a second ping by an ultrasonic transducer assembly; and a step of inferring the presence of bubbles in the sample when the peak amplitude of the first reflected signal exceeds the peak amplitude of the second reflected signal by a processor. A non-transient computer-readable medium may contain instructions that, when executed by the processor, carry out any of the method embodiments described above.

[0010] According to the embodiment, a method for performing sonoporation on a sample containing cells in a container, wherein the container includes a bottom surface having a bottom interface (BB) and a bottom upper interface (TB), the method includes the steps of: transmitting a ping toward the sample by a transducer assembly configured to transmit and receive an acoustic signal; receiving a reflected signal in the transducer assembly, including reflections from the ping, from the TB and from the surface of the sample; measuring the energy of the reflected signal using a processor; and estimating the amount of bubbles using the processor based on the energy of the reflected signal. The step of estimating the amount of bubbles may, at least in part, be based on the energy of the reflection from the TB in the reflected signal. The step of estimating the amount of bubbles may, at least in part, be based on the energy of the reflection from the surface of the sample in the reflected signal. The step of estimating the amount of bubbles may, at least in part, be based on the energy of the reflection from the surface of the sample in the reflected signal. The energy of the reflected signal may correspond to the peak amplitude of the reflected signal with respect to a given reflection. The method may further include the steps of: transmitting a bubble-breaking signal using a transducer assembly configured to break at least a portion of the bubbles; transmitting a second ping toward a sample using a transducer assembly; receiving a second reflected signal in response to the second ping using a transducer assembly, wherein the second reflected signal includes reflections from a TB and reflections from the surface of the sample; measuring the energy of the second reflected signal using a processor; and estimating the amount of a second bubble using a processor based on the energy of the second reflected signal. The step of estimating the amount of a second bubble may at least partially be based on the energy of the reflection from the TB in the second reflected signal. The step of estimating the amount of a bubble may at least partially be based on the energy of the reflection from the surface of the sample in the reflected signal.The step of estimating the amount of the bubbles may be at least partially based on the energy of the reflection from the surface of the sample in the reflected signal. The method may further include the step of transmitting a second bubble destruction signal configured to destroy at least a part of the bubbles, based on the amount of the second bubbles. The second bubble destruction signal may be different from the bubble destruction signal. The second bubble destruction signal may be determined by a processor, at least partially, based on the amount of the second bubbles. The method may further include the step of performing transfection on cells in the sample. The method may further include the step of predicting the efficiency of transfection, at least partially, based on the amount of the bubbles. The system may be configured to implement any one of the foregoing method embodiments. The non-transitory computer-readable medium contains instructions that, when executed by a processor, may implement any one of the foregoing method embodiments. The present invention provides, for example, the following items: (Item 1) A system for analyzing a sample contained in a container, wherein the container includes a bottom surface having a bottom interface (BB) and a bottom upper interface (TB), and the system is A transducer assembly configured to receive a plurality of electron transmission signals and emit corresponding plurality of transmitted acoustic signals toward the container and the sample, and to receive reflected acoustic signals from at least one of the container or the sample and generate corresponding plurality of electron reception signals, A signal transmission network configured to provide the above-mentioned electron transmission signals to the transducer assembly, A signal receiving network configured to receive the above-mentioned electronic receiving signal from the above-mentioned transducer assembly, A processor configured to control the above signal transmission network and to receive information corresponding to the above electronic received signal from the above signal receiving network. Equipped with, The above-mentioned multiple transmitted acoustic signals comprise a sequence of a first ping, a first bubble-breaking signal, and a second ping. The above multiple reflected acoustic signals include a first TB reflection corresponding to the first ping and a second TB reflection corresponding to the second ping. The system is configured to emit a second bubble-breaking signal based on a comparison of the characteristics of the first TB reflection and the characteristics of the second TB reflection. (Item 2) The system according to item 1, wherein the first TB reflection characteristic comprises a first peak amplitude, the second TB reflection characteristic comprises a second peak amplitude, and the system is further configured to emit a second bubble-breaking signal when the first peak amplitude exceeds the second peak amplitude. (Item 3) The system described in item 1, wherein the peak amplitude of the first ping and the peak amplitude of the second ping are substantially the same. (Item 4) The system as described in item 1, wherein the total energy of the first bubble-breaking signal exceeds the total energy of the first ping and also exceeds the total energy of the second ping. (Item 5) The system according to item 1, wherein the transducer assembly is configured to focus acoustic energy to a first height when emitting the first ping, the first bubble burst signal, and the second ping, and the transducer assembly is configured to focus acoustic energy to a second height above the first height when emitting the second bubble burst signal. (Item 6) The first height is predetermined relative to the TB, and the second height is substantially on the surface of the sample, as described in item 5. (Item 7) The above first height is between + / - 6 mm relative to the above TB, as described in item 5 of the system. (Item 8) The first bubble-rupturing signal described above is the system described in item 1, which has a variable frequency. (Item 9) The energy of the second bubble-breaking signal described above is selected so as not to completely eject the droplet from the sample, as in the system described in item 1. (Item 10) The system described in item 1, wherein the second bubble-breaking signal includes a pre-adjustment signal selected to break bubbles and a subsequent adjustment signal selected to break bubbles. (Item 11) The above pre-adjustment signal is a bubble bursting signal, as described in item 10 of the system. (Item 12) The system according to item 1, wherein the transducer assembly is configured to emit the second bubble-rupturing signal while substantially focusing acoustic energy onto the surface of the sample. (Item 13) The above container is contained within a plate comprising multiple containers containing corresponding samples. Each of the above multiple containers contains the corresponding BB and TB. The system further comprises at least one motor configured to move the plate or at least one of the transducer assemblies such that the transducer assembly is positioned directly beneath each of the plurality of containers. The system is further configured to cause the transducer assembly to emit a sequence of the first ping, the first bubble-breaking signal, and the second ping for each of the plurality of containers. The system is further configured to cause the transducer assembly to emit a second bubble-breaking signal for a given number of containers, based on a comparison of the characteristics of the first TB reflection for a given container and the characteristics of the second TB reflection for a given container. The system described in item 1. (Item 14) The system according to item 13, wherein the system is further configured to issue the second bubble-breaking signal for a given of the plurality of containers after the first ping, the first bubble-breaking signal, and the second ping have been issued for each of the plurality of containers. (Item 15) A method for measuring a sample contained in a container, wherein the container includes a bottom surface having a bottom interface (BB) and a bottom upper interface (TB), and the system is In a transceiver assembly, receiving multiple electronic transmission signals, The above transceiver assembly transmits multiple acoustic signals to the container and the sample, wherein the multiple acoustic signals correspond to the multiple electronic transmission signals. In the above transceiver assembly, multiple reflected acoustic signals are received from at least one of the above containers or samples, The above transceiver assembly generates multiple electronically received signals corresponding to the multiple reflected acoustic signals, The signal transmission network provides the above-mentioned plurality of electron transmission signals to the transducer assembly, In the signal receiving circuit network, the plurality of electronic receiving signals are received from the transducer assembly, Using a processor to control the above signal transmission network, In the above processor, information corresponding to the above electronic reception signal is received from the above signal reception network. Includes, The above-mentioned multiple transmitted acoustic signals comprise a sequence of a first