System and method for electrical stimulation of samples

Abstract

A system for electrical stimulation of samples, the system including: a plate lid having a plurality of pairs of electrodes, the plate lid being configured to removably couple with a culture plate having a plurality of wells, such that each well of the culture plate receives a pair of the electrodes, the pair of electrodes being at least partially in contact with a sample in the corresponding well; and a connector head having one or more pairs of electrical connectors configured to electrically connect to an electrical stimulator.

Description

SYSTEM AND METHODFOR ELECTRICAL STIMULATION OF SAMPLESTECHNICAL FIELD

[0001] The present disclosure relates to a system and method for electrical stimulation of a sample for analysis.BACKGROUND

[0002] Engineered culture systems, also known as engineered tissue or organ culture systems, are artificial environments designed to provide a controlled setting where cells or tissues can grow, interact, and behave similarly to how they would in the body.

[0003] In engineered culture systems, electrical stimulation serves as a powerful tool to measure the functional properties of samples, providing valuable insights into their behaviour and response to external stimuli. For example, these engineered culture systems may include 2D and 3D cell cultures, including cultures for skeletal muscle, smooth muscle, cardiac muscle, neurons, and some endocrine cells (e.g. insulin-releasing pancreatic P cells). By applying controlled electrical signals to cultured cells, tissues or organs (referred to below collectively as "cultured samples"), researchers can mimic physiological conditions and study various cellular processes in vitro.

[0004] For instance, in in vitro muscle tissue engineering studies, electrical stimulation can induce and regulate the contraction of muscle cells, allowing for the assessment of muscle function, strength, and fatigue resistance. This approach is instrumental in understanding muscle physiology, investigating mechanisms underlying muscle diseases, and developing novel therapeutic strategies for conditions such as muscular dystrophy or age-related muscle degeneration. Similarly, in in vitro cardiac tissue engineering studies, electrical stimulation can induce and regulate the contraction of cardiomyocytes, allowing for the assessment of cardiac function and the development of therapeutic interventions for heart diseases. In neuroengineering studies, electrical stimulation enables the investigation of neuronal excitability, synaptic transmission, and network connectivity, and is crucial for understanding brain function and dysfunction.

[0005] Existing methods of applying electrical stimulation to cultured cells or tissues usually involve a researcher or laboratory technician (referred to below as the "operator") manually placing the electrodes for delivering electrical signals within the culture system,e.g., in a well of a culture plate where the biological sample is cultured. The electrodes are connected to a suitable stimulation generator or power supply, e.g., through cables or wires.

[0006] Typically, when a multiple-well culture plate is used, the manual application of electrical stimulation requires a series of operational steps to be performed by the operator: placing the electrodes in one well, controlling the application of electrical stimulation, adjusting stimulation and / or monitoring parameters (e.g., the setting of an imaging device such as a microscope), monitoring the response of the sample and recording relevant data, cleaning or sterilising the electrodes, if necessary, and then moving to the next well to repeat the same process. While manual application of electrical stimulation can be a useful tool in researching cultured samples, this approach has a number of disadvantages, including low efficiency, limited accuracy, inconsistency, and increased risk of contamination.

[0007] Manually applying electrical stimulation to samples requires manually moving the electrodes from well to well and constant adjustment of stimulation and / or monitoring parameters, which is time-consuming and labour-intensive, especially when handling large numbers of samples or conducting high-throughput experiments. This limits the efficiency and scalability of experiments, and may not be feasible for high-throughput studies or longterm culture experiments.

[0008] Achieving precise spatial and temporal control over electrical stimulation manually can be difficult. Subtle variations in electrode positioning or manipulation during stimulation may lead to unintended effects on nearby cells or tissues, resulting in low accuracy of the measurement results, and making it difficult to isolate specific cellular responses. Errors made by the operator(s) in placing the electrodes (e.g., placing the electrodes in a wrong well, i.e., a well different from the well to be monitored) may also lead to incorrect results.

[0009] Due to the limited precision of control (which may be caused by inconsistency of operation from the same person or the variability between different operators), manual application of electrical stimulation may result in variations between experiments or even within the same experiment. This inconsistency can introduce variability in the cellular response and hinder the accuracy and reproducibility of results.

[0010] Handling electrodes and stimulation equipment manually, and using the same electrodes from well to well, may increase the risk of contamination, especially in cell culture environments where sterility is crucial. Contaminants introduced during the stimulation process can compromise cell viability and affect the accuracy of experimental outcomes.[OOH] It is desired to address or ameliorate one or more disadvantages or limitations associated with the prior art, or to at least provide a useful alternative.SUMMARY

[0012] According to the present invention, there is provided a system for electrical stimulation of samples, the system including: a plate lid having a plurality of pairs of electrodes, the plate lid being configured to removably couple with a culture plate having a plurality of wells, such that each well of the culture plate receives a pair of the electrodes, the pair of electrodes being at least partially in contact with a sample in the corresponding well; and a connector head having one or more pairs of electrical connectors configured to electrically connect to an electrical stimulator; wherein the connector head is configured to be moved automatically relative to the plate lid by a moving mechanism, such that: at a first time, each pair of the one or more pairs of electrical connectors of the connector head are electrically connected to a corresponding pair of a first one or more pairs of the electrodes of the plate lid, to provide electrical stimulation to the sample(s) in a first group of one or more wells of the culture plate; and at a second time, each pair of the one or more pairs of electrical connectors of the connector head are electrically connected to a corresponding pair of a second one or more pairs of the electrodes of the plate lid, to provide electrical stimulation to the sample(s) in a second group of one or more wells of the culture plate.

[0013] In one embodiment of the present invention, the one or more pairs of electrical connectors of the connector head include a single pair of electrical connectors.

[0014] In one embodiment of the present invention, the one or more pairs of electrical connectors of the connector head include multiple pairs of electrical connectors.

[0015] In one embodiment of the present invention, the moving mechanism is configured to move the culture plate when the plate lid is removably coupled to the culture plate.

[0016] In one embodiment of the present invention, the connector head is configured to couple to an imaging device. In a further embodiment of the present invention, the imaging device includes a microscope, and the connector head is configured to couple to an objective of the microscope. In another embodiment of the present invention, the movement of theconnector head relative to the plate lid is caused at least partially by the movement of the objective of the microscope.

[0017] In another embodiment of the present invention, the connector head further includes a printed circuit board (PCB) for electrically connecting the electrical connectors to the electrical stimulator, wherein the PCB has one or more windows to allow transmission of light.

[0018] In one embodiment of the present invention, the PCB has a gap between each pair of the electrical connectors to allow transmission of light.

[0019] In one embodiment of the present invention, the electrodes of the plate lid are arranged in an array of rows and columns.

[0020] In another embodiment of the present invention, the plate lid has: a lid body with a first side and a second side opposite to each other; wherein each of the electrodes of the plate lid has an elongated shape, one end of which protruding from the first side of the lid body to contact the sample in a corresponding well of the culture plate, the other end of which being exposed on the second side of the lid body to be contactable by a corresponding electrical connector of the connector head to enable electrical connection between the corresponding electrical connector and the electrode.

[0021] In one embodiment of the present invention, each of the electrodes is a rigid pin coated with a conductive metallic layer. In a further embodiment of the present invention, each of the pins is partially coated with an electrically insulating material, exclusive of an end portion of the pin.

[0022] In one embodiment of the present invention, the plate lid and the culture plate form an enclosed containing space when the plate lid is removably coupled to the culture plate.

[0023] In one embodiment of the present invention, each of the electrical connectors of the connector head includes a rollerball mechanism to secure the contact with the corresponding electrodes of the plate lid.

[0024] In one embodiment of the present invention, the sample is a microscopy sample.

[0025] In one embodiment of the present invention, the plate lid has at least one gaseous port for maintaining a controlled gaseous environment in the culture plate during stimulation.

[0026] According to the present invention, there is also provided a connector head for a system for electrical stimulation of samples, the connector head includes:one or more pairs of electrical connectors configured to electrically connect to an electrical stimulator; a coupling mechanism for coupling the connector head to a moving mechanism to automatically move the connector head relative to a plate lid having a plurality of pairs of electrodes, the plate lid being configured to removably couple with a culture plate having a plurality of wells, such that each well of the culture plate receives a pair of the electrodes, the pair of electrodes being at least partially in contact with a sample in the corresponding well; wherein when the connector head is moved relative to the plate lid: at a first time, each pair of the one or more pairs of electrical connectors of the connector head are electrically connected to a corresponding pair of a first one or more pairs of the electrodes of the plate lid received by a first group of one or more wells of the culture plate to provide electrical stimulation to the sample(s) in the first group of one or more wells; and at a second time, each pair of the one or more pairs of electrical connectors of the connector head are electrically connected to a corresponding pair of a second one or more pairs of the electrodes of the plate lid received by a second group of one or more wells of the culture plate to provide electrical stimulation to the sample(s) in the second group of one or more wells.

[0027] According to the present invention, there is further provided a plate lid for electrical stimulation of samples, including: a lid body; and a plurality of pairs of metallic electrodes protruding from the lid body; wherein the lid body is configured to removably couple with a culture plate having a plurality of wells, such that each well of the culture plate receives a pair of the electrodes, and wherein the pair of electrodes is at least partially in contact with a sample in the corresponding well.

