Pressure pulse and electrical pulse membrane perforation

Abstract

Patch clamp techniques are described herein. An example method can include a system transmitting first control instructions to cause a pressure source to apply a negative pressure at a tip of a pipette. The system can transmit second control instructions to cause an amplifier to apply an electrical stimuli at the tip of a pipette contemporaneously to the negative pressure. The system can receive an input indicating that a whole cell configuration of the pipette and a cell has been achieved. The system can transmit third control instructions to cause the pressure source to terminate application of negative pressure at a tip of a pipette based at least in part on the input. The system can transmit fourth control instructions to cause an amplifier to terminate application of the electrical stimuli at a the tip of a pipette based at least in part on the input.

Description

PRESSURE PULSE AND ELECTRICAL PULSE MEMBRANE PERFORATIONCROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 619,665, filed January 10, 2024, the entire contents of which are hereby incorporated by reference.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under R01 GM134110 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0003] Electrical measurements from a single cell can be used to provide useful functional information of the single cell. A patch clamp technique can be used to measure electrical events from the single cell, a cellular compartment, or an isolated membrane patch. The patch clamp technique can involve the formation of a tight seal between a cell membrane and a glass, fluid- filled pipette. After the seal is formed, the membrane may be disrupted within the patch pipette tip without affecting the seal.BRIEF DESCRIPTION OF THE DRAWINGS

[0004] FIG. 1 A is an illustration of an example patch clamp system, according to one or more embodiments.

[0005] FIG. IB is an illustration of an example process for perforating a cell membrane, according to one or more embodiments.

[0006] FIG. 2 is an illustration of an example timing diagram for a pressure pulse and an electrical pulse, according to one or more embodiments.

[0007] FIG. 3 is an illustration of an example patch clamp system, according to one or more embodiments.

[0008] FIG. 4 is an illustration of an example patch clamp system, according to one or more embodiments.

[0009] FIG. 5 is an illustration of an example patch clamp system, according to one or more embodiments.

[0010] FIG. 6 is a process flow for an example patch clamp technique, according to one or more embodiments.

[0011] FIG. 7 is a process flow for an example patch clamp technique, according to one or more embodiments.

[0012] FIG. 8 is a block diagram of an example of a computing device usable for implementing some aspects of the present disclosure.DETAILED DESCRIPTION

[0013] In the following description, various embodiments will be described. For purposes of explanation, specific configurations and details are set forth to provide a thorough understanding of the embodiments. However, it will also be apparent to one skilled in the art that the embodiments may be practiced without the specific details. Furthermore, well-known features may be omitted or simplified in order not to obscure the embodiment being described.

[0014] Patch clamping is an electrophysiological technique that can be used to study ion channel function in a cell (e.g., neuron, muscle fiber, cardiomyocyte, or an oocyte). As an example, a researcher can prepare a solution (e g., a bath solution, such as an artificial cerebrospinal fluid (aCSF)) and an intracellular pipette solution). The researcher can then place tissue (e.g., brain tissue), while submerged in the bath solution, onto a recording stage. The researcher can further fill a pipette with the solution.

[0015] The researcher can use a microscope to study the tissue and identify, for example, a neuron to be studied. The researcher can use a micromanipulator to cause the pipette to enter the bath solution and approach a neuron. The researcher can cause a slight positive pressure to be applied to the tip of the pipette to prevent debris from entering the pipette. As the tip of the pipette interacts with the neuron, the positive pressure can cause a dimple to form on the neuron. The researcher can then cause a slight negative pressure (e.g., suction) at the pipette tip to form a high resistance seal (e.g., a Giga-seal) in the Giga-ohm range between the pipette and theneuron’s membrane. At this stage, the neuron and the patch pipette are in a cell-attached configuration.

[0016] The researcher can then apply a negative pressure to break the membrane of the neuron. Although the membrane is broken, the seal between the pipette and the membrane remains, such that the pipette becomes an extension of the membrane. At this stage, the pipette and the neuron are in a whole cell configuration. The seal can electrically isolate the cell, such that the ions that are flowing in the intracellular space of the cell flow into an intracellular pipette solution can be recorded by an electrode arranged in the patch pipette. The researcher can then apply an electrical stimulus, for example, a voltage or a current via the patch pipette. The researcher can further measure the response to the electrical stimulus to help understand the neuron’s electrophysiological properties.

[0017] This perforation of the patch can be difficult, especially in small cells or substructures, and a failed perforation attempt may result in a failed experiment. One issue that can occur is that the magnitude of the negative pressure or the duration of the negative pressure can rupture the seal or damage the neuron. In this instance, the researcher has to identify another neuron and restart the process of forming the seal and breaking the membrane. Repeating this process can not only be costly and time consuming, and valuable tissue material can be rendered unusable for research purposes. Conventional methods may also be ineffective for studying small compartments or substructures of a cell, such as an axon or dendrite. The techniques and devices described herein allow for precise regulation of an amplitude and a timing of pressure and electrical pulses used for perforation. The techniques and devices described herein can increase the likelihood of a successful patch perforation. This increased efficiency may accelerate the progress of research by facilitating the work of a novice “patch-clamper” recording from a regular cell to an expert recording from a smaller, technically challenging structure.

[0018] Embodiments herein address the above-referenced issues by providing techniques for using negative pressure and a contemporaneous electrical pulse to break a cellular membrane. A computing device can include a controller (e.g., a microcontroller) that is configured to apply negative pressure contemporaneously with an electrical pulse at a pipette. The microcontroller can cause a negative pressure at the pipette. During the application of the negative pressure, the microcontroller can further cause the application of an electrical pulse at the pipette. Thecombination of the negative pressure and the electrical pulse can be more reliable for breaking the cellular membrane than either the negative pressure pulse or the electrical pulse alone. Furthermore, the combination can result in successful patch clamp in smaller structures, such as small diameter axons that extend away from a cell body. In particular, the microcontroller can regulate the pulses such that there is a first offset between the start of the negative pressure pulse and electrical pulse and a second offset between the end of the electrical pulse and the end of the negative pressure pulse. The microcontroller can cause the application of the negative pressure pulses and the electrical pulses to repeat until the membrane perforates. In some embodiments, the computing device can further include a feedback mechanism to cause the negative pressure pulse and the electrical pulses to cease in response to the membrane perforating.