ping, a first bubble-breaking signal, and a second ping. The above multiple reflected acoustic signals include a first TB reflection corresponding to the first ping and a second TB reflection corresponding to the second ping. A method further comprising using the transducer assembly described above to generate a second bubble-breaking signal based on a comparison between the characteristics of the first TB reflection and the characteristics of the second TB reflection. (Item 16) The method according to item 15, further comprising the first TB reflection characteristic having a first peak amplitude, the second TB reflection characteristic having a second peak amplitude, and the transducer assembly emitting the second bubble-breaking signal when the first peak amplitude exceeds the second peak amplitude. (Item 17) The method according to item 15, wherein the peak amplitude of the first ping and the peak amplitude of the second ping are substantially the same. (Item 18) The method according to item 15, wherein the total energy of the first bubble-breaking signal exceeds the total energy of the first ping and also exceeds the total energy of the second ping. (Item 19) When the first ping, the first bubble bursting signal, and the second ping are emitted, the transceiver assembly is used to focus the acoustic energy to a first height. When the second bubble-rupturing signal is emitted, the transceiver assembly is used to focus the acoustic energy to a second height above the first height. The method described in item 15, further including the method described in item 15. (Item 20) The first height is predetermined relative to the TB, and the second height is substantially on the surface of the sample, as described in item 19. (Item 21) The first height mentioned above is between + / - 6 mm relative to the TB, as described in item 15. (Item 22) The first bubble-rupturing signal described above is provided by the method in item 15, which includes a variable frequency. (Item 23) The energy of the second bubble-rupturing signal described above is selected so as not to completely eject the droplet from the sample, as described in item 15. (Item 24) The method according to item 15, wherein the second bubble-breaking signal described above includes a pre-adjustment signal selected to break bubbles and a subsequent adjustment signal selected to break bubbles. (Item 25) The method described in item 24, wherein the second bubble-breaking signal described above includes a pre-adjustment signal selected to break the bubbles. (Item 26) The method according to item 15, further comprising emitting a second bubble-breaking signal while substantially focusing acoustic energy onto the surface of the sample using the above-described transceiver assembly. (Item 27) The above container is contained within a plate comprising a plurality of containers containing a plurality of corresponding samples, each of the plurality of containers containing a corresponding BB and TB, Using at least one motor, move the plate or at least one of the transducer assemblies so that the transducer assembly is positioned directly beneath each of the multiple containers at different times, The transducer assembly generates the sequence of the first ping, the first bubble-breaking signal, and the second ping for each of the multiple containers. The transducer assembly generates a second bubble-breaking signal for a given number of containers based on a comparison of the characteristics of the first TB reflection for a given container and the characteristics of the second TB reflection for a given container. The method described in item 15, further including the method described in item 15. (Item 28) The method according to item 27, further comprising issuing the second bubble-breaking signal for a given container after the first ping, the first bubble-breaking signal, and the second ping have been issued for each of the containers. (Item 29) A system 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 interface (BB) and a bottom upper interface (TB), and a portion of the bubbles in the sample are substantially located at the TB, and the system is A transducer assembly configured to align the focus of an acoustic energy beam, wherein the height of the focus is substantially on the upper surface of the sample. Equipped with, The transducer assembly described above is further configured to emit a bubble-rupturing signal while the height of the focal point is substantially on the upper surface of the sample. The bubble bursting signal described above has a peak amplitude selected to prevent the complete ejection of droplets from the sample. (Item 30) The system described in item 29 is configured to perform at least one measurement and determine the peak amplitude of the bubble burst signal. (Item 31) The above peak amplitude substantially reaches its maximum amplitude before the droplet is completely ejected from the above sample, as described in item 30. (Item 32) The above bubble burst signal is transmitted by the above transducer assembly. A maximum amplitude determination signal selected to determine the maximum amplitude of an acoustic signal by processing the reflection of the maximum amplitude determination signal from the upper surface of the above sample, wherein an acoustic signal having an amplitude greater than the above maximum amplitude will cause a droplet to be ejected from the above sample, and the maximum amplitude determination signal, A bubble reduction signal including a peak amplitude determined according to the maximum amplitude above, and The system described in item 29, further including the transmission of. (Item 33) The maximum amplitude determination signal mentioned above is, A first disturbance signal selected to disturb the upper surface of the above sample, A first measurement signal following the first disturbance signal, wherein the first measurement signal is selected to be reflected by the upper surface of the sample, and thereby the reflection of the first measurement signal is processed to determine a zero-velocity droplet, and A second disturbance signal following the first measurement signal, wherein the second disturbance signal is selected to disturb the upper surface of the sample, and the peak amplitude of the second disturbance signal exceeds the peak amplitude of the first disturbance signal. A second measurement signal following the second disturbance signal, wherein the second measurement signal is selected to be reflected by the upper surface of the sample, and the reflection of the second measurement signal is processed to determine the zero-velocity droplet. The system described in item 32, comprising: (Item 34) The transducer assembly described above is further configured to emit a sequence of additional disturbance signals and additional measurement signals, wherein the additional disturbance signals have increasing peak amplitudes, as described in item 33. (Item 35) The bubble bursting signal system according to item 29 comprises a plurality of signals, each having a peak amplitude determined according to the maximum amplitude. (Item 36) The system as described in item 35, wherein each of the multiple signals in the bubble bursting signal has a duration of at least 8 μs. (Item 37) A method 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 interface (BB) and a bottom upper interface (TB), the bubbles in the sample come into contact with the TB, and the method is The transducer assembly described above is used to align the focus of the acoustic energy beam, wherein the height of the focus is substantially on the upper surface of the sample. While the height of the above focal point is substantially on the upper surface of the above sample, the transducer assembly is used to emit a bubble bursting signal. Includes, The bubble burst signal described above has a peak amplitude selected to prevent the complete ejection of droplets from the sample. (Item 38) The method according to item 37, further comprising performing at least one measurement to determine the peak amplitude of the bubble bursting signal. (Item 39) The method according to item 38, wherein the peak amplitude described above has substantially the maximum amplitude before the droplet would be completely ejected from the sample. (Item 40) The above bubble bursting signal is, A maximum amplitude determination signal selected to determine the maximum amplitude of an acoustic signal by processing the reflection of the maximum amplitude determination signal from the upper surface of the above sample, wherein an acoustic signal having an amplitude greater than the above maximum amplitude will cause a droplet to be ejected from the above sample, and the maximum amplitude determination signal, A bubble-breaking sub-signal including a peak amplitude determined according to the maximum amplitude above, The method described in item 37, further comprising: (Item 41) The maximum amplitude determination signal mentioned above is, A first disturbance signal selected to disturb the upper surface of the above sample, A first measurement signal following the first disturbance signal, wherein the first measurement signal is selected to be reflected by the upper surface of the sample, and thereby the reflection of the first measurement signal is processed to determine a zero-velocity droplet, and A second disturbance signal following the first measurement signal, wherein the second disturbance signal is selected to disturb the upper surface of the sample, and the peak amplitude of the second disturbance signal exceeds the peak amplitude of the first disturbance signal. A second measurement signal following the second disturbance signal, wherein the second measurement signal is selected to be reflected by the upper surface of the sample, and the reflection of the second measurement signal is processed to determine the zero-velocity droplet. The method described in item 40, comprising: (Item 42) The method according to item 41, further comprising a sequence