[0028] According to the present invention, there is further provided a method for electrical stimulation of samples, the method including: coupling a plate lid having a plurality of pairs of electrodes to a culture plate having a plurality of wells, such that each well of the culture plate receives a pair of the electrodes, the pair of electrodes being at least partially in contact with a sample in the corresponding well; andautomatically moving a connector head relative to the plate lid using a moving mechanism, wherein the connector head has one or more pairs of electrical connectors configured to electrically connect to an electrical stimulator; wherein at a first time, each pair of the one or more pairs of electrical connectors of the connector head are electrically connected to a corresponding pair of a first one or more pairs of the electrodes of the plate lid, to provide electrical stimulation to the sample(s) in a first group of one or more wells of the culture plate; and at a second time, each pair of the one or more pairs of electrical connectors of the connector head are electrically connected to a corresponding pair of a second one or more pairs of the electrodes of the plate lid, to provide electrical stimulation to the sample(s) in a second group of one or more wells of the culture plate.BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Some embodiments of the present invention are hereinafter described with reference to the accompanying drawings in which:

[0030] FIG. 1 is a drawing of an example of a system for electrical stimulation of microscopy samples;

[0031] FIG. 2 shows an example of a plate lid of the system for electrical stimulation of microscopy samples;

[0032] FIG. 3 shows an example of a culture plate to be used with the system for electrical stimulation of microscopy samples;

[0033] FIG. 4(a) shows a perspective view of the plate lid coupled to the culture plate;

[0034] FIG. 4(b) is a schematic diagram showing a top view of the plate lid coupled to the culture plate;

[0035] FIG. 4(c) is a schematic diagram showing a cross-sectional view of the plate lid coupled to the culture plate;

[0036] FIG. 5 is a schematic diagram showing a top view of the plate lid;

[0037] FIG. 6(a) is a schematic diagram showing a cross-sectional view of the plate lid along line A-A of FIG. 5;

[0038] FIG. 6(b) is a schematic diagram showing a detailed view of area B of FIG. 6(a);

[0039] FIG. 6(c) is a schematic diagram showing a detailed view of area C of FIG. 6(a);

[0040] FIG. 6(d) is a schematic diagram showing a detailed view of area D of FIG. 5;

[0041] FIG. 6(e) is a schematic diagram showing a cross-sectional view of a portion of the plate lid with a plurality of conductive pins;

[0042] FIG. 6(f) is a schematic diagram showing a cross-sectional view of a portion of a conductive pin in the plate lid;

[0043] FIG. 7 shows an example of the plate lid including the conductive pins with an insulative coating;

[0044] FIG. 8 is a drawing of an example of the connector head;

[0045] FIG. 9 is a schematic diagram showing an example of the system for electrical stimulation of microscopy samples in use;

[0046] FIG. 10(a) and FIG. 10(b) are schematic diagrams showing exemplary trajectories of electrical connectors of the connector head when moving relative to the plate lid;

[0047] FIG. 11 is a schematic diagram showing an example of the system with a motorized stage supporting the culture plate;

[0048] FIG. 12(a) is a schematic diagram showing a side view of a head body of the connector head;

[0049] FIG. 12(b) is a schematic diagram showing a cross-sectional view of the head body along line A-A of FIG. 12(a);

[0050] FIG. 13 is a schematic diagram showing the connector head attached to an objective of a microscope;

[0051] FIG. 14(a) shows an example of a printed circuit board (PCB) of the connector head of FIG. 7;

[0052] FIG. 14(b) is a schematic diagram showing circuit traces in the PCB of FIG. 13(a);

[0053] FIG. 14(c) shows alternative designs of a window area of the PCB;

[0054] FIG. 15 is a schematic diagram showing an example of the connector head with a shield;

[0055] FIG. 16 shows an example of tissue samples placed within wells of the culture plate, being positioned surrounding two poles present within each well;

[0056] FIG. 17 (a) is a plot showing the force of contraction of samples in wells 1-15 of the culture plate measured under electrical stimulation at different currents and a 20Hz frequency;

[0057] FIG. 17(b) is a plot showing the force of contraction of samples in wells 16-33 of the culture plate measured under electrical stimulation at different currents and a 20Hz frequency;

[0058] FIG. 18 is a plot showing the variation in the force measured under electrical stimulation at different currents;

[0059] FIG. 19(a) is a plot showing the force of contraction of samples in wells 1-18 of the culture plate measured in Tissue Centering mode ("LID") at 20mA, 100mA, Navigator mode ("LAS") at 20mA, 100mA, and in Manual mode ("MANUAL") at 20mA;

[0060] FIG. 19(b) is a plot showing the force of contraction of samples in wells 19-34 of the culture plate measured in Tissue Centering mode ("LID") at 20mA, 100mA, Navigator mode ("LAS") at 20mA, 100mA, and in Manual mode ("MANUAL") at 20mA;

[0061] FIG. 20 is a plot showing data variability in the force measured in Tissue Centering mode ("LID") at 20mA, 100mA, Navigator mode ("LAS") at 20mA, 100mA, and Manual mode ("MANUAL") at 20mA;

[0062] FIG. 21(a) is a plot showing the force of contraction measured from Well No. 1 - 18 of the culture plate in Tissue Centering mode ("LID") at 100mA, Navigator mode ("LAS") at 100mA, and Manual mode ("Manual") at 20mA;

[0063] FIG. 21(b) is a plot showing the force of contraction measured from Well No. 19 - 34 of the culture plate in Tissue Centering mode ("LID") at 100mA, Navigator mode ("LAS") at 100mA, and Manual mode ("Manual") at 20mA;

[0064] FIG. 22 is a plot showing data variability in the force measured in Tissue Centering mode ("LID") at 100mA, Navigator mode ("LAS") at 100mA, and Manual mode ("Manual") at 20mA;

[0065] FIG. 23 is a plot showing the correlation of the force measured in Navigator mode ("LAS") at 100mA and Manual mode ("MANUAL") at 20mA;

[0066] FIG. 24(a) is a plot showing the pre-force measured in response to the prestimulation at different currents of stimulation, with and without PTFE coating (insulated);

[0067] FIG. 24(b) is a plot showing the post-force measured in response to the poststimulation at different currents of stimulation, with and without PTFE coating (insulated);

[0068] FIG. 24(c) is a plot showing the relative force (Post Force relative to Pre Force) at different currents of stimulation, with and without PTFE coating (insulated);

[0069] FIG. 24(d) is a plot showing the viability readouts at Post Stimulation relative to Pre stimulation, at different currents of stimulation, with and without PTFE coating(insulated);

[0070] FIG. 25(a) is a plot showing the viability with and without PTFE coating (insulated) (combined data from current 20mA and above);

[0071] FIG. 25(b) is a plot showing the force of contraction shows the force of contraction with and without PTFE coating (insulated) (combined data from current 20mA and above);

[0072] FIG. 26 is a plot showing force measured from tissue only stimulated at the end point (i.e. without Pre-Stimulations) at different currents of stimulation, with and without PTFE coating (insulated);

[0073] FIGs. 27(a) - 27(g) are diagrams showing an alternative example of the PCB of the connector head with multiple pairs of electrical connectors;

[0074] FIGs. 28(a) and 28(b) are diagrams showing an alternative embodiment of the plate lid having a gaseous port in the form of a CO2 inlet;

[0075] FIG. 29 is a diagram showing the relationship between changes in pH in culture media and the resulting media colour shift when the culture media contains a pH indicator;

[0076] FIGs. 30 and 31 are photographs showing a comparison of culture media colour changes in the culture plate observed under different environmental conditions;

[0077] FIGs. 32(a) - 32(d) and 33(a) - 33(d) are plots showing the experimental results of functional assessment of bioengineered skeletal muscle tissue using the system 100; and

[0078] FIGs. 34 (a) - 34(d) are plots showing the experimental results of functional assessment of human cardiac organoids using the system 100.DETAILED DESCRIPTIONDefinitions

[0079] The present invention provides a system and method for automated electrical stimulation of a sample.

[0080] The sample(s) as referred to herein may be any substance having the potential to react to electrical stimulation, which results in measurable functional properties of the sample(s). The sample(s) may include any biological or chemical substance(s).

[0081] In one embodiment, the sample(s) may be microscopy sample(s).

[0082] The term "microscopy sample(s)" used in the present disclosure refers to biological materials that are prepared or cultured in a culture plate, and observed / monitored using an imaging device, such as a microscope.

[0083] The microscopy sample may include, e.g., muscle tissue and / or other type(s) of excitable cells. The term ‘excitable cell(s)’ refers to electrically active cell types including, e.g., muscle cells (skeletal muscle, smooth muscle, and / or cardiac muscle), neurons, and some endocrine cells (e.g. Insulin-releasing pancreatic P cells).

[0084] The microscopy sample has the potential to react to electrical stimulation, which results in measurable functional properties of the sample. For instance, when the microscopy sample is muscle tissue, it may contract in response to electrical stimulation, which can be used for measuring / analysing functional properties of the muscle tissue, e.g., force of contraction, and / or tissue viability. Alternatively, the microscopy sample may be a population of cells which can react to electrical stimulation and be used for calcium / voltage dye imaging.

[0085] According to some embodiments, the system and method described herein significantly improve the efficiency of electrical stimulation of samples and enhance the accuracy and reliability of the measurement results. It also eliminates human errors and variability associated with manual stimulation and reduces the risk of contamination during the stimulation experiments, which further improves the accuracy of the results.

[0086] Further advantages of the embodiments of the system and method are described below in association with specific embodiments or implementations.General structure of the system

[0087] Described herein is a system 100 for automated electrical stimulation of microscopy samples. FIG. 1 shows an exemplary embodiment of the system 100, including a plate lid 120 and a connector head 140.Plate lid

[0088] An example of the plate lid 120 is illustrated in FIG. 2. The plate lid 120 is configured to removably couple with a culture plate 200 that holds microscopy samples. An example of the culture plate 200 is illustrated in FIG. 3. The culture plate 200 has a plurality of wells 202. Each of the wells 202 is configured to hold a microscopy sample 204 (not shown in FIG. 3) cultured in the well or prepared elsewhere in advance and moved into the well. Inone embodiment, the wells may include a plurality of poles to aid in the measurement of the reaction of the microscopy sample to electrical stimulation.

[0089] Referring to FIG. 4, the plate lid 120 has a lid body 124. The lid body 124 is substantially planar, with a shape similar to a top face 206 of the culture plate 200 and a slightly larger size than the top face 206 of the culture plate 200. When the plate lid 120 is coupled to the culture plate 200, the lid body 124 sits on and covers the top of the culture plate 200, thereby covering all of the wells 202 on the culture plate 200. FIG. 4(a) - FIG. 4(c) show an example of the plate lid 120 coupled to the culture plate 200. Preferably, the plate lid 120 and the culture plate 200 form an enclosed space between them when coupled together.

[0090] The lid body 124 is formed of a transparent and electrically insulating material, e.g., Polycarbonate (PC) or any plastic, wherein the transparent and electrically insulating material may be rigid, semi-flexible or flexible.

[0091] In one embodiment, a fastening mechanism 125 is used for releasably locking the plate lid 120 and the culture plate 200 together once they are coupled. The fastening mechanism may include, for example, clips or snap fasteners, preferably provided on at least two sides of the rectangular lid body 124 and the culture plate 200.

[0092] As shown in FIG. 5, the plate lid 120 has a plurality of pairs of electrodes 122 (each pair of electrodes 122 also referred to below as "electrode pair 122a, 122b"), arranged in an array of rows and columns matching the positions of the wells 202 on the culture plate 200. Further details of the plate lid 120 are shown in FIG. 6(a) - FIG. 6(f).

