Method and device for synchronously measuring multiple parameters of contractile biological tissue

Abstract

In order to measure multiple parameters of an object to be measured (2) made of a contractile biological tissue (3), said parameters describing a current dynamic state of the object to be measured (2), the object to be measured (2) is suspended on at least two holding elements (5) which are spaced apart from one another and are movable relative to one another in the direction of their spacing. A first development over time (32) is detected in that a first distribution of a chemical potential over the first region (16) is detected in a time-resolved manner by means of fluorescence microscopic imaging of a first region (16), located between the holding elements, of the object to be measured (2), which is suspended on the holding elements. A second development over time (33) is detected in that a second distribution of an electric field potential over the second region (11) is detected in a time-resolved manner by means of a microelectrode array (12) applied to the object to be measured (2) in a second region (11), located between the holding elements, of the object to be measured (2), which is suspended on the holding elements. A third development over time (34) is detected in that the distance between the holding elements (5) is detected and / or changed in a time-resolved manner and / or a force acting in the direction of the spacing between the holding elements (5) is detected in a time-resolved manner. The first development over time (32), the second development over time (33) and the third development over time (34) are temporally correlated with one another, wherein time marks (29 to 31) are set and registered together with the respective development over time (32, 33 or 34) during the detection of the different developments over time.

Description

[0001] REHBERG HÜPPE + PARTNER - 1 - Originally submitted version 22030PCT 22.09.2025

[0002] RHP Ref.: 22030PCT ZLP6

[0003] Patent application: File number not yet assigned

[0004] Priority: DE 10 2024 127433.6 (23.09.2024)

[0005] Title: Method and device for the synchronous measurement of several

[0006] Parameters of contractile biological tissues

[0007] Applicant: Georg-August-University Göttingen Foundation under Public Law,

[0008] University Medicine

[0009] METHOD AND DEVICE FOR SYNCHRONOUS MEASUREMENT OF MULTIPLE PARAMETERS OF CONTRACTIVE BIOLOGICAL TISSUES

[0010] TECHNICAL AREA OF INVENTION

[0011] The invention relates to a method and a device for measuring parameters of a test object made of contractile biological tissue, which describe a current dynamic state of the test object. The contractile biological tissue can, in particular, be a biological tissue containing cardiac muscle cells in the form of an artificial heart muscle.

[0012] Contractile biological tissues, especially artificial heart muscle, are not only of interest as such, for example for the development of implants, but they can also serve as probes for the effect of pharmaceuticals on a beating heart in the context of so-called drug screening.

[0013] STATE OF THE ART

[0014] From WO 2017 / 207431 A1, a perforated plate with a depression in the upper surface of a plate body is known. An open-topped annular channel is formed in the depression, its inner circumference bounded by a closed, circumferential wall. A horizontal outer circumference of the circumferential wall decreases from bottom to top up to an upper edge of the circumferential wall. Within the horizontal outer circumference, retaining elements connect to the upper edge of the circumferential wall, maintaining a free horizontal distance from one another. The retaining elements are portions of vertically extending retaining fingers that are elastically supported on the plate body. When the perforated plate is used, a suspension of connective tissue cells and other cells is introduced into the annular channel. REHBERG HÜPPE + PARTNER - 2 - Originally submitted version 22030PCT 22.09.2025.The cavity is filled with a nutrient solution up to and above the holding elements, and movements of the free upper ends of the holding elements or of an adjoining pointer section are recorded with a camera. An electrical voltage of variable amplitude can be applied between electrodes on the holding elements and / or electrodes in the nutrient solution. The nutrient solution is color-matched to the holding fingers so that the holding fingers contrast visually with the nutrient solution.

[0015] From EP 3831 925 A1, a bioreactor system for the production of artificial three-dimensional biological tissue constructs is known. The bioreactor system has a cultivation chamber designed for the formation, cultivation, and subsequent testing and / or stimulation of tissue constructs on two support elements. The cultivation chamber has a top plate, a bottom plate, and a side wall extending between the top plate and the bottom plate. The support elements are attached to the side wall and extend from there into a cavity bounded by the top plate, the bottom plate, and the side wall. The vertical distance between the support elements and the bottom plate is between 1 mm and 25 mm. The support elements are designed to allow the formation of an artificial tissue construct in contact with them.The support elements are connected to associated coupling elements, which allow the coupling of a sensor device, a measuring device, and / or a force-generating device to the respective support element. The support elements can perform horizontal movement. The known bioreactor system further includes an electrical pacemaker unit, a source of high-energy blue light for optogenetic applications or optical pacing, and / or a microscope for imaging purposes. The transparent design of the top plate and / or the bottom plate allows the observation of the development and / or stimulation responses of the tissue constructs by wide-field and fluorescence microscopy.