of additional disturbance signals and additional measurement signals, wherein the additional disturbance signals have increasing peak amplitudes. (Item 43) The method according to item 39, wherein the bubble bursting signal comprises a plurality of signals, each having a peak amplitude determined according to the maximum amplitude. (Item 44) The method according to item 43, wherein each of the multiple signals in the bubble burst signal has a duration of at least 8 μs. (Item 45) A system for determining the presence of bubbles in a sample contained within a container in an ultrasonic system, wherein the container includes a bottom surface having a bottom interface (BB) and a bottom upper interface (TB), and the method is: An ultrasonic transducer assembly, wherein the ultrasonic transducer assembly is configured to emit a first ping having a first energy, further configured to emit a second ping having a second energy following the first ping, further configured to emit a higher energy signal following the first ping and prior to the second ping, the higher energy signal having energy greater than the first and second energies, and the ultrasonic transducer assembly is 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, A processor, wherein the processor is configured to infer the presence of bubbles in the sample when the peak amplitude of the first reflected signal exceeds the peak amplitude of the second reflected signal. A system that includes these features. (Item 46) A method for determining the presence of bubbles in a sample contained in a container within an ultrasonic system having an ultrasonic transducer assembly and a processor communicating with the ultrasonic transducer assembly, wherein the container includes a bottom surface having a bottom interface (BB) and a bottom upper interface (TB), and the method is: The ultrasonic transducer assembly above emits a first ping having a first energy, The ultrasonic transducer assembly emits a second ping with a second energy following the bubble-breaking signal. The ultrasonic transducer assembly emits a higher-energy signal following the first ping and prior to the second ping, wherein the higher-energy signal has energy exceeding that of the first and second energies. The ultrasonic transducer assembly receives the first reflected signal reflected from the TB from the first ping, The ultrasonic transducer assembly receives the second reflected signal reflected from the TB from the second ping, The above processor infers the presence of bubbles in the sample when the peak amplitude of the first reflected signal exceeds the peak amplitude of the second reflected signal. Methods that include... (Item 47) A method for performing sonoporation on a sample containing cells in a container, wherein the container includes a bottom surface having a bottom interface (BB) and a bottom upper interface (TB), and the method is: A transducer assembly configured to transmit and receive acoustic signals transmits the ping towards the above sample, In the transducer assembly described above, the reflected signal is received from the ping, the TB, and the surface of the sample, Using a processor, the energy of the reflected signal is measured, Using the above processor, the amount of bubbles is estimated based on the energy of the reflected signal. Methods that include... (Item 48) Estimating the amount of bubbles described above is done according to the method in item 47, at least in part, based on the energy of the reflection from the TB in the reflected signal described above. (Item 49) Estimating the amount of bubbles described above is done, at least in part, based on the energy of the reflection from the surface of the sample in the reflected signal, as described in item 47. (Item 50) Estimating the amount of bubbles described above is done, at least in part, based on the energy of the reflection from the surface of the sample in the reflected signal, as described in item 48. (Item 51) The energy of the reflected signal described above corresponds to the peak amplitude of the reflected signal with respect to a given reflection, as described in item 47. (Item 52) Using the above transducer assembly, a bubble-breaking signal is transmitted which is configured to break at least a portion of the bubbles. Using the above transducer assembly, a second ping is transmitted towards the above sample, The transducer assembly described above is used to receive a second reflected signal in response to the second ping, wherein the second reflected signal includes reflections from the TB and reflections from the surface of the sample. Using the above processor, the energy of the second reflected signal is measured, Using the above processor, the amount of the second bubble is estimated based on the energy of the second reflected signal. The method described in item 47, further including the method described in item 47. (Item 53) Estimating the amount of the second bubble described above is, at least in part, based on the energy of the reflection from the TB in the second reflected signal, as described in item 52. (Item 54) Estimating the amount of bubbles described above is done according to the method described in item 52, at least in part, based on the energy of the reflection from the surface of the sample in the reflected signal described above. (Item 55) Estimating the amount of bubbles described above is done, at least in part, based on the energy of the reflection from the surface of the sample in the reflected signal, as described in item 53. (Item 56) The method according to item 54, further comprising transmitting a second bubble-breaking signal configured to break at least a portion of the bubbles based on the amount of the second bubbles. (Item 57) The second bubble bursting signal described above is different from the bubble bursting signal described above, as described in item 56. (Item 58) The method according to item 56, wherein the second bubble bursting signal is determined by the processor at least partially based on the amount of the second bubble. (Item 59) The method according to item 47, further comprising performing transfection on cells in the above sample. (Item 60) The method according to item 59, further comprising predicting the efficiency of the transfection based at least in part on the amount of bubbles. (Item 61) A system for performing sonoporation on a sample containing cells in a container, wherein the container includes a bottom surface having a bottom interface (BB) and a bottom upper interface (TB), and the system is A transducer assembly, wherein the transducer assembly is configured to transmit and receive acoustic signals, transmit a ping toward the sample, and receive reflected signals from the ping, including reflections from the TB and from the surface of the sample. A processor, wherein the processor is configured to measure the energy of the reflected signal and estimate the amount of bubbles based on the energy of the reflected signal. A system that includes these features. (Item 62) The system according to item 61, wherein the processor is configured, at least in part, to estimate the amount of bubbles based on the energy of the reflection from the TB in the reflected signal. (Item 63) The system according to item 61, wherein the processor is configured, at least in part, to estimate the amount of bubbles based on the energy of the reflection from the surface of the sample in the reflected signal. (Item 64) The system according to item 62, wherein the processor is configured, at least in part, to estimate the amount of bubbles based on the energy of the reflection from the surface of the sample in the reflected signal. (Item 65) The energy of the reflected signal corresponds to the peak amplitude of the reflected signal with respect to a given reflection, as described in item 61 of the system. (Item 66) The transducer assembly is further configured to transmit a bubble-breaking signal configured to break at least a portion of the bubbles, to transmit a second ping toward the sample, and to receive a second reflected signal in response to the second ping, the second reflected signal including reflections from the TB and reflections from the surface of the sample. The processor described above is further configured to measure the energy of the second reflected signal and to estimate the amount of the second bubble based on the energy of the second reflected signal. The system described in item 61. (Item 67) The system according to item 66, wherein the processor is configured, at least in part, to estimate the amount of the second bubble based on the energy of the reflection from the TB in the second reflected signal. (Item 68) The system according to item 66, wherein the processor is configured, at least in part, to estimate the amount of bubbles based on the energy of the reflection from the surface of the sample in the reflected signal. (Item 69) The system according to item 67, wherein the processor is configured, at least in part, to estimate the amount of bubbles based on the energy of the reflection from the surface of the sample in the reflected signal. (Item 70) The system according to item 66, wherein the transducer assembly is configured to transmit a second bubble-breaking signal configured to break at least a portion of the bubbles based on the amount of the second bubbles. (Item 71) The second bubble bursting signal described above is different from the bubble bursting signal described above, as in the system described in item 70. (Item 72) The system according to item 70, wherein the second bubble bursting signal is determined by the processor at least partially based on the amount of the second bubble. (Item 73) The system described in item 61, further comprising performing transfection on cells in the above sample. (Item 74) The system according to item 73, further comprising predicting the efficiency of the transfection based at least in part on the amount of bubbles. [Brief explanation of the drawing]