[0093] As shown in FIG. 6(a), each of the electrodes 122 is secured in the lid body 124, extending from a top side 124a of the lid body 124 to a bottom side 124b of the lid body 124 opposite to the top side 124a. The electrodes 122 protrude from the bottom side 124b, such that when the plate lid 120 is coupled with the culture plate 200, each well 202 of the culture plate 200 receives a corresponding electrode pair 122a, 122b. At least a portion of each of the two electrodes 122a, 122b is protruding into the well 202 and in contact with the microscopy sample 204 in the well 202, as shown in FIG. 4(c).

[0094] The number of electrode pairs 122a, 122b on the plate lid 120 and their arrangement are configured to match the number and arrangement of wells 202 on the culture plate 200. For example, in this exemplary embodiment, the culture plate 200 is a standard 96- well culture plate (having 96 wells). Correspondingly, the plate lid 120 has 96 pairs of (i.e., 192 in total) electrodes 122, such that each well 202 on the culture plate 200 receives a pair of electrodes 122.Pin

[0095] Each of the electrodes 122 includes a rigid, conductive pin, e.g., a metal pin or a rigid pin coated with a conductive metallic layer. In this exemplary embodiment, each of the electrodes 122 is a brass alloy pin with a gold plating finish. In alternative embodiments, each of the electrodes may be a carbon or carbon-based electrode. However, using the metal pin provides the electrodes with higher mechanical strength compared to carbon electrodes. Further, in an alternative embodiment, the electrode can have a plating finish using a different metallic material rather than gold. However, plating with gold is preferable, as gold has high chemical stability and is biocompatible. Each pin is mounted on the lid body 124 through a mounting hole 126. As shown in FIGs. 6(b), 6(e) and 6(f), each pin is provided with a flange 127 at the top end to allow easy assembly of the pin with the lid body 124 and to secure the pin in position.

[0096] Preferably, each of the conductive pins is partially coated with an electrically insulating material, e.g., Polytetrafluoroethylene (PTFE), forming an insulating coat 128. As shown in FIG. 6(c), the insulating coat 128 covers a majority portion of the pin protruding from the lid body 124, exclusive of the end portion 129a of the pin that will be in direct contact with the microscopy sample 204, e.g., the last 1-1.5mm of the pin. This concentrates the electrical current to be applied to the microscopy sample 204 and restricts the cross-exchange of charges between adjacent wells, thereby improving the accuracy of the results to provide targeted stimulation of the microscopy sample within a specific well. In addition, having the insulating coat 128 can also reduce the surface energy of the conductive pins. This may prevent or restrain potential capillary action that causes the media fluid in the well to flow out, thereby preventing or reducing cross-contaminate of samples in nearby wells. Alternatively, each of the conductive pins may be coated with a Polyolefin or any other suitable thermoplastic material or coating with insulative properties (e.g., a Tygon tubing, or a spray insulation). Preferably, the top end portion of the 129b of the pin near the lid body 124 (e.g., the first 0.5 mm protruding from the bottom surface 124b of the lid body 124) is also exposed from the insulating coat 128, to avoid the insulating coat 128 spreading onto the lid body 124 when manufacturing the plate lid 120. An example of the conductive pin(s) with an insulative coating is shown in FIG. 6(c) and FIG. 7.Connector head

[0097] An example of the connector head 140 is shown in FIG. 8. The connector head 140 includes a pair of electrical connectors 142a, 142b secured on a head body 144.

[0098] As shown in FIG. 9, the electrical connectors 142a, 142b are configured to be electrically connected (e.g., through an internal circuit of the connector head 140) to an electrical stimulator 300 that generates electrical pulses / signals. Preferably, the electrical stimulator 300 is a digital stimulator that allows for precise control / adjustment of the parameters (e.g., current, duration, timing) of its output pulses / signals. The electrical stimulator may be any system that can generate a controlled voltage or current pulse, including for example Harvard Apparatus (LE12106 & LE12406TC&OM), Panlab BASIC STIMULATOR LE12006, Digitimer DS2A Isolated Voltage Stimulator, BIOP AC Voltage Stimulators BSLSTMB, BSLSTMA or Constant Voltage Stimulator - Unipolar Pulse STM200.

[0099] Each of the electrodes 122 extends from one side 124a of the lid body 124 to the opposite side 124b of the lid body 124.

[0100] On the bottom side 124b, the electrodes 122 protrude from the lid body 124, such that each electrode pair 122a, 122b can be received by a corresponding well 202 of the culture plate 200. On the top side 124a, the end of each electrode 122 is exposed from the lid body 124.

[0101] The distance between the two electrical connectors 142a and 142b of the connector head 140 is substantially the same as the distance between the two electrodes 122a and 122b of the plate lid 120, such that when the connector head 140 is placed in proximity to the plate lid 120, the two electrical connectors 142a and 142b on the connector head 140 can align with and physically contact the two electrodes 122a and 122b on the plate lid 120, respectively, forming a secure electrical connection between them. In this way, the electrical connectors 142a and 142b can deliver the electrical pulses / signals received from the electrical stimulator 300 to the two electrodes 122a and 122b, to apply electrical stimulation to the microscopy sample 204 in the corresponding well 202 receiving the two electrodes 122a and 122b. Preferably, each of the electrical connectors 142a and 142b is provided with a rollerball mechanism 146 at its tip to secure the physical contact and electrical connection with a corresponding electrode 122 of the plate lid 120. Alternatively, the electrical connectors 142a and 142b may be replaced with a ‘brush’ connector, a U-shaped connector or any other suitably shaped connector to provide contact with the two electrodes 122a and 122b on the plate lid 120.Relative Movement of the Connector Head

[0102] In use, the connector head 140 is configured to be moved relative to the plate lid 120, such that the two electrical connectors 142a and 142b of the connector head 140 are in electrical connection with a different pair of electrodes 122a and 122b at each time, so as to stimulate the microscopy samples in different wells 202 of the culture plate 200 in succession. That is, the electrical stimulation is provided to the microscopy samples in the culture plate 200 well to well.

[0103] For example, as shown in FIG. 9, at a first time, the two electrical connectors 142a and 142b of the connector head 140 are in electrical connection with the pair of electrodes 122a and 122b of the plate lid 120, to stimulate the microscopy sample 204 in a well 202 of the culture plate 200. At a second time, the connector head 140 is moved to a different position (not shown) relative to the plate lid 120, such that the two electrical connectors 142a and 142b of the connector head 140 are in electrical connection with the pair of electrodes 122a' and 122b' of the plate lid 120 to stimulate the microscopy sample 204' in a different well 202' of the culture plate 200. Preferably, the connector head 140 is configured to move from one well to an adjacent well for each step, to traverse all the wells 202 of the culture plate 200. Alternatively, the connector head 140 is configured to move from one well to any second well within plate 200 for each subsequent step, to traverse all the wells 202 of the culture plate 200. Exemplary trajectories showing the movement of the two electrical connectors 142a and 142b of the connector head 140 relative to the plate lid 120 are illustrated in FIG. 10. FIG. 10(a) shows an example where the electrical connectors 142a and 142b move in an intermittent trajectory from row to row, while FIG. 10(b) shows an alternative example where the electrical connectors 142a and 142b move in an intermittent trajectory from column to column. In an alternative embodiment, the movement of the two electrical connectors 142a and 142b of the connector head 140 relative to the plate lid 120 may be in any direction across the plate lid. For example, moving across the plate lid beginning from the top left well to the bottom right well, then to the top right well to the bottom left well, and so on. In a further embodiment, the connector head may move across the plate lid alternating rows, columns or wells, or move across the plate lid in any other trajectory as required.

[0104] An imaging device, e.g., a microscope, is used to capture one or more images of the microscopy sample being electrically stimulated. This may also include one or more video(s), defined as a sequence of images or frames captured in succession. The capturedimage(s) or video(s) is then analysed using existing imaging processing techniques to derive one or more measurable functional properties of the microscopy sample 204. For example, when the microscopy sample 204 is muscle tissue, the measurable functional properties may include the force of contraction. An example of the microscope used is a Leica THUNDER Imager. However, the invention may be implemented to adapt to any other type of microscope suitable for imaging microscopy samples on a culture plate.

[0105] Referring to FIG. 11 showing a motorised stage 400 supporting the culture plate 200 with the plate lid 120. The movement of the connector head 140 is automated by using a moving mechanism. For example, a motorised stage 400 can be configured to support the culture plate 200 with the plate lid 120 removably coupled to it, and to move them relative to the connector head 140.

[0106] The movement of the motorised stage 400 is controlled by a computing device, e.g., an external computer. Preferably, the computing device is in data communication with the imaging device for capturing the image(s) of the microscopy sample 204 being electrically stimulated, such that the movement of the motorized stage 400 is coordinated with the imaging process performed by the imaging device, to synchronise the electrical stimulation process and the imaging process. This ensures that the well being imaged is the same well being stimulated, and that when the motorized stage 400 moves to align the next well with the electrical connectors 142a and 142b of the connector head 140, the imaging device is configured accordingly to capture the image(s) of the next well. This arrangement further improves the accuracy of the measurement / analysis results.Coupling mechanism (to an imaging device)

[0107] Preferably, the connector head 140 is configured to be removably coupled to the imaging device to further improve the synchronisation of the electrical stimulation process and the imaging process.

[0108] For example, when the imaging device is a microscope 500, the connector head 140 is configured to be removably coupled to an objective 510 of the microscope 500 through a coupling mechanism. Preferably, once coupled, the centre of the objective 510 is substantially aligned with a middle point between the two electrical connectors 142a and 142b of the connector head 140, such that the microscopy sample 204 being electrically stimulated is placed in the centre of the field of view (FoV) of the microscope 500 for image / video capturing.

[0109] Referring to FIGs. 12(a) and 12(b), as an example of the coupling mechanism, the head body 144 of the connector head 140 may include a head mount 150 formed with a pair of clamping arms 150a and 150b to attach the connector head 140 to the objective 510 of the microscope 500 in between. The distance between the two clamping arms 150a and 150b can be adjusted by tightening / loosening a bolt to securely fix the connector head 140 to the objective 510. FIG. 13 is a schematic diagram showing the connector head 140 attached to the objective 510 of the microscope 500.Printed Circuit Board (PCB)

[0110] The head body 144 further includes a printed circuit board (PCB) 148 for electrically connecting the electrical connectors 142a, and 142b to the electrical stimulator 300. FIGs. 14(a) and 14(b) show an example of the PCB 148 located within in the connector head 140 shown in FIG. 8. The rigid structure of the PCB 148 also physically supports the electrical connectors 142a, and 142b and secures them in place.