[0019] FIG. 1 A is an illustration of an example patch clamp system, according to one or more embodiments. As illustrated, a patch clamp system 102 is in operable communication (e.g., electrical communication and fluidic communication) with a pipette 104. The tip 106 of the pipette 104 can be arranged on a membrane 108 of a cell 110. The patch clamp system 102 can cause a negative pressure via an opening 112 the pipette 104 to form a seal 112 (e.g., a Gigaseal) between the pipette 104 and the membrane 108. The pipette 104 can include a lumen 114, which can include a hollow volume within the pipette. In some instances, the lumen 114 may be filled with an intracellular solution that permits ions 116. The membrane 108 can act as a barrier to contain ions 116 in the body of an unperforated cell.

[0020] The patch clamp system 102 can further cause a perforation 114 in the membrane 108 using a combination of a negative pressure pulse and an electrical pulse. The patch clamp system 102 can include a controller (e.g., a microcontroller) 118 configured to control a pressure unit 120 and an electrical unit 122. The pressure unit 120 can include a pressure regulator (e.g., one or more valves) for the application of negative pressure via the opening 112. The pressure unit 120 can further include one or more valves. The one or more valves can be arranged between and be in fluidic communication with a pressure source (e.g., one or more of a vacuum pump, vacuum blower, an air pump, an air compressor) and the pipette 104. The one or more valves can further regulate a fluidic communication between the pressure source and the pipette 104 for applying negative pressure pulses.

[0021] The controller 1118 can be configured to control one or more pressure parameters of the pressure unit 120, via the one or more valves. For example, the controller 118 can transmit control instructions to adjust a valve to regulate a negative pressure pulse generated by a pressure source. The controller 118 can use the one or more valves to set various pressure parameters, such as the start time of a pressure pulse, the end time of a pressure pulse, the width of any pressure pulse, the magnitude of any pressure pulse, and the rate of any pressure pulse. The negative pressure, in combination with an electrical stimulus, can cause a patch of the membrane 108 to be pulled into the lumen 114, where the patch may become perforated to achieve continuity between the cell interior and an intracellular fluid solution in the lumen 114 (e.g., a whole cell configuration).

[0022] The electrical unit 122 can include a signal generator (e.g., an amplifier circuit) for generating an electrical stimulus to perforate the membrane 108 via a recording electrode 124 (e.g., a silver chloride electrode). The electrical stimulus can include, for example, a current pulse or a voltage pulse. For example, a signal generator can be configured to generate a current, or the signal generator can be configured to generate a voltage at the pipette. The controller 120 can further adjust one or more electrical parameters of the electrical signal. For example, the controller 120 can adjust the start time of an electrical pulse, the end time of an electrical pulse, the rate of the electrical pulse, the amperage of an electrical current, the voltage, and the width of an electrical pulse. As used herein, the rate can include a quantity of pulses in a given time interval. For example, the rate can include one pulse (pressure or electrical) per second.

[0023] In an alternate embodiment, the electrical stimulus can be delivered via a stimulus electrode. The stimulus electrode can include a conductive wire or filament connected to the electrical unit 122. The stimulus electrode can be arranged in a hollow portion of a wall of the pipette 104. An end of the stimulus electrode can be configured to extend away from the wall at a predetermined distance from the tip 106. As illustrated, the stimulus electrode is configured to extend inward into the lumen 114. The stimulus electrode can further be configured to deliver an electrical stimulus for perforating the membrane at a predetermined distance (e.g., 0-1 micron) from the tip 106 of the pipette 104.

[0024] The controller 118 can generate control instructions to cause both the pressure pulse and the electrical pulse contemporaneously. For example, the controller 118 can generate controlinstructions as to the duration, rate, and magnitude of the pressure pulse and the electrical pulse. The controller 118 can generate control instructions to increase or decrease a duration of the pressure pulse. For example, if membrane 108 is not perforating, a user can provide an input to cause the controller 118 to generate control instructions to increase the duration of the pressure pulse. The controller 118 can also generate control instructions to increase or decrease the rate of the pressure pulses. The controller 118 can further increase or decrease the magnitude of the pressure pulses. Additionally, the controller 118 can set the duration and magnitude of the electrical pulse. For example, if adjusting the duration, rate, and magnitude of the pressure pulses does not result in a successful perforation 114, a user can enter an input to cause the controller 120 to generate control instructions for increasing the duration and magnitude of the electrical pulse. As the rate of the electrical pulses may be the same as the rate of the pressure pulses, adjusting the rate of the pressure pulse may necessarily adjust the rate of the electrical pulses.

[0025] It should be appreciated that the controller 118 can use the pressure unit 120 and the electrical unit 122 to implement various patterns from the pressure pulse and the electrical pulse to effectuate the patch clamp technique. The pattern can be configured to successfully effectuate the perforation 114 in the cell membrane 108. The pattern can be based on, for example, the cell type (e g., neuron, muscle cell, epithelial cell, cardiomyocyte, stem cell, immune cell, or other cell type) the patch clamp recording type, or other appropriate factor. The characteristics can include the physical characteristics of the membrane at the point of perforation. For example, a neuron may have a tougher membrane than other types of cells. Therefore, the controller 118 may be configured to cause a pressure pulse or electrical pulse having parameters corresponding to a neuron. In another example, an immune cell, such as a lymphocyte, may have a more delicate membrane than the neuron. Therefore, the controller 118 can set the pressure pulse parameters based on the cell type. For example, the negative pressure applied by the pressure unit 120 for the immune cell may be less than the negative pressure applied for the neuron. In another example, the controller 118 can cause the width of the pressure pulse to be greater for a neuron than the width of a pressure pulse for an immune cell. The controller 118 can also regulate the electrical pulse parameters based on the cell type. For example, the controller 118 can cause the magnitude of the electrical pulse to be greater than the magnitude for a neuron than an immune cell. The controller 118 can further regulate the pressure pulse parameters and the electrical pulse parameters based on each other’s parameters. The controller 118 can cause thepressure pulse to have a first parameter based on a second parameter of the electrical pulse, and vice versa. For example, the width of the pressure pulse can be based on the magnitude or width of the electrical pulse.