[0011] [Figure 1] Figure 1 shows a representation of the ADE system, including a cross-sectional view of the container plate containing multiple containers (or wells) for holding multiple individual samples, a receiving plate, a transducer assembly, and a block diagram of the electronic circuitry.

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

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

[0014] [Figure 4] Figure 4 shows a top view of a container plate with multiple containers.

[0015] [Figure 5] Figure 5 shows a top view of multiple containers within a container plate, and a flowchart illustrating the sequence for sequentially performing ADE on each container.

[0016] [Figure 6] Figure 6 shows the container for holding the sample.

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

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

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

[0020] [Figure 9B] Figure 9B shows scanning electron microscope images of TB in a sample-holding container, affected by air bubbles.

[0021] [Figure 10] Figure 10 shows a sequence for bubble improvement measures according to one embodiment.

[0022] [Figure 11] Figure 11 shows a top view of a container plate having a container containing a sample with bubbles.

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

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

[0025] [Figure 14] Figure 14 illustrates the stabilization of reflections from the TB inside the sample-holding container, which is affected by air bubbles.

[0026] [Figure 15] Figure 15 illustrates the stabilization of zero-velocity droplets (ZVDs) in a sample-holding container, which is affected by air bubbles.

[0027] [Figure 16] Figures 16A, 16B, and 16C illustrate illustrative examples of Ping's reflection in the presence of different amounts of bubbles according to the embodiment.

[0028] [Figure 17] Figures 17A, 17B, 17C, and 17D illustrate illustrative examples of Ping reflection after different bubble burst signals have been emitted according to the embodiment.

[0029] [Figure 18] Figure 18 shows four microscopic images of cells with attached bubbles after a bubble burst signal has been emitted, according to the embodiment.

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

[0031] [Figure 20] Figure 20 shows a graph of signals illustrating the reduction of bubbles in different samples according to the embodiment.

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

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

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

[0035] The above description of a technique of this application, as well as the following detailed description, will be better understood when read in conjunction with the accompanying drawings. For illustrative purposes, a technique is shown in the drawings. However, it should be understood that the claims are not limited to the arrangements and fixtures shown in the accompanying drawings. Furthermore, the appearance shown in the drawings is one of many decorative appearances that may be adopted to achieve the described function of this system. [Modes for carrying out the invention]

[0036] Detailed explanation Figure 1 depicts an exemplary representation of the ADE system 100, including a cross-sectional view of a container plate 120 (e.g., a microplate) containing multiple containers 122 (e.g., microplate wells) for holding multiple individual samples 101, a receiving plate 130 containing multiple receiving wells for receiving the injected liquid 102 from the samples 101, and a block diagram of electronic equipment 140. The ADE system 100 further includes a transducer assembly 110, a binding fluid 160, an X / Y / Z motor 150 (or a motor with fewer than three dimensions, e.g., a non-vertical (Z) dimension motor), and / or a temperature sensor (not shown). Figure 2 further shows the transducer assembly 110, including a transducer 112 and an acoustic lens 113. The ADE system 100 can characterize the containers 122 and / or the samples 101 and inject the liquid. A given sample 101 is the liquid of interest, held in the corresponding container 122. This disclosure focuses on a container 122, which is a microplate well, but the techniques described herein can be used in conjunction with other containers 122, such as tubes, flasks, and beakers, and any sample 101 contained therein.

[0037] 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 an acoustic lens 113. In the figure, the beam 170 is shown in two dimensions, but the beam 170 is actually three-dimensional. Furthermore, the beam 170 is shown as an equilateral triangle, but the beam 170 can have different shapes. By adapting the configuration of the transducer assembly 110, it may be possible to vary the height of the focal point of the acoustic energy beam 170. For example, the focal length of the beam 170 may be changeable by adapting the transducer assembly 110. Such a transducer assembly 110 is described in U.S. Patent Application No. 16 / 369,780 (U.S. Publication No. 2019 / 0302063) (which is incorporated herein by reference in its entirety). Furthermore, it may be possible to change the height of the focal point 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] As depicted in Figure 1, the beam 170 is focused onto the upper surface of sample 101, which may be at the interface between sample 101 and the air above sample 101. First, the beam 170 passes through the bonding liquid 160, the bottom wall 123 of container 122, and then through the depth of sample 101 to reach the surface 103 of sample 101.

[0039] The electronic network 140 includes a processor 143, a motor controller 142, a signal transmission network 144, a signal receiving network 145, and a temperature sensor network 141. Although shown as separate components for illustrative purposes, the parts of the electronic device 140 may be combined or integrated. Furthermore, some of the components shown may include multiple different subcomponents, which are not specifically shown. For example, the processor 143 may include multiple processors (e.g., multiple processors distributed in different locations).

[0040] The processor 143 generates or controls an analog electrical signal (an electronic transmission signal such as a radio frequency (RF) signal) to be communicated to the transducer 112 via the signal transmission network 144. 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 (for example, an acoustic signal reflected from the container 120 or sample 101 in response to an emitted acoustic signal) and vibrate in tune with it. This may generate an analog electrical signal (an electronically received signal), which is then communicated to the signal receiving network 145. The processor 143 may receive information corresponding to the received acoustic signal from the signal receiving network 145 in the form of an electronically received signal. The information in the electronically received signal will be analyzed by the processor 143.

[0041] The processor 143 can 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 receiving plate 130 to control the relative movement between the transducer assembly 110, the container plate 120, and / or the receiving plate 130. The processor 143 can control one or more motors 150 to position the transducer assembly 110 directly beneath a given container 122 in the container plate 120 (for example, the transducer assembly 110 is centered relative to the center of a given container 122), and then move the transducer assembly 110 directly beneath another given container 122 in the container plate 120. Alternatively, or in addition, the container plate and / or receiving plate 130 may be translated in one or more X, Y, and Z dimensions using motors or other translation devices.

[0042] In some embodiments, the ADE system 100 may include a temperature sensor (not shown) which may be located in the binding fluid 160 in the region between the container plate 120 and the receiving plate 130 or elsewhere. The temperature sensor network 141 receives signals (e.g., electrical or wireless) from the temperature sensor and communicates with the processor 143 so that the temperature (e.g., of the binding fluid 160, container 122, sample 101, and air temperature) can be measured.

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

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

[0045] Figure 4 shows a top view of a container plate 120 having multiple containers 122. The container plate 120 shown is a 384-well microplate (e.g., a polypropylene microplate, designated as 384-PP). Figure 5 shows a top view of the multiple containers 122 and an exemplary pattern (a meandering pattern) for performing ADE and / or other ultrasonic techniques (such as bubble breaking) on ​​each container 122 and the sample 101 therein, as described with respect to Figure 3. In this embodiment, a motor 150 moves the transducer assembly 110 along the x and y axes, positioning it beneath the various containers 122. Any other suitable pattern may be used (e.g., a raster pattern). It may also be possible to move the container plate 120 or a combination of the container plate 120 and the transducer assembly 110 to achieve similar results.

[0046] Figure 6 shows a container 122 (or well) holding the sample 101, which has a surface 103 (i.e., an upper surface or free surface). The bottom wall 123 of the container 122 defines the bottom upper interface (TB) 124 and the bottom upper interface (BB) 125.

[0047] Figure 7 shows the reflected signal and its corresponding envelope, where the reflected signal is received in the transceiver assembly 110 in response to the emitted signal. BB indicates the reflection from BB125. TB indicates the reflection from TB124. SR (Surface Reflection) indicates the reflection from surface 103 of sample 101. The total time over which the reflection appears is called time of flight or TOF. In Figure 7, there is a different individual TOF for each reflection BB, TB, and SR. When the distance between the transducer and the BB, TB, or surface 103 is generally known, a particular reflection can be identified within the overall reflected signal based on its TOF.