[0016] A method and a bioreactor for in vitro cardiac simulation are known from WO 2021 / 094749 A1. The bioreactor has an actuator for moving an actuator rod within a bioreactor vessel to exert a mechanical force on a tissue sample. The bioreactor also has a fixed seat for the actuator. A force sensor detects the mechanical force exerted by the tissue sample, and the actuator is controlled by a controller based on this detected force. To stimulate the tissue cyclically, the actuator is moved during a REHBERG HÜPPE + PARTNER - 3 - Originally submitted version 22030PCT 22.09.2025

[0017] The stimulation cycle is controlled based on the force detected during a previous cycle. A sensor and electrical stimulator are also provided for the electrical stimulation of the tissue sample. A mechanical waveform and an electrical waveform of the stimulation are synchronized. With each application of a pulsed electrical stimulus, a trigger signal is generated, and the mechanical force is applied in response to this signal.

[0018] Calcium imaging is a microscopy technique used to optically measure calcium levels. 2+ The concentration of calcium in a tissue, also known as calcium potential, is measured using calcium indicators, which are fluorescent molecules whose fluorescence properties indicate the binding of calcium. 2+-ions react. Calcium imaging is typically performed in wide-field reflected-light fluorescence microscopy, also known as epifluorescence microscopy.

[0019] To detect electrical field potentials from artificial heart tissue, the use of microelectrode arrays is known; see, for example, the overview of current techniques in electrophysiology and contraction force measurements in J. Park et al.: Heart-on-Chip for Combined Cellular Dynamics Measurements and Computational Modeling Towards Clinical Applications, Annals of Biomedical Engineering, Vol. 50, No. 2, February 2022.

[0020] TASK OF INVENTION

[0021] The invention is based on the objective of demonstrating a method and a device for measuring parameters of a measurement object made of a contractile biological tissue, which capture a current dynamic state of the measurement object more accurately than previously known methods and devices and thereby provide better foundations for the development of such biological tissues and their use as probes in drug screening.

[0022] SOLUTION

[0023] The object of the invention is achieved by a method with the feature of independent claim 1 and a device with the features of independent claim 11. Preferred embodiments of the method and the device are defined in the dependent claims.

[0024] DESCRIPTION OF THE INVENTION

[0025] In the inventive method for measuring several parameters of a test object made of contractile biological tissue, wherein the several parameters together describe a current dynamic state of the test object, the test object is suspended from at least two mutually movable holding elements in the direction of their separation relative to each other. The mobility of the holding elements is active and / or passive. For example, one of the holding elements can be movable against a restoring force. The restoring force can be an elastic restoring force applied by a spring. Alternatively or additionally, the same or another of the holding elements is movable by means of an actuator in order to specify a defined distance or a defined temporal development of the distance between the holding elements.

[0026] In the method according to the invention, a first temporal development is recorded by using fluorescence microscopy to capture a first distribution of a chemical potential over a first region of the measuring object suspended between the holding elements. Furthermore, in the method according to the invention, a second temporal development is recorded by using a microelectrode array in contact with the measuring object to capture a second distribution of an electric field potential over a second region of the measuring object suspended between the holding elements.Furthermore, in the method according to the invention, a third temporal development is recorded by capturing and / or changing the distance between the elements with time resolution and / or capturing a force acting in the direction of the distance between the holding elements with time resolution. The first, second, and third temporal developments are correlated with each other. For this purpose, time markers are set during the acquisition of the various temporal developments and recorded together with the respective temporal development.