[0111] More specifically, the connector head 140 includes a pair of input terminals 152a and 152b provided on the head mount 150, as shown in FIG. 9, where the input terminals 152a, and 152b are electrically connected to the PCB 148 (see FIG. 14(b)). The input terminals 152a, and 152b are configured to be connected respectively to the positive and negative terminals of the external electrical stimulator 300 that generates electrical pulses / signals. The substrate of the PCB 148 has a window area 154 (shown by the dashed line in FIG. 14(b)) with two rib structures 156a, and 156b and one or more openings 158. The one or more openings 158 allow for the transmission of light during imaging of the stimulated muscle tissue or conductive cells. Preferably, the overall shape and size of the window area 154 are configured to substantially match the shape and size of the objective 510 of the microscope 500, which maximizes the visible field of the microscope 500 when capturing the image / video of the microscopy sample 204 being electrically stimulated.

[0112] As shown in FIG. 14(b), the two electrical connectors 142a and 142b are positioned preferably at an inner end of the two rib structures 156a and 156b, respectively, such that a gap is formed between the two inner ends of the two rib structures 156a and 156b to allow transmission of light. This further increases the amount of light transmitted from the microscopy sample 204 being electrically stimulated to the objective 510 of the microscope 500, thereby improving the quality of the image / video captured, as well as the accuracy of the measurement / analysis results of the functional properties. As shown in FIG. 14(b), the circuittraces 159 in the PCB 148 pass along the two rib structures 156a, and 156b to provide an electrical connection between the electrical connectors 142a and 142b to the input terminals 152a and 152b. The PCB 148 is secured to the head mount 150 through fastening mechanisms, e.g., threaded inserts, screws and bolts, or the like.

[0113] Preferably, the clamping arms 150a and 150b of the head mount 150 have an inner profile that matches the shape and size of the window area 154 of the PCB 148, to allow for the transmission of light while providing protection to the PCB 148. As shown in FIG. 12(a) and (b), the head mount 150 also has a pair of rib structures 160a and 160b, protruding from the two clamping arms 150a and 150b, respectively. Each of the rib structures 160a and 160b of the head mount 150 is shaped to match at least a portion of the rib structures 156a and 156b of the PCB 148, to protect the rib structures 156a and 156b and improve their mechanical strength to support the electrical connectors 142a and 142b.

[0114] The inner end of each rib structure 160a / 160b of the head mount 150 extends to where a corresponding electrical connector 142a / 142b is positioned, such that a gap is formed between the two inner ends of the two rib structures 160a and 160b to allow for transmission of light. This further increases the amount of light transmitted from the microscopy sample 204 being electrically stimulated to the objective 510 of the microscope 500, thereby improving the quality of the image / video captured, as well as the accuracy of the measured / analysed results of the functional properties of the microscopy sample 204.

[0115] Preferably, the head mount 150 is formed with a rigid material with sufficient mechanical strength for supporting / protecting the PCB 148 and attaching the connector head 140 to the imaging device such as the microscope. For example, the head mount 150 can be formed with a metallic alloy, such as an aluminium alloy. Alternatively, the head mount 150 may be formed of a plastic material, a metal material, or a combination thereof.

[0116] In the embodiments described above, the substrate of the PCB 148 has the window area 154 with two rib structures 156a, and 156b (connecting to the two electrical connectors 142a, 142b, respectively) and one or more openings 158.

[0117] The shape and arrangement of the rib structures and the one or more openings 158 may vary, as long as they allow transmission of sufficient light during imaging of the stimulated muscle tissue or conductive cells, and the rib structures provide sufficient mechanical strength for supporting the electrical connectors 142a, and 142b.

[0118] Preferably, the one or more openings 158 include a gap between the electrical connectors 142a and 142b to allow transmission of light from the centre of the well (which typically is the centre or approximate to the centre of the microscope sample).

[0119] Some alternative designs of the shape of the window area 154 are shown in FIG. 14(c).Shield

[0120] Referring to FIG. 14, the PCB 148 is accommodated in the head mount 150, and the connector head 140 further includes a shield 162 covering and protecting the PCB 148.

[0121] The shield 160 is formed of a transparent and rigid material, any transparent plastic or glass-based material, or a combination thereof, for example, Polycarbonate (PC).

[0122] A pair of holes is formed on the shield 162 to allow the electrical connectors 142a and 142b to pass through and protrude from the surface of the shield 162.Exemplary dimensions

[0123] As an example, for the system 100 to be used with a typical 96-well culture plate (measures approximately 128 mm in length, 86 mm in width, and 15 mm in height, with the wells arranged in an 8 x 12 grid, each well having a diameter of about 6.4 mm and a depth of about 10.8 mm), the plate lid 120 has a length of 128 mm, a width of 86 mm, and a total height of 13 mm (including the lid body with a thickness of 4 mm, and a portion of the pin electrodes protruding for 9mm from the bottom side 124b of the lid body 124). At the top end, the pin head is substantially flush with the top side 124a of the lid body 124, with an allowable protrusion of up to 0.02mm.

[0124] The metal pin electrodes are arranged on the lid body 124 in a l6 x l2 grid (each pair of pins in the adjacent rows being received in the same well of the culture plate), with the distance between two adjacent columns being 9 mm, and the distance between the adjacent rows being approximately 4.5mm (Alternatively, the rows may have alternating pitches, with 4.59 mm between the two rows aligning with the same row of wells on the culture plate, and 4.41 mm between the two rows aligning with different rows of wells on the culture plate).

[0125] Each pin has a total length of 12.95 mm, with the portion above the flange being 0.79 mm (including 9 mm protruding from the lid body 124). The flange has a diameter of 1.83 mm, while the portion of the pin immediately below the flange has a diameter of 1.42 mm and tapers to approximately 1.35 mm (see examples as shown in FIG. 6(e) and FIG. 6(f)). As described above, the pin is made of brass alloy, with a gold plating finish having a thickness of 0.25pm.

[0126] Each pin is coated with a PTFE coating except the first 0.5 mm protruding from the lid body 124, and the last 1.5 mm of the bottom end portion of the pin (which will be in direct contact with the microscopy sample 204), the PTFE coating has a thickness of 0.05 mm.

[0127] The connector head 140 has a length of 89.5 mm, a width of 50.1 mm, and a total height of 28 mm (including the head mount 150 being 24 mm thick and the shield 162 being 4 mm thick, with the PCB 148 accommodated in the head mount 150). The electrical connectors 142a, and 142b protrude from the surface of the connector head for approximately 2.4 mm. The window area 154 has a diameter of 40.10 mm. The electrical connectors 142a, and 142b are located in the middle of the window area 154, with a distance between the two electrical connectors being approximately 4.59 mm to match the distance between the two corresponding metal pin electrodes on the lid body 124.Process of applying stimulation

[0128] A process for applying electrical stimulation to microscopy samples using the system 100 includes: a) coupling the plate lid 120 to the culture plate 200. Each of the wells 202 of the culture plate 200 holds a microscopy sample 204. The microscopy samples have been captured in the well, or pre-prepared elsewhere in advance and moved into the well. When the plate lid 120 is coupled to the culture plate 200, each well 202 of the culture plate 200 receives a pair of the electrodes 122, and each pair of electrodes 122 is at least partially in contact with the microscopy sample 204 in the corresponding well. b) automatically moving the connector head 140 relative to the plate lid 120 by using the moving mechanism as described above. For example, the moving mechanism may include the motorized stage 400 which supports the culture plate 200 with the plate lid 120 removably coupled to it, and moves the culture plate 200 with the plate lid 120 relative to the connector head 140, such that:• at a first time, the two electrical connectors 142a and 142b of the connector head 140 are in electrical connection with the pair of electrodes 122a and 122b of the plate lid 120 (as shown in FIG. 8), to stimulate the microscopy sample 204 in a well 202 of the culture plate 200; and• at a second time, the connector head 140 is moved to a different position relative to the plate lid 120, such that the two electrical connectors 142a and 142b of the connector head 140 are in electrical connection with the pair of electrodes 122a' and 122b' of the plate lid 120 to stimulate the microscopy sample 204 in a different well 202' of the culture plate 200.[0129J When the microscopy sample 204 in a well is being stimulated, the behaviour of the microscopy sample 204 in response to the electrical stimulation is recorded by an imaging device (e.g., the microscope 500) that captures one or more image(s) / video(s) of the sample.[0130J The captured image(s) or video(s) is then analysed using existing imaging processing techniques to measure / derive one or more functional properties of the microscopy sample 204.

[0131] In this way, as the connector head 140 is moved automatically from well to well, the electrical connectors 142a and 142b are connected to different pairs of electrodes 122 in sequence, to stimulate and image microscopy samples in different wells.Advantages

[0132] According to at least some embodiments, the system 100 described above allows fast and automated selection of the well and the microscopy sample therein to be electrically stimulated, which significantly reduces the time and labour required for applying electrical stimulation to microscopy samples in a culture plate with multiple walls, especially when handling large numbers of samples or conducting high-throughput studies. As a result, the system 100 improves the efficiency of applying electrical stimulation to microscopy samples. For instance, as described in further detail in the "Experimental Results" below, for the same experiment on a full 96-well culture plate, the manual stimulation method requires approximately 3 hours to complete the stimulation and imaging for the whole plate, while with the system 100 it only requires 15 - 20 minutes.

[0133] According to at least some embodiments, the system 100 described above also improves the precision in positioning the electrodes and controlling the stimulation signals, ensuring consistent and accurate delivery of stimulation to each well. This reduces variations in the cellular response and improves measurement accuracy.

[0134] According to at least some embodiments, the system 100 described above further eliminates human errors and variability associated with manual stimulation, andensures greater consistency and reproducibility of experimental results. This also allows standardised stimulation protocols to be easily implemented and replicated across multiple experiments or experimental setups, enhancing the reliability of the results.

[0135] Furthermore, according to at least some embodiments, the system 100 described above reduces the risk of contamination during the stimulation experiments, which further improves the accuracy of the results.