[0048] Figure 8 shows a scanning electron microscope image of the inner surface of an exemplary container 122, the inner surface including TB124. Surface texturing on a scale of approximately 5–10 μm may be observed (it is a faint, irregular shape). Such texturing does not appear in all containers 122, and it has been observed that plate 120 may be influencing the containers 122. As shown in Figure 11, affected containers are referred to as affected containers 126, and affected containers 126 (which may be a subset of containers 122) may occur at locations A11, A14, P11, P14, B11, B14, O11, or O14, but other locations are also possible. Such locations may be close to or adjacent to the injection molding ports when the container plate 120 is formed. There may be 1–6 affected containers 126, but it has been observed that plate 120 may also have more affected containers 126. Such texturing can be caused by the separation of plastic components during molding and / or molding process parameters (e.g., temperature and pressure gradients). However, the techniques described herein are not necessarily specific to the texture shown in Figure 8. Rather, the techniques described herein are applicable to a variety of conditions in which one can benefit from bubble reduction measures.

[0049] As shown in Figures 9A and 9B, texturing can occur only within the affected container 126. Figure 9A shows a micrograph of the TB124 of an unaffected container 122 containing sample 101. Figure 9A shows that there are few, if any, bubbles. Figure 9B shows a similar micrograph of the TB124 of the affected container 126 containing sample 101. In Figure 9B, it can be seen that bubbles 104 have formed in the TB124. As used herein, the affected container 126 is also a certain type of container 122.

[0050] Bubbles 104 are observed in the affected container 126, which is filled with sample 101 containing, for example, Milli-Q® water, 1x phosphate-buffered saline (PBS), a critical micelle concentration of Triton® X-100 in 1x PBS ≤ 14%, or 70%–80% dimethyl sulfoxide in water. Bubbles 104 may have a diameter of 1.5–5 μm, but the techniques described herein are not limited to specific bubble 104 sizes, as will be recognized.

[0051] The bubbles 104 in TB124 can affect the acoustic injection process. For example, bubbles 104 can increase acoustic reflection from TB124. Referring back to Figure 7, the acoustic energy beam is refracted through BB125 and TB124 and reaches the surface 103 of sample 101. Once it reaches the surface 103, a liquid bulge is generated, and then the liquid 102 is injected. However, in the affected container 126, bubbles 104 can cause greater reflection from TB124 than usual. This may be due to a greater acoustic impedance mismatch between the affected container 126 material (e.g., polypropylene) and the gas trapped within the bubbles 104 compared to the acoustic impedance mismatch between the container 126 material and sample 101 without bubbles 104. In the affected container 126, the bubble 104 may not cover 100% of the area, and the acoustic energy beam intersects with TB124, but the bubble 104 may be relatively uniformly dispersed across TB124. The reflected signal may include reflections from areas of TB124 where the bubble 104 is absent and areas of TB124 where the bubble 104 is present. With respect to reflections from portions of the beam that do not intersect with the bubble 104, the reflected energy may have a typical amplitude expected from TB124. With respect to reflections from portions of the beam that intersect with the bubble 104, the emitted signal may actually strike two interfaces: firstly, the TB124 / gas interface at the lower surface of the bubble 104 adhered to TB124, and secondly, the gas / fluid interface at the top of the bubble 104. The reflections from these two interfaces can be almost simultaneous, assuming they are separated in time by approximately several tens of nanoseconds. The combined reflection originating from bubble 104 can be substantially higher in energy than the reflection from TB124 without any bubble 104.

[0052] It has been observed that the energy reflected from TB124 in the affected container 126, which contains sample 101, is three times higher than that reflected in the unaffected container 122, which also contains sample 101. In some cases, the energy received from TB124 in the transducer assembly 110 can be so high that it saturates the signal receiving network 145 (exceeding the acceptable operating range). This can lead to problems such as false identification of an empty container 122. When the energy reflected from TB124 saturates (or otherwise sufficiently high) the signal receiving network 145, it can identify (or assist in identifying) an empty container 122. However, if bubbles 104 are present, a given container 122 may be falsely identified as not containing sample 101. Another problem with the affected container 126 is the incorrect assessment of sample 101 for calibration, taking into account the impedance signatures of container 122 and / or sample 101.

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

[0054] One known technique to address the interference of bubbles 104 is to reduce the amplitude of the emitted signal (e.g., survey ping). However, the reflection from TB124 in the affected container 126 may remain higher in the unaffected container 122. This could mean that the challenges for signature calibration are not addressed. Signature calibration may rely on TB124 and BB125 reflections to determine the properties of the bottom wall 123 and sample 101 (e.g., the acoustic impedance of sample 101), such as those related to ADE.

[0055] As an example, we assume a first amplitude for the emitted signal and peak reflections from BB125 (1,000 counts), peak reflections from TB124 in unaffected container 122 (1,000 counts), and peak reflections from TB124 in affected container 126 (3,000 counts) (counts are increments on the analog-to-digital (A / D) converter). In this specification, “count” refers to the envelope value being fitted. In this example, the A / D converter has a maximum range of 2,048 counts, and therefore a signal with 3,000 counts would saturate the A / D converter. If the amplitude of the emitted signal is reduced to a second value, the peak amplitude of the reflected energy can be halved (500, 500, and 1,500 counts from BB125 in unaffected container 122, TB124, and TB124 in affected container 126, respectively). In this embodiment, the A / D converter can no longer be saturated by the peak amplitude of the signal reflected from TB124 in the affected container 126. However, the ratio of peak amplitude reflections from TB124 to peak amplitude reflections from BB125 in the affected container is still relatively high (3 times), which can lead to incorrect identification of fluid properties by signature.

[0056] In addition, even if the affected container 126 cannot be considered empty and the process proceeds to ADE transfer, these transfers may suffer from incorrect volume and / or poor placement because the instability of the "zero-velocity droplet" (ZVD) (e.g., not repeatedly measured to be substantially constant) is not addressed. MIP may enable the calculation of the ZVD (the amplitude of the acoustic signal required to just detach droplet 102, but without the energy required to separate droplet 102 from the surface 103 of sample 101). Thus, droplet 102 may fall back into sample 101. Once determined, the ZVD value may be scaled to the amplitude required to completely eject droplet 102 from sample 101 and container 122. ZVD instability can be caused by changing the damping as bubbles 104 burst. If bubbles 104 are substantially absent in TB124, MIP may yield a stable value. Similarly, if bubble 104 is present in TB124 acting as an attenuator, but bubble 104 is not destroyed, MIP may determine ZVD to a higher value (to compensate for the attenuation), but the system may still be able to consistently (and reproducibly) eject droplet 102. Instability may arise from the fact that MIP assesses the system using an additional attenuator (in this case, bubble 104), but the attenuation of this additional attenuator is changing (bubble 104 is destroyed). As a result, ZVD may become too high, and droplet 102 may be ejected unintentionally.

[0057] Figure 10 illustrates a sequence 1000 for bubble 104 improvement according to one embodiment. 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, such as a predetermined height above the TB124 and / or a predetermined height relative to the TB124. Such a predetermined height may be -4 to +4 mm below or above the TB124, such as +2.5 mm above the TB124. Between steps 1010, 1020, and 1030, it may be possible to vary the height of the focal point (or otherwise vary the focal length). It may be beneficial to adapt the position and / or shape of the acoustic energy beam 170 (either by moving the transducer assembly 110 along the z-axis or by varying the focal length using 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 ​​TB 124. In steps 1040 and 1050, the focal point of the acoustic energy beam 170 is shifted to the surface 103 of the sample 101. Alternatively, the focal point may be between 4 mm above and 16 mm below the surface 103.