[0026] The controller 120 can generate patterns that respectively include a desired duration, desired rate, and desired magnitude of the pressure pulse and electrical pulse. The pattern can be predetermined pattern or dynamically generated pattern based on real-time feedback information. For example, as indicated above, a controller 120 can be configured to implement various cell type patterns, such as a neuron pattern, a muscle cell pattern, an epithelial cell pattern, a cardiomyocyte pattern, a stem cell pattern, an immune cell pattern, or other appropriate cell type pattern. Each pattern can include a distinct set of pulse parameters that can be implemented by the pressure unit 122 and the electrical unit 124.

[0027] The controller 120 can be configured to operate in different modes for the patch clamp system 102. For example, a first mode can include a single pressure pulse and a single electrical pulse with a given duration. The respective duration of the pressure pulse and the electrical pulse can be manufacturer-determined duration, or a duration set by a user. A second mode can include a series of pressure pulses and electrical pulses with a set rate. The respective rate of each of the pressure pulse and the electrical can be a manufacturer-determined rate, or a rate set by a user.

[0028] A third mode can include a series of pressure pulses and electrical pulses that each have a variable duration and a set rate. The third mode can be referred to as a “ramp” mode. The controller 120 can cause a series of pressure pulses and electrical pulses. The controller 120 can cause the duration of each successive pressure pulse to increase. In some instances, the controller 120 can cause the duration of both the pressure pulse and the electrical pulse to increase with each successive pulse. In other embodiments, only one of the durations of the pressure pulse or the electrical pulse increases with each successive pulse. In some embodiments, the electrical pulse can be in the form of a voltage step. As each successive pulse is applied to the cell membrane, the respective pressure parameters and electrical parameters can be recorded along with a state of the cell membrane (e.g., perforated, partially perforated, not perforated). The cell’s electrical response to an electrical pulse can be indicative of whether the membrane has been perforated. For example, the shape of the current deflection following the voltage step changes from a simple small square wave to a rapid transient with an exponential decay to a newhigher steady state. The peak of the transient is inversely proportional to the series resistance. Based on detecting this change, the controller 120 can determine that the membrane has been perforated. In other embodiments, a researcher can, with the assistance of a microscopic tool, visually determine whether the membrane has been perforated. The respective duration of the pressure pulse and the electrical pulse can be manufacturer-determined (e.g., predetermined) duration, or a duration set by a user. In some instances, a user can use the patch clamp system 102 to enter an input to manually override a manufacturer-determined duration. For example, as the user is observing the cell membrane perforate, the user can enter an input to cause the duration of a pressure pulse or electrical pulse to decrease with each successive pulse to not damage the cell.

[0029] A fourth mode can include a series of x-number of pulses (e.g., two pulses or three pulses) having a set duration and at a given rate. For example, the controller 120 can cause a first series of x-number of pressure pulses and electrical pulses that each have a first duration. The controller 120 can then cause a second series of y-number of pressure pulses and electrical pulses that have a second duration. The y-number of pressure pulses and electrical pulses can be the same number of pulses as the x-number of pressure pulses and electrical pulses, or y-number of pressure pulses and electrical pulses can be a different number of pulses as the x-number of pressure pulses and electrical pulses. The second duration can be the same as the first duration, or the second duration can be different than the first duration.

[0030] A fifth mode can be a manual mode, in which a user can set one or more of the pressure pulse parameters and electrical pulse parameters. In this mode, the user can observe the patch clamp process and use the patch clamp system 102 to manually adjust one or more of the pulse parameters.

[0031] The dynamically generated pattern can be based on feedback received by the controller 120. For example, the patch clamp system 102 can include a feedback unit 128 for intermittently or continuously collecting feedback information during the patch clamping process. For example, the feedback unit 128 can include one or more amplifiers for amplifying current generated by the cell, and collecting a voltage feedback signal. The patch clamp system 102 can also operate on various modes, such as voltage-clamp mode and current-clamp mode. For example, in a voltage-clamp mode, the electrical unit 124 can set a desired voltage; and thecurrent can be adjusted to maintain the voltage as feedback. In a current-clamp mode, the electrical unit 124 can apply a constant current and measure a voltage as feedback. In another example, the patch clamp system 102 can include a sensor for monitoring the pressure inside the pipette. The change in pressure can be considered feedback. In each of these instances, the controller 120 can use the feedback to regulate the parameters of the pressure pulse and the electrical pulse. For example, the controller 120 can implement decision tree logic to intermittently or continuously determine the appropriate pulse parameters based on the feedback.

[0032] In another embodiment, the feedback can be in the form of data to be used by a machine learning model. For example, the patch clamp system 102 can include a sensor (e.g., a microscope system) for intermittently or continuously monitoring a cell. The sensor can collect information, such as image data, and transmit the data to controller 120 of the patch clamp system 102. The controller 120 can convert the sensor data into machine learning inputs. The controller 120 can further access a machine learning model that is trained to use the sensor data to generate predictions for pressure pulse parameters and electrical pulse parameters. The machine learning model can be implemented by a neural network, such as a convolutional neural network (CNN) or transformer network. For example, a first machine learning model can be trained to use image processing techniques to extract features from the image data to determine the various determinations of a cell perforation process. For example, how likely is the cell to become perforated, how likely is the perforation going to cause the cell to be unusable, or other appropriate determination. The first machine learning model can combine its input with a second machine learning model (e.g., ensemble learning). In the instance of ensemble learning, the outputs of the first machine learning model and the second machine learning model are combined to generate a single output. The first machine learning model can also transmit its output to be used as an input for the second machine learning model. In either case, the output can include a prediction as to the appropriate pressure pulse and electrical pulse parameters. The controller 120 can receive the output and adjust the pressure pulse and electrical pulse parameters in real-time. In other embodiments, the first and second machine learning models can be trained to use the feedback to generate an output as to appropriate pressure pulse and electrical pulse parameters, including termination of both pulses. Again, the controller 120 can intermittently or continuously receive the output and adjust the pressure pulse and electrical pulse parameters in real-time based on the feedback.