[0136] Preferably, according to at least some embodiments, each of the electrodes 122 is a metal pin, e.g., a brass alloy pin with a gold plating finish. While the electrodes 122 may alternatively take other suitable forms, e.g., carbon electrodes with a rectangular plate shape, using the metal pin as electrodes 122 provides the electrodes with higher mechanical strength compared to using electrodes with alternative materials, e.g., carbon electrodes, thereby allowing the electrodes to be designed and made in a much smaller size compared to a plate lid with carbon electrodes. This enables the plate lid 120 and the system 100 to be used with culture plates with a large number of wells, e.g., 48-well culture plates, and 96-well culture plates, thereby allowing higher throughput functional screening / experiment. Alternatively, the embodiments of the invention can be implemented to be used with culture plates with any other suitable number of wells, e.g., a 6-well, 12-well or 24-well plate, or a culture plate with more than 96 wells.

[0137] By contrast, carbon electrodes can only be made in much larger dimensions due to the inherent fragileness of the material, and therefore would be too big for the well size in a 48-well culture plate or 96-well culture plate would be too small to accommodate, thus providing a much more limited throughput.

[0138] Furthermore, as the metal pin electrodes have higher mechanical strength compared to carbon electrodes, they are also easier to replace and clean, and more robust against potential damage.

[0139] In addition, using the system 100 described above, the electrical stimulation and imaging of the microscopy samples can be repeated multiple times during the culturing process, without compromising the sterility of the sample. As such, the system 100 not only can be used for end-point measurement, but also before and during the treatment / culturing of the sample.

[0140] Preferably, the plate lid 120 and the culture plate 200 form an enclosed space between them when coupled together, this further ensures the sterility of the sample, and extends the potential duration and flexibility of the experiment.

[0141] In addition, in at least some embodiments, the movement of the motorized stage 400 is coordinated with the imaging process performed by the imaging device. That is, the electrical stimulation process and the imaging process are synchronised. This ensures that the well being imaged is the same well being stimulated, thereby improving the accuracy of the measurement results. Furthermore, coordinating the stimulation process and the imaging process also allows for reducing the time required for adjusting the settings of the imaging device, thereby enabling rapid imaging of the samples upon electrical stimulation.

[0142] In addition, in at least some embodiments, each of the metal pin electrodes is partially coated with an electrically insulating material, e.g., Polytetrafluoroethylene (PTFE) to cover a majority portion of the pin protruding from the lid body 124. Advantageously, this restricts the cross-exchange of charges between adjacent wells and eliminates unintended stimulation of the samples in nearby wells, thereby improving the accuracy of the results. This also prevents or reduces the potential capillary action of the media fluid in the well flowing out, thereby preventing cross-contaminate of samples in nearby wells.

[0143] In addition, in at least some embodiments, the PCB 148 has a window area 154 with one or more openings 158 to allow transmission of light during imaging of the stimulated muscle tissue or conductive cells. This enlarges the visible field of the microscope 500 when capturing the image / video of the microscopy sample 204 being electrically stimulated, and allows sufficient light to pass from the microscopy sample 204 to the microscope 500 to ensure the imaging quality and accurate downstream functional analysis.

[0144] In addition, in the system 100 described above, the selection of the microscopy sample 204 to be electrically stimulated is achieved through the relative movement of the connector head 140 to the plate lid 120 (e.g., by using a motorized stage 400). This not only enables a rapid selection and change in the well to be stimulated, but also eliminates the need to use a separate PCB or electrical circuit in the plate lid 120 for selecting the electrodes 122 to be electrically connected (to an external electrical stimulator), thus ensuring a more cost- effective design solution of the plate lid 120 with less manufacturing complexity.Variations / Alternative embodiments[Selecting multiple wells]

[0145] The embodiments of the present invention can be implemented to be used with culture plates with any suitable number of wells, e.g., 6-well plates, 12-well plates, 24-well plates, 48-well plates, 96-well plates, or culture plates with more than 96 wells.

[0146] In some embodiments, the connector head 140 has a single pair of electrical connectors 142a, 142b used for selecting a single well at a time to apply the stimulation. The connector head 140 may alternatively be configured to have multiple pairs of electrical connectors that can connect to and stimulate multiple wells at the same time.

[0147] For example, at a first time, the multiple pairs of electrical connectors on the connector head 140 are electrically connected to a first group of electrode pairs on the plate lid 120, to provide electrical stimulation to the microscopy samples in a first group of wells on the culture plate 200. At a second time, the multiple pairs of electrical connectors on the connector head 140 are electrically connected to a second group of electrode pairs on the plate lid 120, to provide electrical stimulation to the microscopy samples in a second group of wells on the culture plate 200, the second group of wells are different from the first group of wells.

[0148] Preferably, the microscope 500 used is capable of imaging multiple wells at the same time, or in a short period of time without substantially changing the settings.

[0149] To further increase the speed and efficiency of experiments, the relative movement of the connector head 140 to the plate lid 120 (e.g., through the motorized stage 400 that supports the culture plate 200 to which the plate lid 120 is removably coupled) can be configured based on the number of wells selected / stimulated at each time, to avoid / reduce redundancy and maximize the efficiency of experiment / screening.

[0150] This is particularly advantageous when used with culture plates with a large number of wells, to allow high-throughput experiments or screening, and increase experimental efficiency and scalability.

[0151] Preferably, the number of pairs of electrical connectors is a divisor of the number of wells on the culture plate 200 that the connector head is to be used with. For example, if the culture plate 200 is a 96-well plate (8 rows, 12 wells in each row), the connector head 140 may have 2 or 4 pairs of electrical connectors, which can double or quadruple the speed and efficiency of experiment / screening. Alternatively, the connector head 140 may have 12 pairs of electrical connectors, which would allow stimulating a whole row of 12 wells at a time, therefore improving the experiment / screening speed and efficiency by 12 times.

[0152] Alternatively, the electrical connectors 142a and 142b may be in the form of a brush connector or U-shape electrical connector, or any such form as to provide a means to electrically connect to an electrode pair 122a and 122b to provide electrical stimulation to the microscopy samples in one or more wells on the culture plate 200.

[0153] Simultaneously stimulating multiple wells in a culture plate at the same time may also allow easy and efficient observation, comparison and / or analysis of microscopy samples of different types (for example 2D cardiomyocytes, 3D muscle tissues and 2D neurons on the same plate) or under different conditions, e.g., different types of cells, different media compositions, and / or different preparation procedures / conditions.

[0154] An exemplary embodiment of the connector head 140 having multiple pairs of electrical connectors is shown in FIGs. 27(a) - 27(g).

[0155] For simplicity, FIGs. 27(a) - 27(g) only illustrate an example of the PCB 148 within the connector head 140, having input terminals 152a and 152b and multiple pairs of electrical connectors 142a and 142b. The remaining portions of the connector head 140, such as the remaining parts of the head body 144, are not shown.

[0156] In this embodiment, the PCB 148 is provided with 32 pairs of electrical connectors 142a and 142b, allowing stimulation of up to 32 wells (4 columns x 8 rows) of the culture plate 200. For example, if the culture plate 200 is a 96-well culture plate, this design enables simultaneous stimulation of approximately one-third of the wells.

[0157] FIGs. 27(e) and 27(f) show a fabricated PCB 148 with such a design. FIG. 27(g) shows the PCB 148 in use, i.e., when it is mounted onto the plate lid 120 and electrically connected to a corresponding plurality of pairs of electrodes 122 on the plate lid 120.

[0158] Preferably, each of the electrical connectors 142a and 142b is a spring-loaded pin, also known as a pogo pin (as shown in FIG. 27(b)), which ensures a secure electrical connection with the corresponding electrode 122 (being a conductive pin) on the plate lid 120. As shown in FIGs. 27 (c) and 27(d), a central viewing hole 149 (e.g., approximately 1.3 mm x 3.9 mm) is provided on the PCB 148 between each pair of electrical connectors 142a and 142b to allow light to pass through from the microscopy sample 204 during imaging.

[0159] The input terminals 152a and 152b, in the form of banana plugs, can be connected to the positive and negative terminals of an external electrical stimulator 300 that generates electrical pulses / signals. As shown in FIG. 27(d), the electrical connectors 142a and 142b are electrically connected to the input terminals 152a and 152b, such that the current provided by the stimulator 300 can pass sequentially (as shown by the dotted line in FIG. 27(d)) through each well of the culture plate 200, thereby applying multi-well stimulation to the samples in those wells.

[0160] This design allows stimulation and imaging of multiple wells simultaneously, for example, by using a microscope equipped with a multi -array camera.

[0161] This design is also advantageous when applying chronic or prolonged stimulation to the samples - e.g., applying a 30-minute treatment every 2 days to muscle tissues / cells to mimic exercise - as it allows stimulating and imaging multiple wells without moving the PCB 148.

[0162] While FIGs. 27(a) - 27(g) illustrate a PCB design with 32 pairs of electrical connectors 142a and 142b (4 columns x 8 rows), this approach may be adapted to provide a PCB design with any other suitable number of electrical connector pairs, enabling stimulation of any desired number of wells across the multi-well culture plate 200 (e.g., from 2 wells up to 96 wells for a 96-well culture plate).[Moving mechanism]

[0163] In the embodiments described above, the relative movement between the connector head 140 and the plate lid 120 is achieved by moving the plate lid 120, and more specifically, by using the motorized stage 400 that supports the culture plate 200 to which the plate lid 120 is removably coupled.

[0164] Alternatively, this relative movement may be achieved by moving the connector head 140. For example, some microscopes are capable of automatically moving the objective from well to well to scan multiple wells of a culture plate, capturing images from each well. As such, when the connector head 140 is attached to the objective of the microscope, the movement of the objective of the microscope can cause the connector head 140 to move from well to well, such that the electrical connectors of the connector head 140 can electrically connect to different electrodes pairs on the plate lid 120, to provide stimulation to samples in different wells. In this way, the synchronisation between the electrical stimulation process and the imaging process is achieved in a simple, time-efficient and cost-effective manner (as the motorized stage 400 is not required). This also provides a more accurate synchronisation and ensures the precise control of the position of the connector head 140 relative to the plate lid 120. This further secures the electrical connection between the electrical connectors on the connector head 140 and the corresponding electrodes on the plate lid 120, which improves the quality of the electrical signal applied to the sample, and reduces "non-responders" due topoor electrical connection, thereby enhancing the accuracy of the experiment / screening results.