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

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

[0060] The first bubble-breaking signal may be selected to break (or burst) at least some bubbles 104. Figure 10 shows such broken bubbles as burst bubbles 105, which are filled black circles. The first bubble-breaking signal may impart energy to bubbles 104 in TB124. One bubble-breaking mechanism may be bubble cavitation. The energy input to a given bubble 104 at the relevant frequency results in the expansion of the bubble 194. As the radius of bubble 104 increases, the surface tension can no longer be sufficient to sustain the bubble 104. Bubble 104 may increase in size until the signal stops, and then bubble 104 may break into many substantially smaller bubbles (i.e., bubble-breaking). Another bubble-breaking mechanism may be through the bubble being pushed upward and removed from TB124.

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

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

[0063] TB124 reflects a portion of the second ping as a second TB reflection. The transducer assembly 110 receives the second TB reflection, and the information may be processed by the processor 143. Since some bubbles 104 were burst by the first bubble bursting signal in step 1020, a smaller fraction of acoustic energy than that related to the first ping in step 1010 may be reflected from TB124.

[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. Embodiments of such characteristics include overall energy or peak amplitude. If the characteristic for each is a peak amplitude, such a peak amplitude may be for the signal itself or for a corresponding envelope for 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 when bubble 104 or other types of interference are not present), the processor 143 infers that bubble 104 is present in TB 124. For example, the first ping and the second ping may be the same. Thus, theoretically, the first TB reflection and the second TB reflection should be the same under ideal conditions. However, if the second TB reflection is less than the first TB reflection (e.g., has a lower peak amplitude or overall energy), then, based on the reflection principle described above, it can be inferred that bubble 104 is present in TB 124. In this embodiment, the second TB reflection was expected to be the same as the first TB reflection in the absence of bubbles (or other interference). The criteria / criteria for inferring the presence of bubble 104 may vary depending on the situation, such as the calibration of a given system.

[0065] Examples of such criteria / multiple criteria for inferring the presence of bubbles 104 are various ratios such as TB'' / TB' or (TB'' / BB'') / (TB' / BB'), where BB' and TB' are reflections from BB125 and TB124 from a first ping, and BB'' and TB'' are reflections from BB125 and TB124 from a second ping. If such a ratio falls below a given threshold (e.g., 0.6 to 0.95), the presence of bubbles 104 can be inferred.

[0066] In step 1030, if the presence of bubble 104 in TB124 is inferred, flowchart 1000 proceeds to step 1040. Otherwise, sequence 1000 may skip step 1040 and proceed to step 1050. According to embodiments, steps 1020 and / or 1030 may be repeated before step 1040, either in the same manner or with parameters that vary as discussed above.

[0067] In step 1040, a second bubble burst signal is emitted by the transducer assembly 110. The second bubble burst signal may be selected to burst substantially all of the bubbles 104 (or substantial portions thereof) in TB124. Embodiments of the second bubble burst signal are described below in the context of Figures 12, 13, and 15.

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

[0069] The pre-adjustment signal may have an amplitude and frequency selected to prevent liquid from being ejected. The amplitude may be fixed. The amplitude may be determined in advance, for example, by sweeping a range of frequencies and amplitudes within the test sample for a given calibration. The pre-adjustment may include a shorter signal (e.g., with a duration similar to the first bubble burst signal) that is emitted repeatedly (e.g., 50 times). During this iteration process, the reflection of acoustic energy from TB124 may decrease until it stabilizes at a substantially constant value (e.g., within an acceptable range). As bubbles 104 continue to burst in TB124, less and less acoustic energy is reflected for the reasons discussed above. The TB reflection may be monitored by system 100 to determine, for example, when such reflections are repeated at a substantially constant value (e.g., within an acceptable range). If the stabilization of the TB reflection occurs before the maximum number of iterations, the pre-adjustment signal may be terminated. Figure 14 illustrates the stabilization of the reflection from TB124 in the exemplary affected container 126 as the iteration continues. The y-axis represents the ratio of the peak amplitude of the reflection from TB124 to the peak amplitude of the reflection from BB125. The reflection from BB125 may remain substantially constant throughout the bubble 104 breaking process.

[0070] The adjustment signal may be used to remove substantially any remaining bubbles 104 in TB124. Pre-adjustment does not have to use the substantially maximum amplitude of the acoustic energy so that injection can be avoided. Instead, pre-adjustment may use a fixed amplitude, as discussed above. Adjustment may, in contrast, involve a process in which the maximum amplitude is experimentally determined for each vessel 122, and then the acoustic energy is emitted at the substantially maximum amplitude.

[0071] The influence of the remaining bubbles 104 may be a factor in the MIP process. The adjustment signal may stabilize the MIP solution. The adjustment signal may include MIP imaging, followed by multiple droplet "false transfers" (e.g., 20 droplets are mistakenly ejected) without scaling the ZVD to actually eject the liquid. The multiple false transfers (no droplets ejected) after the MIP imaging process are repeated until the measured ZVD value stabilizes to a substantially constant value (e.g., within an acceptable range). According to one embodiment, with respect to a given iteration, MIP imaging is performed, followed by 20 false transfers, although more or fewer false transfers are also possible. If the ZVD value stabilizes before the maximum number of iterations (e.g., 20 sequences of (MIP imaging + multiple false transfers)), the adjustment signal may be terminated. The stabilization of the ZVD over 20 iterations in an exemplary affected container 126 is shown in Figure 15. The y-axis represents the ZVD value calculated from each MIP measurement. For each iteration, surface 103 is disturbed with a repeating signal of increasing amplitude, and the raised bulge is measured by measuring the width of the returned surface reflection (SR). The width of the SR corresponds to the size of the bulge. Once a predetermined size is reached, the ZVD can be calculated, i.e., the amplitude required to theoretically detach the droplet so that it falls back into sample 101 without being ejected. With respect to ADE, the amplitude of the acoustic signal is increased to a value greater than the ZVD so that the droplet would be ejected. In contrast, spurious transport is performed with an amplitude that is the experimentally determined ZVD so that there is no possibility of the droplet being ejected.

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

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

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

[0075] Another embodiment of the adjustment signal is illustrated in Figure 13 in flowchart 1300. In step 1310, bulge imaging is performed. In step 1320, multiple sham transfers are performed. In steps 1330, 1340, and 1350, bulge imaging and multiple sham transfers (e.g., 20 sham transfers) are repeated until the ZVD measurement stabilizes (e.g., within an acceptable tolerance). Steps 1330, 1340, and 1350 may each be repeated at least a predetermined number of times (e.g., 3 times) until the ZVD measurement stabilizes over them. Alternatively, or in addition, steps 1330, 1340, and 1350 may be repeated a predetermined maximum number of times (e.g., 10 times), regardless of whether the ZVD measurement stabilizes at the predetermined number of times. In step 1360, ADE is performed based on the determined ZVD.

[0076] Returning to Figure 10, in step 1050, the transducer assembly 110 emits a signal, causing droplet 102 to be ejected from sample 101 according to the ADE principle. Several embodiments of ADE are disclosed in U.S. Publication No. 2021 / 0394171 or PCT Publication No. WO2020 / 092407 (both of which are incorporated herein by reference as a whole).