[0033] In some embodiments, the patch clamp system 102 can be configured to permit a user to manually regulate the pressure pulse and the electrical pulse. As indicated above, the patch clamp system 102 can include a sensor (e.g., an image capturing device) that can collect live data as the user is attempting to create a perforation 114 in the membrane 108. The user can enter an input to cause the controller to adjust one or more parameters of the pressure pulse and the electrical pulse. For example, as the user is viewing live image data, the user can enter an input to adjust the width of the pressure pulse or the width of the electrical pulse. The user can also enter an input into the patch clamp system to cause the pressure pulses and the electrical pulses to stop.

[0034] The patch clamp system 102 can further include a recording unit 130. The recording unit 130 can be configured to record the measured flow of ions 118 through a channel 132. The recording unit 130 can be in operable communication with a recording electrode 132 for recording the electrical properties of the cell 110. For example, ions 118 in cellular cytosol can traverse the perforation 114 and enter the lumen 116. Furthermore, the recording electrode 132 can be used to measure various electrophysiological parameters of the cell 110.

[0035] FIG. IB is an illustration of an example process for perforating a cell membrane, according to one or more embodiments. At To, a researcher can use a micromanipulator to cause a pipette 150 (e.g., pipette 104) to enter a bath solution and approach a membrane 152 (e.g., membrane 108). The pipette 150 can include a lumen (e.g., lumen 114) that has been filled with an intracellular solution 154. The researcher can then cause a slight negative pressure (e.g., suction) at the pipette 150 to form a seal 156 (e.g., a seal 112) between the pipette 150 and the membrane 152. At this stage, the membrane 152 and the pipette 150 are in a cell-attached configuration. The membrane 152 can include one or more ion channels 158. In FIG IB, the ion channels 158 are illustrated outside the seal 156. It should be appreciated that in a real-world scenario, it is likely that an ion channel 158 is on a part of the membrane 152 that is surrounded by the seal 156.

[0036] At Ti a controller (e.g., controller 118) can cause a pressure unit (e.g., pressure unit 120) to create a negative pressure via an opening 160 (e.g., opening 112). The negative pressure can be in the form of pressure pulses and the controller can be configured to set the duration, magnitude, and rate of the pressure pulses. The controller can also cause an electrical unit (e.g.,electrical unit 122) to create electrical stimuli via a recording electrode 162 (e.g., recording electrode 124). The electrical stimuli can be the form of electrical pulses. Each electrical pulse can include a voltage step, and the controller can be configured to control the duration, magnitude, and rate of the electrical pulses. The controller can control the pressure pulses and electrical pulses, such that each electrical pulse can correspond to a pressure pulse. The correspondence is described with more particularity with respect to FIG. 2.

[0037] The controller can cause the pressure pulses and the corresponding electrical pulses to perforate the membrane 152. As illustrated, the negative pressure caused by the pressure pulses is causing the membrane 152 to be pulled into the lumen of the pipette 150. The mechanical strain on the membrane 152 can deteriorate the structure of the membrane 152. Furthermore, the electrical pulses can further deteriorate the structure of the membrane 152. The controller can continue to cause the pressure pulses and the electrical pulses until the membrane 152 perforates.

[0038] At T2, the pressure pulses and electrical pulses can create a perforation 164, and the controller can cause the pressure pulses and the electrical pulses to cease. As illustrated, the perforation 164 is within the seal 156 to create a whole-cell configuration.

[0039] FIG. 2 is an illustration 200 of an example timing diagram for a pressure pulse and an electrical pulse, according to one or more embodiments. As indicated above, a controller (e.g., controller 120) can set the pulse parameters of a pressure pulse and the electrical pulse. The controller can configure the pressure pulse to have a first width (wl) 202 extending from a start time of Ti to a stop time of T4. As used herein, a pulse width can include the duration between a start time and a stop time. In some instances, the first width 202 can be set by a user. For example, the user can manually enter an input to set the first width 202. In other instances, the first width 202 can be a predetermined first width 202 of a set of first widths, and the user can select the first width 202 from a plurality of available first widths. The first width 202 can be configurable by the controller and the length of the first width 202 can be based on various factors, such as a magnitude of the pressure pulse, a width of a corresponding electrical pulse, a magnitude of the corresponding electrical pulse, a cell type, patch clamp recording type, a likelihood that the cell membrane is going to perforate, or another appropriate factor. During the first width 202, the controller can cause a pressure generator to apply a negative pressure to the pipette. For example, at Ti , the pressure generator can begin generating a negative pressure, andat T4, the pressure generator can cease generating the negative pressure until the next pressure pulse. In some embodiments, Ti can correspond to the rising edge of the pressure pulse, and T4 can correspond to the falling edge of the pressure pulse.

[0040] The controller can further configure the electrical pulse to have a second width (w2) 204 extending from a start time of T2 to a stop time of T3. In some instances, the second width 204 can be set by a user. For example, the user can use the patch clamp system 102 to manually enter an input to set the second width 204. In other instances, the second width 204 can be a predetermined second width 204 of a set of second widths, and the user can select the second width 204 from a plurality of available second widths. In some embodiments, T2 can correspond to the rising edge of the electrical pulse and T3 can correspond to the falling edge of the electrical pulse. As illustrated, the electrical pulse can correspond to the pressure pulse as the second width 204 occurs during the first width 202. In addition to configuring the duration of the second width 204, the controller set a first offset 206 extending from Ti to T2, and a second offset 208 extending from T3 to T4.

[0041] The second width 204 is configurable by the controller and the duration of the second width 204 can be based on various factors, such as a magnitude of the electrical pulse, a width of a corresponding pressure pulse, a magnitude of the corresponding pressure pulse, a cell type, a likelihood that the cell membrane is going to perforate, or another appropriate factor. The controller can set the first offset 206 and the second offset 208 based on similar considerations, such as a magnitude of the electrical pulse, a magnitude of the pressure pulse, a duration of a pressure pulse, a width of an electrical pulse, a cell type, a likelihood that the cell membrane is going to perforate, or another appropriate factor. It should be appreciated that the first offset 206 and the second offset 208 can be any time interval (e.g., 0-x time units). In other words, the first offset 206 and the second offset 208 can be used to cause the pressure pulse and the electrical pulse to both have the same start time, have the electrical pulse have a start time after the pressure pulse start time, or both have the same stop time.