[0165] Alternatively, the relative movement between the connector head 140 and the plate lid 120 may be achieved by coordination of the motorized stage 400 and a movable objective of a microscope, for example, by using the motorized stage 400 to move the culture plate 200 with the plate lid 120 close to a desired position, and then using the microscope with the movable objective for fine-tuning the position of the connector head 140 to more precisely align the electrical connectors of the connector head 140 with the corresponding electrodes on the plate lid 120.

[0166] In some embodiments, the relative movement between the connector head 140 and the plate lid 120 is achieved by combining automated control and manual operation. For example, the movement in the horizontal directions (i.e., the x-axis and y-axis) is controlled by a computing device that controls the automated movement of the objective of the microscope, and the movement in the vertical direction (i.e., the z-axis) is controlled by an operator manually lower the connector head (attached to the obj ective of the microscope) onto the plate lid. Alternatively, the relative movement in all directions may be automated, to improve efficiency and minimize the potential inconsistency introduced by manual operation.[Gaseous port]

[0167] In some embodiments, the plate lid 120 further includes at least one gaseous port configured to maintain a controlled gaseous environment in the culture plate during stimulation and / or imaging.

[0168] In some embodiments, the gaseous port is a carbon dioxide (CO2) inlet that enables regulated delivery of CO2 into the culture plate 200 while the plate lid 120 is in use.

[0169] In many cell culture applications, a bicarbonate-CCh buffering system is employed to maintain a stable physiological pH, typically around 7.0-7.4. In this system, dissolved CO2 in the medium is in equilibrium with carbonic acid and bicarbonate ions, which work together to counteract pH changes caused by cellular metabolism. The pH achieved is dependent on both the bicarbonate concentration in the medium and the partial pressure of CO2 in the surrounding atmosphere. Accordingly, most culture media are formulated for use within controlled CO2 environments, such as incubators operating at approximately 5% CO2. When cultures are removed from such conditions (e.g., during imaging outside an incubator),the reduced CO2 availability disrupts the buffering equilibrium and causes the media pH to rise rapidly, potentially impairing cell viability and function.

[0170] Maintaining such pH conditions requires a continuous supply of CO2. Without CO2, the buffering capacity of the bicarbonate system diminishes and the media pH rapidly increases.

[0171] To address this problem, in some embodiments, the plate lid 120 includes a CO2 inlet 170 (as shown in FIGs. 28(a) and 28(b)) that allows insertion of a CO2 delivery tube to provide controlled delivery of CO2 into the headspace above the wells of the culture plate 200. This configuration helps maintain appropriate media pH for an extended period, e.g., during stimulation and imaging procedures performed outside a standard cell culture incubator.

[0172] For example, regulated CO2 delivery through the inlet 170 can maintain cell culture media pH for at least 30 minutes or 60 minutes, similar to conditions inside a standard incubator. This prolongs the usable duration of stimulation and imaging experiments without introducing confounding effects from pH drift.

[0173] Conventional culture media typically include phenol red as a pH indicator. The media colour shifts from bright yellow at lower pH (approximately pH 6) to dull pink at higher pH (approximately pH 8), as shown in the colour chart of FIG. 29.

[0174] FIGs. 30 and 31 compare the media colour observed under four environmental conditions: (a) inside a cell culture incubator (control), (b) inside a microscope chamber at 37 °C without CO2 supply, (c) inside a microscope chamber at 37 °C with CO2 delivered through the inlet 170, and (d) on the laboratory bench at ambient laboratory temperature (approximately 25 °C). After 30 minutes (FIG. 30(b)) and 60 minutes (FIG. 31(b)), media in the microscope chamber without CO2 supplementation show a noticeable shift toward pink, indicating pH increase. By contrast, when CO2 is supplied through the inlet 170 (FIG. 30(c) and (FIG. 31(c))), the media retain colouration consistent with physiologically appropriate pH, similar to that maintained under standard incubator conditions.

[0175] These results demonstrate that the incorporation of the CO2 inlet 170 enables the plate lid 120 to maintain a controlled microenvironment that preserves physiological pH during extended stimulation and imaging. Consequently, this configuration mitigates media alkalinization and supports cell viability and function during prolonged out-of-incubator experiments.

[0176] In alternative embodiments, the gaseous port may be configured to deliver other gases such as oxygen (O2), nitrogen (N2), or additional gases depending on the experimental objective.

[0177] In some embodiments, the gaseous port may include both a gaseous inlet (e.g., a CO2 inlet) and a gaseous outlet to help maintain stable internal pressure within the culture plate 200.

[0178] In some further embodiments, the gaseous port may include a plurality of inlets configured to deliver multiple types of gases into the culture plate 200.Application / Usage

[0179] The system and method described above may be used for automated electrical stimulation of a variety of types of microscopy samples, e.g., electrically active cells including but not limited to muscle cells (skeletal muscle, smooth muscle, and / or cardiac muscle), neurons, and some endocrine cells (e.g. insulin-releasing pancreatic P cells); engineered / cultured tissues and organs.

[0180] By providing an automated and efficient solution to electrically stimulate and observe microscopy samples, the described system and method enhance the speed, consistency, reproductively and scalability of high-throughput experiments and screening, also leading to more accurate and reliable experiment / screening results.

[0181] This technique can be used for various purposes across different fields of research, and provides a powerful tool for studying cellular and tissue physiology, investigating cell-material interactions, and advancing various fields of biomedical research.Experimental Results

[0182] A series of experiments has been conducted to demonstrate the technical effects of the system and method of the present invention. Some of these experiments refer to the use of plates with wells comprising ‘poles’ (see FIG. 16 for an illustrated example). Wells comprising poles are one example for use of the invention(s) described herein, however these may be used in combination with any suitable well or culture plate, dependent upon the requirements of the microscopy sample. For example, cardiac or skeletal muscle tissues would typically require poled wells for analysis, however, this would not be required for a 2D microscopy sample.

[0183] In some of the experiments described hereinafter, the microscopy samples are bioengineered skeletal muscles, generated using existing methods, e.g., techniques described in Mills et al, Development of a human skeletal micro muscle platform with pacing capabilities. Biomaterials, 198, pp.217-227 wa olendijk, J. et al. ‘Proteome-Wide Systems Genetics Identifies UFMylation as a Regulator of Skeletal Muscle Function ’. ELIFE, vol. 11, 2022.

[0184] A digital microscope is used to capture images and videos of the muscle tissue in response to electrical stimulation applied, using either a manual method or the system 100. The videos are then analysed using existing imaging processing techniques to derive one or more measurable functional properties of the microscopy samples, e.g., the force of contraction or tissue viability. The videos may also be analysed to measure or detect how certain tissue, cell or protein responds to electric stimulation, e.g., in marker expression analysis.

[0185] Movement of the plate in each experiment is achieved by the motorized stage and is controlled by for example the software of the imaging device.

[0186] Experiments 1 and 2 as described hereinafter compare the performance of the electrical stimulation of muscle tissue samples at different currents in the following three alignment modes:1) Manual stimulation mode (also referred to below as the "Manual mode")

[0187] This technique requires manually placing two wires connected to a digital stimulator on the microscopy sample in each well of the culture plate.

[0188] FIG. 16 shows an illustrated example of the microscopy sample 204 within the well 202, being positioned surrounding two poles 164a (i.e. the left pole) and 164b. The tissue appearing on the screen of the microscope will be centred based on the location of the left pole of the well, marked physically with masking tape on the monitor. This alignment may be achieved through manual / visual means (i.e. marked physically with masking tape on the monitor), or alternatively through the use of software to highlight a region of interest.

[0189] After each stimulation, the wire is physically removed from the sample, and placed into the next well, with the view of the microscope adjusted.

[0190] For a full plate with 96 wells, the whole process takes approximately 2 hours for both 1Hz and 20Hz stimulations.2) Visual centring stimulation mode (also referred to below as the "Tissue Centring mode")

[0191] This is an adaptation of the manual stimulation mode, wherein the lid 120 and contact head 140 are utilized to stimulate the microscopy sample. As above, this technique aims to visually position the tissue sample within each well of interest in the same place in the field of view every time, to ensure consistency of the automated analysis. For each well, the microscope records a 5s video. For a full plate with 96 wells, the whole process takes approximately 8 minutes of videoing, 16 minutes total for two different stimulation rates of 1Hz and 20Hz. Plus, time to move between wells and adjust focus (additional ~15 minutes).3) Microscope navigation mode (also referred to below as the "Navigator mode")

[0192] In this mode, the "LAS X Navigator" software provided by Leica Microsystems is utilised to control the relative position of the lid 120 and contact head 140, which allows locating the edges of the wells of a 96-well plate to determine the centre of each well. This automatically aligns the electrical connectors 142a and 142b of the connector head 140 with the metal pin electrodes on the plate lid 120 to enable the delivery of electrical stimulation.

[0013] The "LAS X Navigator" software is able to generate coordinates for any number of locations throughout a well for unbiased imaging. In the present experiment, one view in the centre of the predetermined well size is selected, this ensures the precise alignment between the electrical connectors of the connector head 140 and the corresponding electrodes on the plate lid 120. The process moves to each well and records a 5s video.

[0194] For a full plate with 96 wells, the whole process is estimated to take 8 minutes, 16 minutes in total for 1Hz and 20Hz, plus approximately 15 minutes additional time to set up the LAS X Navigator and for the microscope to focus (approximately 30 minutes in total).[Experiment 1] Comparing Tissue Centring mode with Manual mode

[0195] A series of studies was conducted to compare the Tissue Centring mode to the Manual mode at different currents, the results of which are shown in FIG. 17(a), FIG. 17(b), FIG. 18, and Table 1.

[0196] In these studies, the centring stimulation was set up in the same way as the manual stimulation, centring the tissue on the monitor screen when applying the stimulation.

[0197] FIG. 17(a) shows the force of contraction measured from Well No. 1 - 15 in Tissue Centring mode when the stimulation current is 20mA, 50mA and 100mA compared to Manual mode at 20mA, with a frequency of 20Hz.

[0198] FIG. 17(b) shows the force of contraction measured from Well No. 16 - 33 in Tissue Centring mode at 20mA, 50mA and 100mA compared to Manual mode at 20mA, with a frequency of 20Hz.

[0199] FIG. 18 shows the variability in the force measured in Tissue Centring mode at 20mA, 50mA and 100mA compared to Manual mode at 20mA.