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

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

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

[0080] Figure 19 shows a flowchart 1900 for a method of bursting bubbles according to an embodiment. This method may be carried out by components described in the context of Figures 1 and 2 (e.g., processor 143 and transducer assembly 110). This method may be similar to those disclosed in Figures 10, 12, and / or 13. It may not be necessary to perform all steps. For example, steps 1910 and / or 1950 may be bypassed and / or omitted. Further steps may be performed in parallel, partially or completely. For example, steps 1910 and 1920 may be performed in parallel. The method corresponding to flowchart 1900 may be used for bubble bursting techniques in which ADE is absent, such as the transfection embodiment described below. For example, pre-adjustments 1220 and / or adjustments 1230, as described in the context of Figure 12, may not be used.

[0081] In step 1910, the 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 the identification of reflections from physical sample features, including BB, TB, and SR, which are collected within a pulsed echo ultrasonic signal. For example, the volume of the sample may be determined by measuring the time of the SR reflection and determining the dimensions of the container (which may be a standard for a given type of plate).

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

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

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

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

[0086] Figures 16A–16C illustrate examples of Ping reflection in the presence of different amounts of bubbles. Ping is emitted toward the sample as described in the context of Figures 10 and 19. In Figure 16A, the sample has a smaller number of bubbles, and the amount of bubbles gradually increases in samples 16B and 16C. The y-axis is shown as count (count is an increment on an analog / digital (A / D) converter), but is essentially arbitrary and intended to provide a way to objectively compare each of Figures 16A–16C with each other. The x-axis is time. As can be seen from each figure, the peak amplitude of the BB reflection is identical. However, the peak amplitude of the TB reflection increases as the number of bubbles in the sample increases. With respect to transfection (discussed below), many bubbles can bind to cells, and cells are heavier than the bulk liquid in the sample, and as a result tend to settle toward or toward the TB. Bubbles form gas pockets, and more acoustic energy will be reflected where these pockets are located. Furthermore, as the number of bubbles increases, the peak amplitude of 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 surface reflection can also be caused by relatively disordered or irregular surface geometry resulting from bubbles suspended in the bulk liquid of the sample and, possibly, bubbles floating on the surface.

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

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

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

[0090] Depending on the embodiment, the bubble improvement process described herein can be used in conjunction 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 method of carrying out transfection involves sonoporation. Sonoporation refers to the use of sound within the ultrasonic range to increase the permeability of the cell plasma membrane. Increasing the permeability of the cell plasma membrane allows for the uptake of large molecules into the cell, such as DNA, which is desirable during transfection. Sonoporation employs acoustic cavitation of microbubbles to improve the delivery of these large molecules. The bubbles used for transfection may have a diameter of, for example, 5 μm.

[0091] Information generated by assessing and / or controlling the amount (number and / or concentration) of bubbles in a sample can have certain advantages usable for the transfection process. For example, this may be useful for quality control. One advantage is to verify sufficient bubble adhesion to cells prior to sonoporation, potentially using, for example, SR and / or TB reflection information to distinguish free / bound bubbles and assess (e.g., quantify) the binding efficiency in a given sample. A further advantage is verifying the proper removal rate 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 poor results. A further advantage is enabling verification that the appropriate sample (including cells, liquids, and bubbles) is in the expected container. This can flag pipetting errors that could otherwise lead to experimental failure and / or allow for their correction.

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

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

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

[0095] Different groups of bubbles may exist that can be detected using the processes described herein. One group is cell-bound microbubbles. As discussed, these can localize toward the bottom (TB) of the container, leading to the formation of a gas layer. This gas can produce a greater reflected signal from the TB compared to reflections from a substantially bubble-free sample (i.e., without gas). Other groups of microbubbles may include those suspended in the bulk of the fluid or on the fluid surface. Microbubbles in these locations will not affect the signal from the TB, but they can still affect the signal from surface reflections (SR) via acoustic scattering.

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

[0097] Figures 20-23 illustrate the effect and effectiveness of a technique disclosed herein on a sample containing bubbles and mammalian cells. The sample being assessed was a suspension of mammalian cells at various densities with microbubbles attached. An ultrasonic ping was transmitted to the sample by a transducer assembly, and the reflection therefrom was received by the transducer assembly. The ping was transmitted, and the corresponding reflection was assessed before and after the transmission of the bubble-breaking signal. The reflected data was interpreted by reading all the stored data using a custom MATLAB® script, and then determining the peak amplitude of the reflection from the container BB, container TB, and the sample surface.

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

[0099] In addition to the differences observed within the TB reflection signal, there are also likely differences, induced by bubbles, within the signal reflected by the surface of a given sample. SR reflection can also potentially be evaluated as described above, but was not considered for the generation of the data depicted in FIGS. 20-23.

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

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

[0102] Figure 22 shows a graph according to the embodiment, where the x-axis represents transfection efficiency as a percentage and the y-axis represents bubble removal rate as a percentage. Samples with A549 cells were assessed. Removal rates were measured as described with respect to Figure 21. Transfection efficiency for the samples was measured using flow cytometry. Figure 22 shows the correlation between the bubble removal rate measured during transfection and the percentage of cells successfully transfected with green fluorescent protein (GFP) encoded mRNA. Transfection efficiency was assessed using flow cytometry and the expression rate of GFP in individual cells was measured. A similar correlation was observed using HEK-293 cells, as shown in Figure 23. These correlations suggest that pre- and post-disruption bubble assessment can be used as a predictor of transfection success for one or more cell types, thereby enabling real-time process improvements and compensating for insufficient microbubble removal rates through dynamic regulation of the number, amplitude, and / or duration of bubble disruption signals.

[0103] Many of the embodiments described herein may be implemented on or in conjunction with a computer storage product, which includes a non-transient computer-readable medium (which may also be referred to as a non-transient processor-readable medium) having instructions or computer code thereon, for performing various computer implementation operations, as will be understood. These embodiments may include those with a processor 143 (or a relevant part of such an embodiment). The medium may include one or more distinctly different mediums. The code may be executed on one or more processors such as the processor 143 (which themselves may include multiple processors). The computer-readable medium (or processor-readable medium) is non-transient in the sense that it does not itself contain transient propagating signals (e.g., propagating electromagnetic waves that carry information on a transmission medium such as space or cable). The medium and the computer code (which may also be referred to as code) may be designed and constructed for a specific purpose or for a combination of purposes. Examples of non-transient 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 discs / digital video discs (CD / DVDs), compact disc-read-only memory (CD-ROMs), and holographic devices; magneto-optical storage media such as optical discs; carrier signal processing modules; and hardware devices specifically configured to store and execute program code, such as application-specific integrated circuits (ASICs), programmable logic devices (PLDs), read-only memory (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 code, as discussed herein.

[0104] Some embodiments and / or methods described herein can be implemented by software (running 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 (running on hardware) may be C, C++, Java®, Ruby, Visual Basic TM , and / or other object-oriented, procedural, or other programming languages ​​and development tools can be represented in a variety of software languages ​​(e.g., computer code). Embodiments of computer code include, but are not limited to, microcode or microinstructions, machine instructions such as those produced by a compiler, code used to produce web services, and files containing high-level instructions executed by a computer using an interpreter. For example, embodiments may be implemented using imperative programming languages ​​(e.g., C, Fortran, etc.), functional programming languages ​​(e.g., Haskell, Erlang, etc.), logic programming languages ​​(e.g., Prolog), object-oriented programming languages ​​(e.g., Java®, C++, etc.), interpreted languages ​​(JavaScript, Typescript, Perl), or other preferred programming languages ​​and / or development tools. Additional embodiments of computer code include, but are not limited to, control signals, encrypted code, and compressed code.