[0042] In some embodiments, the controller can also set the pressure pulse duty cycle 210 and an electrical pulse duty cycle 212. In some instances, the pressure pulse duty cycle 210 and an electrical pulse duty cycle 212 can be set by a user. In other instances, a user may select a duty cycle from a set of candidate duty cycles. For example, the user can use the patch clamp system102 to manually select a desired duty cycle. The duty cycle can include a ratio of a time from when a circuit is on to when the circuit is off. For example, the pressure pulse duty cycle 210 can include a ratio of a length of time that a pressure unit (e.g., pressure unit 122) is on to a length of time that the pressure unit is off. For example, the pressure pulse duty cycle 210 can include a ratio of the first width 202 to a third width (ws) 214, where the third width 214 can extend from T4 to Ts. As illustrated, T4 can include a stop time of a first pressure pulse and Ts can include a start time of a second pressure pulse. Similar, the electrical duty cycle 212 can include the ratio of the second width 204 to a fourth width (w4) 216, where the fourth width 216 extends from T3 to Te. AS illustrated, T3 can include a stop time of a first electrical pulse, and Te can include a start time of a second electrical pulse. Similarly, the user can use the patch clamp system 102 to manually input a desired rate for the pressure pulse and the electrical pulse. In other instances, the user can select the rate from a set of candidate rates.

[0043] In some embodiments, the patch clamp system 102 can include the functionality that permits the user to parameterize the pressure pulses and the electrical pulses. For example, the patch clamp system 102 can include a graphical user interface (GUI) that permits a user to interact with the controller. The GUI can include a visual representation of options that the user can select to parameterize the pressure pulses and the electrical pulses. For example, the GUI can be implemented by on display and include icons that the user can select. In some embodiments, the display is a touch screen display and the user can interact with the GUI via a touch. In other embodiments the user can interact with the GUI using a peripheral device, such as a mouse or keypad. The user can user the GUI to set various parameters of the electrical pulses and the pressure pulses. For example, the user can use the GUI to select the respective duration, magnitudes, and rates of the pressure pulses and the electrical pulses.

[0044] Although not illustrated, the controller can further set the magnitude of each of the pressure pulse and the electrical pulse. As used herein, the magnitude refers to the size of the unit (e.g., the number of Pascals (Pa) or the number of amperes (A)). The amplitude can refer to a strength of the waveform used to generate the pulse. In some instances, the respective magnitude of the pressure pulse and the electrical pulse can be set by a user. For example, the user can use the patch clamp system 102 to manually select a desired magnitude for each of the pressure pulse and the electrical pulse.

[0045] FIG. 3 is an illustration 300 of an example patch clamp system, according to one or more embodiments. As illustrated, a controller 302 (e.g., controller 120) can be in operable communication with various elements of a patch clamp system (e.g., patch clamp system 102). The controller 302 can be in operable communication with various input elements. Although illustrated as buttons and knobs, the input elements can assume various forms, such as buttons, knobs, switches, and other appropriate input elements. For example, the controller 302 can be in operable communication with a valve input element 304 and a mode input element 306. The valve input element 304 can be used by a user to manually cause a pressure unit (e.g., pressure unit 122) to apply a negative pressure. The valve input element 304 can include a trigger to cause the path clamp system to cyclically transmit pulses directed toward a membrane of a cell. The valve input element 304 can also be used to cause the pressure unit to close a valve and cease applying the negative pressure. The electrical input element 308 can be used by a user to manually cause an electrical unit (e.g., electrical unit 124) to deliver an electrical stimulus at a pipette, or cease delivering an electrical stimulus. A user can use a mode input element 308 to set a mode for the patch clamp system. Examples of modes for the patch clamp system have been described above.

[0046] The controller 302 can further be in operable communication with various pulse parameter input elements. An amplitude input element 310 can be used by a user to set the amplitude of a pressure pulse or an electrical pulse. It should be appreciated that only one amplitude input element 310, duration input element 312, and rate input element 314 are illustrated, the patch clamp system can include additional input elements for pressure pulse and electrical pulse. For example, a user can use one input element for the pressure pulse and another input element for the electrical pulse. A user can use the amplitude input element 310 to adjust the amplitude of the pressure pulses and electrical pulses after the patch clamp system has begun applying the pressure pulses and electrical pulses. A user can use the duration input element 312 to adjust the duration of the pressure pulses and electrical pulses after the patch clamp system has begun applying the pressure pulses and electrical pulses. A user can use the rate input element 314 to set the rate of the pressure pulses and electrical pulses before the patch clamp system has begun applying the pressure pulses and electrical pulses. A user can also use the rate input element 314 to adjust the rate of the pressure pulses and electrical pulses after the patch clamp system has begun applying the pressure pulses and electrical pulses. A user can use the initialduration input element 316 to set the duration of the pressure pulses and electrical pulses before the patch clamp system has begun applying the pressure pulses and electrical pulses. As indicated above, the patch-clamp system can include a GUI. In these embodiments, one or more of the mode input element 308, amplitude input element 310, duration input element 312, rate input element 314, and the initial duration input element 316 can be a respective interactive element of the GUI.

[0047] The controller 302 can be in operable communication with one or more sensors 318. The one or more sensors can include various sensors and be used for various purposes. For example, one or more sensors can be used to monitor, in real-time, the parameters of the pressure applied by the patch clamp system. For example, a pressure sensor can be arranged between a valve and the pipette. The pressure sensor can be used to measure the pressure applied by the patch clamp system. One or more other sensors can be used to monitor, in real-time, parameters of the electrical stimulus applied by the patch clamp system. For example, a voltage meter or ammeter arranged at the pipette can be used to measure electrical stimulus applied by the patch clamp system. Yet one or more other sensors can be used to assist the user to observe the patch clamp process. For example, an image capturing device can be used to generate an image of the pipette and the cell. The image can be presented on a display for the user.