[0200] Table 1 shows descriptive statistics comparing Tissue Centring mode at 20mA, 50mA and 100mA and Manual mode at 20mA.Table 1: Descriptive statistics comparing Tissue Centring mode and Manual mode

[0201] In the Manual mode, manual stimulation of the 33 wells was completed in approximately 40 minutes (plus the time to move between wells and adjust the focus of the microscope, which was approximately 15 minutes).

[0202] In the Tissue Centring mode, stimulation of 33 wells was completed in approximately 10 minutes in total plus the time to move between wells and adjust the focus of the microscope (~15 minutes).

[0203] These studies show that the Tissue Centring mode can be completed much faster than the Manual mode.[Experiment 2] Comparing Manual mode, Centring mode and Navigator mode

[0204] A series of studies were further conducted to compare the Manual mode, Tissue Centering mode (centering the tissue on the screen utilizing the system of the present invention for stimulation of the microscopy sample) and Navigator mode (utilizing the centre coordinates set by the "LAS X Navigator" software, and utilizing the system of the present invention for stimulation of the microscopy sample), respectively. The results are shown in FIG. 19(a), FIG. 19(b), FIG. 20, and Table. 2.

[0205] FIG. 19(a) shows the force of contraction measured from Well No. 1 - 18 in Tissue Centring mode ("LID") at 20mA, 100mA, Navigator mode ("LAS") at 20mA, 100mA, and Manual mode ("MANUAL") at 20mA.

[0206] FIG. 19(b) shows the force of contraction measured from Well No. 19 - 34 in Tissue Centring mode ("LID") at 20mA, 100mA, Navigator mode ("LAS") at 20mA, 100mA, and Manual mode ("MANUAL") at 20mA.

[0207] FIG. 20 shows the variability in the force measured in Tissue Centring mode ("LID") at 20mA, 100mA, Navigator mode ("LAS") at 20mA, 100mA, and Manual mode ("MANUAL") at 20mA.

[0208] Table 2 shows descriptive statistics of the results in Tissue Centring mode ("LID") at 20mA, 100mA, Navigator mode ("LAS") at 20mA, 100mA, and Manual mode ("MANUAL") at 20mA.Table 2: Descriptive statistics of contractile force (pN) comparing Manual mode, Tissue Centring mode and Navigator mode

[0209] The results of further experiments comparing the Tissue Centring mode at 100mA, the Navigator mode at 100mA, and the Manual mode at 20mA are shown in FIG. 21(a), FIG. 21(b), FIG. 22, and Table 3.

[0210] FIG. 21(a) shows the force of contraction measured from Well No. 1 - 18 in Tissue Centring mode ("LID") at 100mA, Navigator mode ("LAS") at 100mA, and Manual mode ("Manual") at 20mA.

[0211] FIG. 21(b) shows the force of contraction measured from Well No. 19 - 34 in Tissue Centring mode ("LID") at 100mA, Navigator mode ("LAS") at 100mA, and Manual mode ("Manual") at 20mA.

[0212] FIG. 22 shows the variability in the force measured in Tissue Centring mode ("LID") at 100mA, Navigator mode ("LAS") at 100mA, and Manual mode ("Manual") at 20mA.

[0213] FIG. 23 shows the data correlation of the force measured in Navigator mode ("LAS") at 100mA and Manual mode ("MANUAL") at 20mA.

[0214] Table 3 shows descriptive statistics of the results in Tissue Centring mode ("LID") at 100mA, Navigator mode ("LAS") at 100mA, and Manual mode ("Manual") at 20mA.Table 3: Descriptive statistics of contractile force (pN) comparing Manual mode, Tissue Centring mode and Navigator mode

[0215] These studies across Experiments 1 and 2 show that while the Tissue Centring mode and Navigator mode, both utilising the lid 120 and contact head 140 of the present invention, can complete the stimulation significantly faster than the Manual mode, whilst the Navigator mode results in much fewer non-responders relative to the Tissue Centring mode. This demonstrates that the Navigator mode allows more precise control of the position of theconnector head 140 relative to the plate lid 120, which secures the electrical connection between the electrical connectors on the connector head 140 and the corresponding electrodes on the plate lid 120. This improves the quality of the electrical signal applied to the sample, and reduces "non-responders" due to poor electrical connection, thereby enhancing the accuracy of the experiment / screening results.[Experiment 3]

[0216] This experiment compares the performance of the system 100 when the metal pin electrodes of the plate lid 120 are configured with and without the PTFE insulating coat.

[0217] In this experiment, electrical stimulations are applied to the tissue twice: once at a start point ("Pre Stimulations") of a study, and once at an end point ("Post Stimulations ") of the study. The tissue force of contraction in response to the electrical stimulation is measured at the start point ("Pre Force") and the end point ("Post Force"), respectively.

[0218] The study timeline is shown in Table 4 below.Table 4: Study Timeline for Experiment 3

[0219] FIG. 24(a) shows the Pre Force measured in response to the Pre Stimulation at different currents of stimulation, with and without PTFE coating.

[0220] FIG. 24(b) shows the Post Force measured in response to the Post Stimulation at different currents of stimulation, with and without PTFE coating.

[0221] FIG. 24(c) shows the relative force (Post Force relative to Pre Force) at different currents of stimulation, with and without PTFE coating.

[0222] FIG. 24(d) shows the tissue viability readouts at Post Stimulation relative to Pre stimulation, at different currents of stimulation, with and without PTFE coating.

[0223] FIG. 25(a) shows the overall tissue viability and FIG. 25(b) shows the force of contraction, with and without PTFE coating (combined data from current 20mA and above). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, **p < 0.0001.

[0224] In addition, FIG. 26 shows the force of contraction measured from tissue only stimulated at the end point (i.e., without Pre Stimulation) at different currents of stimulation, with and without PTFE coating.

[0225] The descriptive statistics for the relative force shown in FIG.24c are summarised in Table 5 below.Table 5: Descriptive statistics for relative force

[0226] The descriptive statistics for tissue viability normalised to Pre Stimulation shown in FIG. 24(d) is summarised in Table 6 below.Table 6: Descriptive statistics for tissue viability normalised to Pre Stimulation

[0227] As shown in FIG. 24(c), irrespective of the current that was applied during these studies, the normalised force was around 1.0.

[0228] While some variability was noted, the current of 20mA with PTFE insulation produces the most consistent results (mean = 1.069 and SD = 0.5856), as shown in Table. 5 and FIG. 24(c).

[0229] While raw force production was reduced at 5mA stimulation, currents greater than 20mA produced a similar amount of force. As shown in FIG. 25(a) and FIG. 25(b), the PTFE insulation improved the force production.

[0230] Tissue viability decreased as the current that was applied increased (FIG. 24(d)).20mA with insulation had reasonably consistent viability (Mean = 1.759 and SD = 0.6946; Table 6) and viability readout on par with controls. It also appears that using the 100mA stimulation was damaging the tissues beyond repair at the Pre Stimulation, which caused poor responses at the end point.

[0231] During this experiment, for the metal pin electrodes without PTFE coating, some capillary action was observed when inserting the electrodes of the plate lid 120 into the tissue culture media, resulting in media ‘flowing’ out of the wells. On the other hand, the pins insulated with PTFE coating appear to effectively prevent the capillary action, with no media flowing out of its cell. These results show that insulating the metal pin electrodes can significantly reduce the surface energy of the metal pin electrodes, which prevents or reduces the media fluid in the well from flowing out due to capillary action, thereby preventing the cross-contaminate of samples in nearby wells.Summary of Experiments 1 to 3

[0232] The above experimental results show that the system 100 described herein (in both the Tissue Centring mode and the Navigator mode) significantly reduces the time and labour required for applying electrical stimulation to microscopy samples in a culture plate with multiple walls, and improves the efficiency and scalability of experiment / screening.

[0233] In addition, by coordinating the relative movement of the plate lid 120 and the connector head 140 with the scanning ability of the imaging device (Navigator mode), the electrical connectors on the connector head 140 and be aligned more precisely with the corresponding electrodes on the plate lid 120, which secures the electrical connection between them and improves the quality of the electrical signal applied to the microscopy sample 204. This effectively reduces the number of "non-responders" due to poor electrical connection, thereby enhancing the accuracy of the experiment / screening results.

[0234] Furthermore, the experimental results also show that having an insulating coat (e.g., PTFE coating), on the metal pin electrodes can effectively reduce the surface energy of the metal pin electrodes, which prevents or reduces the media fluid in the well from flowing out due to capillary action, thereby preventing cross-contaminate of samples in nearby wells.[Experiment 4] Stimulation of Bioengineered Human Skeletal Muscle Tissues

[0235] This experiment assesses the system 100’s ability to perform controlled electrical pacing and quantitative measurement of contractile function in engineered muscle tissues and organoids. More specifically, it establishes that the system 100 can be used for assessing pharmacological modulators and characterising their effects on tissue force generation and contraction kinetics.

[0236] In this experiment, the system 100 is applied to bioengineered skeletal muscle tissues (PMID: 30527761) to assess functional responses to pharmacological modulators. Fast skeletal muscle troponin activators Tirasemtiv (Tir) and Reldesemtiv (Rel) increase calcium binding affinity to troponin-C, enhancing the force-calcium relationship and increasing contraction force and length (PMID: 24634285). The skeletal muscle myosin-2 inhibitor MPH-220 selectively binds ATP sites on myosin heads, preventing power stroke initiation and reducing contraction force (PMID: 33035452). By using the system 100, the effects of these compounds on skeletal muscle contractile function and kinetics can be assessed.

[0237] In particular, baseline effects of these modulators on twitch (1 Hz) and tetanic (20 Hz) force responses were evaluated over a dose range of 0.1 pM, 1 pM, and 10 pM during a 60-minute treatment. Tir and Rel increased both twitch and tetanic force at 10 pM, whereas MPH-220 reduced twitch and tetanic force at the same concentration. Contraction kinetics, including 50% activation time (Ta_50) and 50% relaxation time (Tr_50), were quantified. No significant shifts in activation time were observed; however, Tir at 10 pM significantly prolonged relaxation time, indicating a slower return to baseline force. MPH-220 causedapproximately a 20% decrease in relaxation time across all doses, indicating faster relaxation, even when maximal contraction force remained unchanged at lower concentrations.