[0027] The time markers set in the method according to the invention can be set without reference to the contractions of the contractile biological tissue, for example, at fixed time intervals. REHBERG HÜPPE + PARTNER - 5 - Originally filed version 22030PCT 22.09.2025. However, the time markers can also be triggered by reaching a specific criterion during one of the first, second, or third temporal developments. This makes it possible to easily select the relevant parts of the first, second, and third temporal developments by selecting time periods that follow or are arranged around the time markers and discarding the remaining temporal developments as irrelevant. This significantly reduces the volume of data to be analyzed and stored.Specifically, the partial periods for which the temporal developments are correlated and then stored can represent less than 50% of the total time of their recording, or even less than 25%.

[0028] Specifically, the same or at least time-synchronized time markers can be set, and these can be recorded as parts of the three temporal developments. While it is also possible to set time markers that are not time-synchronized but only have a defined temporal relationship to each other, this is generally less preferred unless the recording of one temporal development has an inherent delay compared to the recording of another, which could be compensated for by a temporal offset of the time markers. Such compensation can also be performed later during temporal correlation. The time markers can be recorded directly as parts of the three temporal developments or together with them but separately, for example, on a different channel of a multiple signal.

[0029] Insofar as the recorded temporal developments are numbered here as first temporal development, second temporal development, third temporal development, this numbering serves solely to distinguish the temporal developments. It does not document any chronological sequence or ranking.

[0030] In the method according to the invention, the first temporal development, the second temporal development and the third temporal development can be analyzed with respect to the time markers in order to capture the current dynamic state of the object being measured in a multidimensional time-resolved manner.

[0031] In the method according to the invention, not only are all parameters of the respective contractile biological tissue currently considered relevant recorded, i.e., a chemical potential, such as in particular a Ca 2+The measurement not only captures the potential, the electric field potential, and at least one current length or force acting in the length direction of the contractile biological tissue, but also time-synchronously records temporal developments. This allows for both a multiparametric description of the current dynamic state of the object being measured and a significant reduction in errors in describing the dynamic state. This reduction is beneficial both from a statistical perspective, because more measurements are available for each point in time, and from the perspective of error compensation, because in partial periods where one of the temporal developments is not recorded—for whatever reason—this omission does not result in a complete loss of data in the overall result, i.e., the description of the current dynamic state of the object being measured, due to the other time-synchronously recorded temporal sequences.

[0032] The second region, over which the distribution of the electric field potential is recorded with time resolution, is, in the ideal case—though often only theoretical—identical to the first region, over which the first distribution of the chemical potential is recorded with time resolution. In practice, however, simultaneous recording of the electric field potential and the chemical potential in the same spatial region proves difficult, if not impossible, because the corresponding measurement methods mutually interfere with each other. For example, the microelectrode array used to record the electric field potential obstructs fluorescence microscopy imaging, such as epifluorescence light microscopy, at least with its individual microelectrodes.Where possible, at least a partial overlap of the second region, over which the distribution of the electric field potential is recorded with time resolution, with the first region, over which the first distribution of the chemical potential is recorded with time resolution, should be aimed for. If this is also not practical, the regions should at least partially, and preferably completely, overlap in a projection in the main direction of the fluorescence microscopy image.

[0033] In the method according to the invention, the distance between the holding elements and / or the force acting in the direction of the distance between the holding elements and / or an external voltage applied to an electrode or electrically stimulating the object being measured can be repeatedly and actively changed, i.e., by external control, with a fourth temporal development. In one embodiment of the method according to the invention, the fourth temporal development is defined relative to the time markers and / or relative to the first temporal development or trigger signals generated therefrom and / or relative to the second temporal development or trigger signals generated therefrom and / or relative to the third temporal development and / or trigger signals generated therefrom.The force acting in the direction of the distance between the holding elements, which is repeatedly and actively changed externally with the fourth temporal development, is regularly such a force that puts the measured object under tensile stress.

[0034] In particular, the fourth temporal development can be defined in relation to the time markers and / or to at least one of the first, second, and third temporal developments or trigger signals generated therefrom in such a way that an externally imposed or trained dynamic state of interest is applied to the object of measurement. This imposition or training can serve the purpose of repeatedly inducing the same defined dynamic state in the same or different contractile biological tissues, in order to directly compare measurements performed under specific boundary conditions. Furthermore, very specific dynamic states can be imposed or trained, which are of interest, for example, in drug screening. These specific dynamic states could, for instance, be contraction disorders when searching for a substance that reduces such disorders.