[0048] The controller 302 can further be in operable communication with one or more manual input device(s) 320. For example, a manual input device can include a manual trigger that a user can use to manually to control one or more parameters of the pressure pulses and the electrical pulses. The controller 302 can be connected to a manual input device for the electrical pulses and a manual input device for the pressure pulses. For example, in the instance that the manual input device includes a trigger for the pressure pulses, the user can depress the trigger to cause negative pressure to be created, and release the trigger to cause the negative pressure to cease. Therefore, the user can manually control the duration of the pressure pulse via the trigger. Similarly, in the instance that the manual input device includes a trigger for the electrical pulse, the user can depress the trigger to cause negative pressure to be created, and release the trigger to cause the negative pressure to cease.

[0049] FIG. 4 is an illustration 400 of an example patch clamp system, according to one or more embodiments. A patch clamp system (e.g., patch clamp system 102) can include acontroller 402 arranged inside a housing 404. The patch clamp system of FIG. 4 is similar to the patch clamp system of FIG. 3. The patch clamp system of FIG. 3 can differ from the patch clamp system of FIG. 4. However, it should be appreciated that each of the elements of the patch clamp system described in FIG. 3 can be incorporated into the patch clamp system of FIG. 4, and each of the elements of the patch clamp system described in FIG. 4 can be incorporated into the patch clamp system of FIG. 3. The controller 402 can be in operable communication with a mode input element 406 (e.g., mode input element 308) and a valve input element 408 (e.g., valve input element 304). The valve input element 408 can include a trigger to cause the path clamp system to cyclically transmit pulses directed toward a membrane of a cell. The controller 402 can further be in operable communication with an On / Off Switch 410. The patch clamp system can receive power from a battery or receive power externally via a power cable 412. The controller 402 can further be in operable communication with a sensor 414 (e.g., sensor 318). As illustrated, the sensor 414 can be a pressure sensor connected to a valve 416 for applying positive pressure or negative pressure. The patch clamp system can be used to successfully create a perforation in a cell membrane and to seal the cell membrane to a pipette as described above.

[0050] FIG. 5 is an illustration 500 of an example patch clamp system, according to one or more embodiments. A patch clamp system (e.g., patch clamp system 102) can include a controller (e.g., controller 120) arranged inside a housing 502 (e.g., housing 404). The controller can be in operable communication with an initial duration switch 504 (e.g., initial duration input element 316), which can be used to set the initial duration of a pressure pulse or an electrical pulse. The controller can further be in operable communication with a duration input element 506 (e.g., duration input element 312). A user can use the duration input element 506 to adjust the duration of the pressure pulses and electrical pulses after the patch clamp system has begun applying the pressure pulses and electrical pulses. The controller can further be in operable communication with a rate input element 508 (e.g., rate input element 314). A user can also use the rate input element 508 to adjust the rate of the pressure pulses and electrical pulses after the patch clamp system has begun applying the pressure pulses and electrical pulses.

[0051] FIG. 6 is a process flow 600 for an example patch clamp technique, according to one or more embodiments. While the operations of processes 600 and 700 are described as being performed by generic computers, it should be understood that any suitable device may be used toperform one or more operations of this process. Processes 600 and 700 (described below) are illustrated as logical flow diagrams, each operation of which represents a sequence of operations that can be implemented in hardware, computer instructions, or a combination thereof. In the context of computer instructions, the operations represent computer-executable instructions stored on one or more computer-readable storage media that, when executed by one or more processors, perform the recited operations. Generally, computer-executable instructions include routines, programs, objects, components, data structures, and the like that perform particular functions or implement particular data types. The order in which the operations are described is not intended to be construed as a limitation, and any number of the described operations can be combined in any order and / or in parallel to implement the process.

[0052] At 602, the method can include a computing system transmitting first control instructions to cause a pressure source to apply a negative pressure at a pipette.

[0053] At 604, the method can include the computing system transmitting second control instructions to cause an amplifier to apply an electrical stimulus at the pipette, the control instructions setting the electrical stimulus to occur contemporaneously with the negative pressure.

[0054] At 606, the method can include the computing system determining that a cell membrane has been perforated. The determination can be based on a user-based input. The input can also be received via feedback mechanism that monitors the patch clamp process in real-time. A sensor can provide sensor-based data to the controller indicating that a membrane of a cell has been perforated.

[0055] At 608, the method can include the computing system transmitting third control instructions to cause the pressure source to terminate the application of negative pressure at a pipette based at least in part on the input. At 610 the method can include the computing system transmitting fourth control instructions to cause an amplifier to terminate the application of the electrical stimulus at the pipette based at least in part on the input.

[0056] FIG. 7 is a process flow 700 for an example patch clamp technique, according to one or more embodiments. At 702, the method can include a computing system (e.g., the patch clamp system 102) determining parameters of a plurality of pressure pulses to be applied to amembrane of a cell. The parameters can include the duration, magnitude, and rate of the pressure pulses. The parameters can be based on user input or pre-defined parameters.

[0057] At 704, the method can include the computing system determining parameters of a plurality of electrical pulses to be applied to a membrane of the cell. The parameters can include the duration, magnitude, and rate of the pressure pulses. The parameters can be based on user input or pre-defined parameters. Each electrical pulse of the plurality of electrical pulses can be configured to correspond to a pressure pulse of the plurality of pressure pulses.

[0058] At 706, the method can include the computing system applying the plurality of pressure pulses to the membrane of the cell. The computing system can configure the pressure pulses to exhibit the determined parameters. The plurality of pressure pulses can be applied to perforate the membrane of the cell.

[0059] At 708, the method can include the computing system applying the plurality of electrical pulses to the membrane of the cell. The computing system can configure the plurality of electrical pulses to exhibit the determined parameters. The plurality of electrical pulses can be applied in conjunction with a corresponding plurality of pressure pulses to perforate the membrane of the cell.