[0238] Force-frequency (FF) relationships were assessed following 60 minutes of treatment at 10 pM. Tir and Rel increased the FF relationship, with Rel producing a stronger effect, approximately doubling force relative to control. MPH-220 reduced the FF relationship, with maximal force decreased by approximately 66% relative to vehicle-treated tissues. Fatigability was evaluated under submaximal (10 Hz) stimulation for 10 seconds, with no significant differences observed across treatments.

[0239] FIGs. 32(a) - 32(d) and 33(a) - 33(d) show the results of functional assessment of bioengineered skeletal muscle tissue using the system 100. FIG. 32(a) shows the measured twitch force at 1 Hz stimulation; FIG. 32(b) shows the measured tetanic force at 20 Hz stimulation; FIGS. 32(c) and 32(d) show representative twitch and tetanus force curves, respectively; FIG. 33(a) shows the 50% activation time (Ta_50) during 1 Hz twitch stimulation; FIG. 33(b) shows the 50% relaxation time (Tr_50) during 1 Hz tetanic stimulation; FIG. 33(c) shows the force-frequency curve measured at 1, 2, 5, 10, 20, 40, and100 Hz; and FIG. 33(d) illustrates the change in force during submaximal 10 Hz stimulation for 10 seconds.

[0240] These results demonstrate that the system 100 enables quantitative measurement of compound-induced alterations in skeletal muscle contractile function and kinetics.[Experiment 5] Stimulation of Human Cardiac Organoids

[0241] The following experiment further evaluates the capability of the system 100 to electrically pace and measure contractile function in human engineered tissues, specifically human cardiac organoids (PMID: 28916735). Human cardiac organoids exhibit spontaneous contractions in culture, and controlled electrical pacing allows normalisation of intrinsic beat rate variability and enables standardised measurement of function.

[0242] In this experiment, the effects of cardiac myosin activators Omecamtiv Mecarbil (OM) and Danicamtiv (Dan) on contractile performance were assessed. The system 100 successfully paced the cardiac organoids at 1 Hz and 2 Hz, corresponding to physiological heart rates of approximately 60-120 beats per minute. Under 1 Hz pacing conditions, OM and Dan increased contraction force by approximately 45% and 36%, respectively, when compared with vehicle controls.

[0243] Force-frequency responses were evaluated by pacing the tissues at 2 Hz following baseline assessment at 1 Hz. Vehicle-treated organoids demonstrated an approximate 14% increase in force at 2 Hz relative to 1 Hz. In contrast, OM- and Dan-treated tissues did not exhibit an additional increase in force at 2 Hz, and OM-treated tissues showed a reduction in contraction force relative to the 1 Hz condition.

[0244] Contraction kinetics were assessed by measuring the 50% contraction duration (CD 50) under both pacing conditions. OM prolonged contraction duration, increasing CD 50 by approximately 30% at 1 Hz and 18% at 2 Hz, whereas Dan did not produce significant changes in CD 50. Under all treatment conditions, an overall reduction in contraction duration was observed during 2 Hz pacing, indicative of lusitropic behaviour.

[0245] FIGs. 34 (a) - 34(d) show the results of functional assessment of human cardiac organoids using the system 100. FIG. 34(a) shows the force of contraction during pacing at 1 Hz and 2 Hz following myosin activator treatment; FIG. 34(b) shows the contraction force at 2 Hz relative to the contraction force at 1 Hz; FIG. 34(c) shows the 50% contraction duration (CD 50) during pacing at 1 Hz and 2 Hz; FIG. 34(d) shows the contraction duration at 2 Hz relative to that at 1 Hz.

[0246] These results demonstrate that the system 100 can precisely control electrical pacing of human cardiac organoids and quantitatively measure functional and kinetic effects of pharmacological agents on cardiac contractility.Interpretation

[0247] The FIGs. included herewith show aspects of non-limiting representative embodiments in accordance with the present disclosure, and particular structural elements shown in the FIGs. may not be shown to scale or precisely to scale relative to each other. The depiction of a given element or consideration or use of a particular element number in a particular FIG. or a reference thereto in corresponding descriptive material can encompass the same, an equivalent, an analogous, categorically analogous, or similar element or element number identified in another FIG. or descriptive material associated therewith. The presence in a FIG. or text herein is understood to mean “and / or”, i.e., “X / Y” is to mean “X” or “Y” or “both X and Y”, unless otherwise indicated. The recitation of a particular numerical value or value range herein is understood to include or be a recitation of an approximate numerical value or value range, for instance, within + / - 20%, + / - 15%, + / - 10%, + / - 5%, + / - 2.5%, + / - 2%, + / - 1%, + / - 0.5%, or + / - 0%. The term “essentially all” or “substantially” canindicate a percentage greater than or equal to 50%, 60%, 70%, 80%, or 90%, for instance, 92.5%, 95%, 97.5%, 99%, or 100%.

[0248] Many modifications will be apparent to those skilled in the art without departing from the scope of the present invention.

[0249] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0250] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that the prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A system for electrical stimulation of samples, the system including: a plate lid having a plurality of pairs of electrodes, the plate lid being configured to removably couple with a culture plate having a plurality of wells, such that each well of the culture plate receives a pair of the electrodes, the pair of electrodes being at least partially in contact with a sample in the corresponding well; and a connector head having one or more pairs of electrical connectors configured to electrically connect to an electrical stimulator; wherein the connector head is configured to be moved automatically relative to the plate lid by a moving mechanism, such that: at a first time, each pair of the one or more pairs of electrical connectors of the connector head are electrically connected to a corresponding pair of a first one or more pairs of the electrodes of the plate lid, to provide electrical stimulation to the sample(s) in a first group of one or more wells of the culture plate; and at a second time, each pair of the one or more pairs of electrical connectors of the connector head are electrically connected to a corresponding pair of a second one or more pairs of the electrodes of the plate lid, to provide electrical stimulation to the sample(s) in a second group of one or more wells of the culture plate.

2. The system of claim 1, wherein the one or more pairs of electrical connectors of the connector head include a single pair of electrical connectors.

3. The system of claim 1, wherein the one or more pairs of electrical connectors of the connector head include multiple pairs of electrical connectors.

4. The system of any one of claims 1 - 3, wherein the moving mechanism is configured to move the culture plate when the plate lid is removably coupled to the culture plate.

5. The system of any one of claims 1 - 4, wherein the connector head is configured to couple to an imaging device.

6. The system of claim 5, wherein the imaging device includes a microscope, and the connector head is configured to couple to an objective of the microscope.

7. The system of claim 6, wherein the movement of the connector head relative to the plate lid is caused at least partially by the movement of the objective of the microscope.

8. The system of any one of claims 1 - 7, wherein the connector head further includes a printed circuit board (PCB) for electrically connecting the electrical connectors to the electrical stimulator, wherein the PCB has one or more windows to allow transmission of light.

9. The system of claim 8, wherein the PCB has a gap between each pair of the electrical connectors to allow transmission of light.

10. The system of any one of claims 1 - 9, wherein the electrodes of the plate lid are arranged in an array of rows and columns.

11. The system of any one of claims 1 - 10, wherein the plate lid has: a lid body with a first side and a second side opposite to each other; wherein each of the electrodes of the plate lid has an elongated shape, one end of which protruding from the first side of the lid body to contact the sample in a corresponding well of the culture plate, the other end of which being exposed on the second side of the lid body to be contactable by a corresponding electrical connector of the connector head to enable electrical connection between the corresponding electrical connector and the electrode.

12. The system of claim 11, wherein each of the electrodes is a rigid pin coated with a conductive metallic layer.

13. The system of claim 12, wherein each of the pins is partially coated with an electrically insulating material, exclusive of an end portion of the pin.

14. The system of any one of claims 1 - 13, wherein the plate lid and the culture plate form an enclosed containing space when the plate lid is removably coupled to the culture plate.

15. The system of any one of claims 1 - 14, wherein each of the electrical connectors of the connector head includes a rollerball mechanism to secure the contact with the corresponding electrodes of the plate lid.

16. The system of any one of claims 1 - 15, wherein the sample is a microscopy sample.

17. The system of any one of claims 1 - 16, wherein the plate lid has at least one gaseous port for maintaining a controlled gaseous environment in the culture plate during stimulation.

18. A connector head for a system for electrical stimulation of samples, the connector head includes: one or more pairs of electrical connectors configured to electrically connect to an electrical stimulator; a coupling mechanism for coupling the connector head to a moving mechanism to automatically move the connector head relative to a plate lid having a plurality of pairs of electrodes, the plate lid being configured to removably couple with a culture plate having a plurality of wells, such that each well of the culture plate receives a pair of the electrodes, the pair of electrodes being at least partially in contact with a sample in the corresponding well; wherein when the connector head is moved relative to the plate lid: at a first time, each pair of the one or more pairs of electrical connectors of the connector head are electrically connected to a corresponding pair of a first one or more pairs of the electrodes of the plate lid received by a first group of one or more wells of the culture plate to provide electrical stimulation to the sample(s) in the first group of one or more wells; and at a second time, each pair of the one or more pairs of electrical connectors of the connector head are electrically connected to a corresponding pair of a second one or more pairs of the electrodes of the plate lid received by a second group of one or more wells of the culture plate to provide electrical stimulation to the sample(s) in the second group of one or more wells.

19. A plate lid for electrical stimulation of samples, including: a lid body; anda plurality of pairs of metallic electrodes protruding from the lid body; wherein the lid body is configured to removably couple with a culture plate having a plurality of wells, such that each well of the culture plate receives a pair of the electrodes, and wherein the pair of electrodes is at least partially in contact with a sample in the corresponding well.

20. A method for electrical stimulation of samples, the method including: coupling a plate lid having a plurality of pairs of electrodes to a culture plate having a plurality of wells, such that each well of the culture plate receives a pair of the electrodes, the pair of electrodes being at least partially in contact with a sample in the corresponding well; and automatically moving a connector head relative to the plate lid using a moving mechanism, wherein the connector head has one or more pairs of electrical connectors configured to electrically connect to an electrical stimulator; wherein at a first time, each pair of the one or more pairs of electrical connectors of the connector head are electrically connected to a corresponding pair of a first one or more pairs of the electrodes of the plate lid, to provide electrical stimulation to the sample(s) in a first group of one or more wells of the culture plate; and at a second time, each pair of the one or more pairs of electrical connectors of the connector head are electrically connected to a corresponding pair of a second one or more pairs of the electrodes of the plate lid, to provide electrical stimulation to the sample(s) in a second group of one or more wells of the culture plate.

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