[0035] To impose, train, or generally establish a dynamic state of interest, alternative or additional physical and / or chemical or pharmacological stimuli can be applied.

[0036] As already indicated, the dynamic states of interest can be pathogenic compared to the natural states of contractile biological tissue. For example, a sustained contraction disorder can be achieved through irregular electrical stimulation that does not follow the spontaneous frequency of the contracting biological tissue. Reducing tissue preload by decreasing the distance between the holding elements near the contraction minimum can induce fibrosis. In this way, various acquired cardiac muscle weaknesses can be simulated. Conversely, increasing the preload between the holding elements near the contraction maximum can mimic the natural model and be used to increase the maturity of the contractile biological tissue. REHBERG HÜPPE + PARTNER - 8 - Originally submitted version 22030PCT 22.09.2025

[0037] In the imposed or trained dynamic state, the object under test can then be exposed to an active substance to record its effect on the dynamic state of interest. The same dynamic state of interest can be set for a large number of identical objects to statically validate the effect of the active substance on the dynamic state of interest and / or to compare the effects of different active substances on the dynamic state of interest.

[0038] As already mentioned, the contractile biological tissue in the inventive method is often cardiac muscle tissue in order to investigate heart diseases and active substances against them.

[0039] An inventive device for measuring several parameters of a test object made of contractile biological tissue, which together describe a current dynamic state of the test object, is particularly intended for carrying out the inventive method. The inventive device has at least two spaced-apart holding elements that are actively and / or passively movable relative to each other in the direction of their spacing and are designed to suspend the test object. A first measuring device of the inventive device comprises a fluorescence light microscope and is designed to detect a first temporal evolution by capturing a first distribution of a chemical potential over a first region of the test object suspended from the holding elements, located between the holding elements, with time resolution, through fluorescence microscopy.The device according to the invention further comprises a second measuring device, which includes a microelectrode array and is configured to detect a second temporal development by detecting, with time resolution, a second distribution of an electric field potential over the second region of the object suspended from the holding elements, located between the holding elements, using the microelectrode array in contact with the object. Furthermore, the device according to the invention comprises a third measuring device, which is configured to detect a third temporal development by detecting and / or changing the distance between the holding elements with time resolution and / or by detecting, with time resolution, a force acting in the direction of the distance between the holding elements.Finally, the device according to the invention has a correlation device which is designed to determine the first temporal REHBERG HÜPPE + PARTNER - 9 - Originally filed version 22030PCT 22.09.2025.

[0040] The development, the second temporal development and the third temporal development are correlated with each other temporally, whereby it sets time markers during the recording of the different temporal developments, which are registered by the first measuring device, the second measuring device and the third measuring device together with the respective temporal development.

[0041] Specifically, in the device according to the invention, the holding elements can be arranged on opposite side walls of the measuring chamber, while the fluorescence light microscope of the first measuring device is arranged to image the measuring object suspended on the holding arms through a transparent bottom of the measuring chamber, and while the microelectrode array of the second measuring device is connected to attach itself from above to the measuring object suspended on the holding elements.

[0042] Specifically, the device according to the invention can include a positioning device for positioning the microelectrode array of the second measuring device relative to the holding elements and for releasing the positioned microelectrode array in a release position above a target position on the object being measured, which is suspended from the holding elements. From the release position, the microelectrode array can descend to the target position on the object being measured, which is suspended from the holding elements. Contact between the microelectrode array and the object being measured can be facilitated by treating the microelectrode array to achieve a particularly low interfacial energy relative to the object being measured, for example, by hydrophilizing it.

[0043] Furthermore, the device according to the invention can include an actuator assembly designed to actively change the distance between the holding elements and / or the force acting in the direction of the distance between the holding elements. Alternatively or additionally, the device according to the invention can include an electrode assembly comprising one or more electrodes and designed to actively apply an external voltage to the electrode, electrically stimulating the object being measured.

[0044] Advantageous further developments of the invention are described in the patent claims, the description, and the drawings. REHBERG HÜPPE + PARTNER - 10 - Originally filed version 22030PCT 22.09.2025

[0045] The advantages of features and combinations of features mentioned in the description are merely exemplary and can have an effect alternatively or cumulatively, without the advantages necessarily having to be achieved by embodiments according to the invention.