[0060] At 710, the method can include the computing system determining whether the membrane has been perforated. The computing system can determine that the membrane has been performed based on various information. For example, the computing system can include a feedback system that measures an electrical response of the membrane and makes the determination based on the response. In other embodiments, a user can visually determine that the membrane has been perforated. In these embodiments, the user may enter an input to cease the application of the plurality of pressure pulses and the plurality of electrical pulses. The computing system can treat this input as an indication that the membrane of the cell has been perforated.

[0061] If the computing system determines that the membrane of the cell has not been perforated, the method can return to step 702. The computing system can then re-determine the parameters of a plurality of pressure pulses and the plurality of electrical pulses.

[0062] If, however, the computing system determines that the membrane of the cell has been perforated, the method process to step 712. At 712, the computing system can terminate the application of the plurality of pressure pulses to the membrane of the cell. At 714, the computing system can terminate the application of the electrical pulses to the membrane of the cell.

[0063] FIG. 8 is a block diagram of an example of a computing device 800 usable for implementing some aspects of the present disclosure. The computing device 800 includes a processor 802 coupled to a memory 804 via a bus 812. The processor 802 can include one processing device or multiple processing devices. Examples of the processor 802 include a Field- Programmable Gate Array (FPGA), an application-specific integrated circuit (ASIC), a microprocessor, or any combination of these. The processor 802 can execute instructions 806 stored in the memory 804 to perform operations. In some examples, the instructions 806 can include processor-specific instructions generated by a compiler or an interpreter from code written in any suitable computer-programming language, such as C, C++, C#, Python, or Java.

[0064] The memory 804 can include one memory device or multiple memory devices. The memory 804 may be non-volatile and include any type of memory device that retains stored information when powered off. Examples of the memory 804 can include electrically erasable and programmable read-only memory (EEPROM), flash memory, or any other type of nonvolatile memory. At least some of the memory 804 includes a non-transitory computer-readable medium from which the processor 802 can read instructions 806. A computer-readable medium can include electronic, optical, magnetic, or other storage devices capable of providing the processor 802 with computer-readable instructions or other program code. Examples of a computer-readable medium include magnetic disks, memory chips, ROM, random-access memory (RAM), an ASIC, a configured processor, optical storage, or any other medium from which a computer processor can read the instructions 806.

[0065] The computing device 800 may also include other input and output (VO) components. The input components 808 can include a mouse, a keyboard, a trackball, a touch pad, a touchscreen display, or any combination of these. The output components 810 can include a visual display, an audio display, a haptic display, or any combination of these. Examples of a visual display can include a liquid crystal display (LCD), a light-emitting diode (LED) display, and atouch-screen display. An example of an audio display can include speakers. Examples of a haptic display may include a piezoelectric device or an eccentric rotating mass (ERM) device.

[0066] The above description of certain examples, including illustrated examples, has been presented only for the purpose of illustration and description and is not intended to be exhaustive or to limit the disclosure to the precise forms disclosed. Modifications, adaptations, and uses thereof will be apparent to those skilled in the art without departing from the scope of the disclosure. For instance, any examples described herein, can be combined with any other examples.

[0067] Although specific embodiments have been described, various modifications, alterations, alternative constructions, and equivalents are also encompassed within the scope of the disclosure. Embodiments are not restricted to operation within certain specific data processing environments but are free to operate within a plurality of data processing environments. Additionally, although embodiments have been described using a particular series of transactions and steps, it should be apparent to those skilled in the art that the scope of the present disclosure is not limited to the described series of transactions and steps. Various features and aspects of the above-described embodiments may be used individually or jointly.

[0068] Further, while embodiments have been described using a particular combination of hardware and software, it should be recognized that other combinations of hardware and software are also within the scope of the present disclosure. Embodiments may be implemented only in hardware, or only in software, or using combinations thereof. The various processes described herein can be implemented on the same processor or different processors in any combination. Accordingly, where components or modules are described as being configured to perform certain operations, such configuration can be accomplished, e.g., by designing electronic circuits to perform the operation, by programming programmable electronic circuits (such as microprocessors) to perform the operation, or any combination thereof. Processes can communicate using a variety of techniques, including but not limited to conventional techniques for inter process communication, and different pairs of processes may use different techniques, or the same pair of processes may use different techniques at different times.

[0069] The specification and drawings are, accordingly, to be regarded in an illustrative rather than a restrictive sense. It will, however, be evident that additions, subtractions, deletions, andother modifications and changes may be made thereunto without departing from the broader spirit and scope as set forth in the claims. Thus, although specific disclosure embodiments have been described, these are not intended to be limiting. Various modifications and equivalents are within the scope of the following claims.

[0070] The use of the terms “a” and “an” and “the” and similar referents in the context of describing the disclosed embodiments (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. The term “connected” is to be construed as partly or wholly contained within, attached to, or joined together, even if there is something intervening. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate embodiments and does not pose a limitation on the scope of the disclosure unless otherwise claimed. No language in the specification should be construed as indicating any nonclaimed element as essential to the practice of the disclosure.

[0071] Disjunctive language such as the phrase “at least one of X, Y, or Z,” unless specifically stated otherwise, is intended to be understood within the context as used in general to present that an item, term, etc., may be either X, Y, or Z, or any combination thereof (e.g., X, Y, and / or Z). Thus, such disjunctive language is not generally intended to, and should not, imply that certain embodiments require at least one of X, at least one of Y, or at least one of Z to each be present.

[0072] Preferred embodiments of this disclosure are described herein, including the best mode known for carrying out the disclosure. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. Those of ordinary skill should be able to employ such variations as appropriate, and the disclosure may bepracticed otherwise than as specifically described herein. Accordingly, this disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the disclosure unless otherwise indicated herein.