[0105] It will be understood by those skilled in the art that various modifications may be made without departing from the scope of the novel techniques disclosed herein, and that equivalents may be substituted. In addition, many modifications may be made without departing from the scope to adapt specific situations or materials to the teachings of the novel techniques. Thus, it is intended that the novel techniques are not limited to the specific techniques disclosed, but rather include all techniques that fall within the scope of the appended claims.

Claims

1. A system for analyzing a sample (101) contained in a container (122), wherein the container (122) includes a bottom surface (123) having a bottom interface (BB) (125) and a bottom upper interface (TB) (124), and the system is A transducer assembly (110) is configured to receive a plurality of electron transmission signals and emit a plurality of corresponding transmitted acoustic signals toward the container (122) and the sample (101), and to receive reflected acoustic signals from at least one of the container (122) or the sample (101) and generate a plurality of corresponding electron reception signals. A signal transmission network (144) configured to provide the aforementioned electron transmission signal to the transducer assembly (110), A signal receiving network (145) configured to receive the electronic receiving signal from the transducer assembly (110), A processor (143) is configured to control the signal transmission network (144) and to receive information corresponding to the electronic reception signal from the signal reception network (145). Equipped with, The plurality of transmitted acoustic signals each include at least a first ping and a first bubble-breaking signal. The plurality of reflected acoustic signals each include at least a first TB reflection corresponding to the first ping, The system is configured to emit the first bubble-breaking signal based on the characteristics of the first TB reflection.

2. The plurality of transmitted acoustic signals comprises a sequence of the first ping, the first bubble bursting signal, and the second ping, The plurality of reflected acoustic signals further comprises a second TB reflection corresponding to the second ping, The system is further configured to emit a second bubble-breaking signal based on a comparison of the characteristics of the first TB reflection and the characteristics of the second TB reflection. The system according to claim 1.

3. The system according to claim 2, wherein the characteristics of the first TB reflection have a first peak amplitude, and the characteristics of the second TB reflection have a second peak amplitude, and the system is further configured to emit a second bubble bursting signal when the first peak amplitude exceeds the second peak amplitude.

4. The system according to claim 2, wherein the peak amplitude of the first ping and the peak amplitude of the second ping are substantially the same.

5. The system according to claim 1, wherein the total energy of the bubble bursting signal exceeds the total energy of the ping.

6. The transducer assembly (110) is configured to focus acoustic energy to a first height when emitting the first ping, the first bubble burst signal, and the second ping, and is configured to focus acoustic energy to a second height above the first height when emitting the second bubble burst signal, preferably, (i) The first height is predetermined relative to the TB (124), and the second height is substantially on the surface (103) of the sample (101), or (ii) The system according to claim 2, wherein the first height is between + / - 6 mm relative to the TB(124).

7. The second bubble-breaking signal includes a pre-adjustment signal selected to break bubbles and a subsequent adjustment signal selected to break bubbles, preferably, The system according to claim 2, wherein the pre-adjustment signal is a bubble bursting signal.

8. The container (122) is contained within a plate (120) comprising a plurality of containers (122) containing a plurality of corresponding samples (101). Each of the plurality of containers (122) includes a corresponding BB (125) and a TB (124), The system further comprises at least one motor (150) configured to move at least one of the plate (120) or the transducer assembly (110) such that the transducer assembly (110) is positioned directly beneath each of the plurality of containers (122). The system according to any of the above claims.

9. The system is further configured to cause the transducer assembly (110) to emit a sequence of the first ping, the first bubble-breaking signal, and the second ping for each of the plurality of containers (122), The system is further configured to cause the transducer assembly (110) to generate a second bubble-breaking signal for a given number of containers (122) based on a comparison of the characteristics of the first TB reflection for a given container (122) and the characteristics of the second TB reflection for a given container (122), preferably, The system according to claim 8, further configured to issue the second bubble-breaking signal for a given of the plurality of containers (122) after the first ping, the first bubble-breaking signal, and the second ping have been issued for each of the plurality of containers (122).

10. A method for analyzing a sample (101) contained in a container (122), wherein the container (122) includes a bottom surface (123) having a bottom interface (BB) (125) and a bottom upper interface (TB) (124), and the system is In the transceiver assembly (110), receiving multiple electronic transmission signals, The transceiver assembly (110) transmits a plurality of transmitted acoustic signals toward the container (122) and the sample (101), wherein the plurality of transmitted acoustic signals correspond to the plurality of electron transmission signals. In the transceiver assembly (110), a plurality of reflected acoustic signals are received from at least one of the container (122) or the sample (101), The transceiver assembly generates a plurality of electronically received signals corresponding to the plurality of reflected acoustic signals, The signal transmission network (144) provides the plurality of electron transmission signals to the transducer assembly (110), In the signal receiving circuit network (145), the plurality of electronic receiving signals are received from the transducer assembly (110), The processor (143) is used to control the signal transmission network (144), The processor (143) receives information corresponding to the electronic reception signal from the signal receiving network (145). Includes, The plurality of transmitted acoustic signals each include at least a first ping, The plurality of reflected acoustic signals each include at least a first TB reflection corresponding to the first ping, A method further comprising using the transducer assembly (110) to generate at least a first bubble-breaking signal based on the characteristics of the first TB reflection.

11. The plurality of transmitted acoustic signals further comprises a second ping, The plurality of reflected acoustic signals further comprises a second TB reflection corresponding to the second ping, The method further includes the transducer assembly (110) generating a second bubble-breaking signal based on a comparison of the characteristics of the first TB reflection and the characteristics of the second TB reflection. The method according to claim 10.

12. The method according to claim 11, further comprising the first TB reflection characteristic having a first peak amplitude, the second TB reflection characteristic having a second peak amplitude, and the transducer assembly (110) emitting the second bubble-breaking signal when the first peak amplitude exceeds the second peak amplitude.

13. The method according to claim 11, wherein the peak amplitude of the first ping and the peak amplitude of the second ping are substantially the same.

14. When the first ping, the first bubble bursting signal, and the second ping are emitted, the transceiver assembly (110) is used to focus the acoustic energy to a first height, When the second bubble-rupturing signal is emitted, the transceiver assembly (110) is used to focus the acoustic energy to a second height above the first height. It further includes, preferably, (i) The first height is predetermined relative to the TB, and the second height is substantially on the surface of the sample, or (ii) The method according to claim 11, wherein the first height is between + / - 6 mm relative to the TB.

15. The container (122) is contained within a plate (120) comprising a plurality of containers (122) containing a plurality of corresponding samples (101), each of the plurality of containers (122) containing a corresponding BB (125) and TB (124), Using at least one motor (150), move at least one of the plate (120) or the transducer assembly (110) so that the transducer assembly (110) is positioned directly beneath each of the plurality of containers (122) at different times, The transducer assembly (110) emits a sequence of the first ping, the first bubble-breaking signal, and the second ping to each of the plurality of containers (122), The transducer assembly (110) generates the second bubble-breaking signal for a given number of containers based on a comparison of the characteristics of the first TB reflection for a given container and the characteristics of the second TB reflection for a given container. It further includes, preferably, The method according to any one of claims 11 to 14, further comprising issuing the second bubble-breaking signal for a given container (122) after the first ping, the first bubble-breaking signal, and the second ping have been issued for each of the containers (122).

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