[0073] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0074] In the foregoing specification, aspects of the disclosure are described with reference to specific embodiments thereof, but those skilled in the art will recognize that the disclosure is not limited thereto. Various features and aspects of the above-described disclosure may be used individually or jointly. Further, embodiments can be utilized in any number of environments and applications beyond those described herein without departing from the broader spirit and scope of the specification. The specification and drawings are, accordingly, to be regarded as illustrative rather than restrictive. 1

Claims

CLAIMSWhat is claimed is:

1. A system, comprising: a controller comprising: one or more processors; and one or more computer-readable media having stored thereon a sequence of instructions, when executed, cause the one or more processors to: set a first duration for a negative pressure pulse to be applied at a pipette, the negative pressure pulse to cause negative pressure; and set a second duration of an electrical pulse to be applied at the pipette, the electrical pulse to cause an electrical stimulus, the second duration set to occur between a start time and a stop time of the first duration; a pressure unit configured to apply the negative pressure pulse at the pipette for the first duration based at least in part on a first control instruction from the controller; and an electrical unit configured to apply the electrical pulse at the pipette for the second duration based at least in part on a second control instruction from the controller.

2. The system of claim 1, wherein the sequence of instructions, when executed, further cause the one or more processors to cause the negative pressure pulse to have a first magnitude, and cause the electrical pulse to have a second magnitude.

3. The system of claim 1, wherein the sequence of instructions, when executed, further cause the one or more processors to: receive an indication that a cell membrane is sealed to the pipette has been perforated; cause the pressure unit to terminate application of the negative pressure pulse based at least in part on the indication; andcause the electrical unit to terminate application of the electrical stimulus based at least in part on the indication.

4. The system of claim 1, further comprising: an electrode in operable communication with the controller, wherein the electrode is configured to be arranged within a lumen of the pipette, and wherein the electrode is configured to measure an electrophysiological parameter of a cell.

5. The system of claim 1, wherein the pressure unit comprises a valve in operable communication with the controller, and wherein the valve is configured to be in fluidic communication with a lumen of the pipette and a pressure source.

6. The system of claim 1, wherein the electrical unit comprises an electrical signal generator in operable communication with the controller.

7. The system of claim 1, further comprising: a pressure sensor arranged to be in fluidic communication with a lumen of the pipette, wherein the pressure sensor is configured to measure a pressure within the lumen.

8. The system of claim 1, wherein the controller is further configured to transmit third control instructions to the pressure unit to set a rate of a plurality of negative pressure pulses, wherein the plurality of negative pressure pulses comprises the negative pressure pulse, wherein each negative pressure pulse of the plurality of negative pressure pulses corresponds to a respective electrical pulse.

9. The system of claim 1, wherein the controller is further configured to transmit third control instructions to the electrical unit to set a rate of a plurality of electrical pulses, wherein the plurality of electrical pulses comprises the electrical pulse, wherein each electrical pulse of the plurality of electrical pulses corresponds to a respective negative pressure pulse.

10. A system, comprising: a controller coupled to a valve situated in fluidic communication between a pipette and a pressure source, the controller having a trigger which, when activated, causes thecontroller to actuate a valve to cause a plurality of negative pressure pulses having a predetermined rate and a predetermined duration to cyclically apply negative pressure to a cell membrane in contact with the pipette; an electrode arranged at a pipette and configured to deliver a plurality of electrical stimuli to the cell membrane, wherein the controller is configured to cause delivery of each electrical stimulus of the plurality of the electrical stimuli to respectively occur during the predetermined duration of a negative pressure pulse of the plurality of negative pressure pulses.

11. A method, comprising: applying, via a pipette, a plurality of negative pressure pulses at a membrane of a cell, a first negative pressure pulse of the plurality of negative pressure pulses configured to have a first duration, the plurality of negative pressure pulses having a first rate; applying, via the pipette, a plurality of electrical pulses at the membrane of the cell, each electrical pulse of the plurality of electrical pulses occurring contemporaneously with a negative pressure pulse of the plurality of negative pressure pulses, a first electrical pulse of the plurality of electrical pulses having a start time offset from a start time of the first negative pressure pulse, the first electrical pulse having a second duration; and terminating application of the plurality of negative pressure pulses and the plurality of electrical pulses based on perforating the membrane.

12. The method of claim 11, wherein each negative pressure pulse of the plurality of negative pressure pulses has the first duration.

13. The method of claim 11, wherein the method further comprises: applying, via the pipette, a second negative pressure pulse of the plurality of negative pressure pulses to the membrane of the cell, wherein the second negative pressure pulse is successive to the first negative pressure pulse, and wherein the second negative pressure pulse has a second duration greater than the first duration.

14. The method of claim 13, wherein the method further comprises: applying, via the pipette, a second electrical pulse of the plurality of negative pressure pulses to the membrane of the cell, the second electrical pulse occurringcontemporaneously to the second negative pressure pulse, wherein the second electrical pulse has the second duration.

15. One or more non-transitory computer-readable media having stored thereon a sequence of instructions that, when executed by one or more processors, cause a system to: transmit first control instructions to cause a pressure source to apply a negative pressure at a pipette; transmit second control instructions to cause a signal generator to apply an electrical stimulus at the pipette, the second control instructions setting the electrical stimulus to occur contemporaneously to the negative pressure; determine that a membrane of a cell has been perforated; transmit third control instructions to cause the pressure source to terminate application of negative pressure at the pipette based at least in part on the determination; and transmit fourth control instructions to cause the signal generator to terminate application of the electrical stimulus at the pipette based at least in part on the determination.

16. The one or more non-transitory computer-readable media of claim 15, wherein the first control instructions comprise a first duration of a first negative pressure pulse used to apply to the negative pressure.

17. The one or more non-transitory computer-readable media of claim 16, wherein the sequence of instructions that, when executed by one or more processors , further cause the system to: receive sensor-based data indicating a state of the membrane; access a machine learning model based at least in part on receiving the sensorbased data; generate, using the machine learning model a prediction for a second duration of a second negative pulse; and transmit fifth control instructions to apply negative pressure via a second negative pressure pulse based at least in part on the prediction.

18. The one or more non-transitory computer-readable media of claim 15, wherein the determination is based at least in part on a user-based input.

19. The one or more non-transitory computer-readable media of claim 15, wherein the first control instructions comprise a duration, rate, and magnitude of negative pressure pulses to be used to apply the negative pressure.

20. The one or more non-transitory computer-readable media of claim 19, wherein the duration, rate, and magnitude are based at least in part on a type of the cell.

Images