[0046] Regarding the disclosure content—not the scope of protection—of the original application documents and the patent, the following applies: Further features can be seen in the drawings—in particular, the depicted geometries of several components relative to one another, as well as their relative arrangement and functional connection. The combination of features from different embodiments of the invention or features from different claims is also possible, deviating from the chosen cross-references in the claims, and is hereby encouraged. This also applies to features that are illustrated in separate drawings or mentioned in their description. These features can also be combined with features from different claims. Likewise, features listed in the claims can be omitted for further embodiments of the invention, but this does not apply to the independent claims of the granted patent.

[0047] The features mentioned in the claims and the description are to be understood, with regard to their number, as meaning that exactly that number or a greater number than the stated number is present, without the need for the explicit use of the adverb "at least". Thus, for example, if a measurement object is mentioned, this is to be understood as meaning that exactly one measurement object, two measurement objects, or more measurement objects are present. The features listed in the claims may be supplemented by further features or may be the only features that the subject matter of the respective claim possesses.

[0048] The reference numerals contained in the patent claims do not constitute a limitation of the scope of the subject matter protected by the patent claims. They merely serve the purpose of making the patent claims easier to understand.

[0049] BRIEF DESCRIPTION OF THE FIGURES

[0050] The invention is further explained and described below with reference to preferred embodiments illustrated in the figures. REHBERG HÜPPE + PARTNER - 11 - Originally filed version 22030PCT 22.09.2025

[0051] Fig. 1 is a schematic representation of an embodiment of the device according to the invention for carrying out the method according to the invention.

[0052] Fig. 2 illustrates a first measuring device comprising a fluorescence light microscope of the device according to the invention as shown in Fig. 1.

[0053] Fig. 3 illustrates a second measuring device of the device according to the invention, comprising a microelectrode array.

[0054] Fig. 4 illustrates a third measuring device of the device according to the invention as shown in Fig. 1, which includes a force measuring system.

[0055] Fig. 5 illustrates a synchronous measurement of several parameters of a contractile biological tissue using the device according to Fig. 1 and time markers; and

[0056] Fig. 6 shows a synchronous measurement of several parameters of a contractile biological tissue, showing only the partial time periods following a peak of the electric field signal for the second contraction shown in Fig. 5.

[0057] FIGURE DESCRIPTION

[0058] The device 1 shown in Fig. 1 for measuring several parameters of a test object 2 made of contractile biological tissue 3, in particular cardiac muscle tissue, comprises a measuring cell 4 in which the test object 2 is suspended by mutually movable holding elements 5 in the direction of their relative distance. The measuring cell 4 is filled with culture medium 6, which serves to supply the contractile biological tissue 3. The test object 2 can be formed directly in the measuring cell 4, for example from cardiac muscle cells and hydrogel, on the holding elements 5, or it can be formed elsewhere and then transferred into the measuring cell 4. The measuring cell 4 with the holding elements 5 is part of a measuring device 7 for measuring contraction forces exerted by the contractile biological tissue 3 on the holding elements 5. In addition, the measuring device 7 detects the distance between the mutually movable holding elements 5.REHBERG HÜPPE + PARTNER - 12 - Originally submitted version 22030PCT 22.09.2025.

[0059] The measuring device 7 is connected to a correlation device 9 via a signal amplifier 8. A further measuring device 10, for recording the distribution of an electric field potential in an area 11 of the object 2 located between the holding elements 5, is also connected to the correlation device 9 via a signal amplifier 13. This measuring device 10 has a microelectrode array 12 (not shown in detail here). Additionally, a measuring device 14 with a fluorescence light microscope 15, designed as an epifluorescence light microscope, is connected to the correlation device 9. This device uses fluorescence light microscopy to image an area 16 of the object 2 between the holding elements 5 in order to record a chemical potential of the contractile biological tissue with time resolution, specifically an oscillating intracellular calcium ion concentration at the surface of the contractile biological tissue 3.Lines 17 to 19, through which measuring devices 7, 10, and 14 are connected to the correlation device 9, serve not only to transmit the temporal developments of the electric field potential and the chemical potential recorded by the individual measuring devices to the correlation device 9, but also to transmit time markers 29, 30, and 31 to the measuring devices 14, 10, and 7. The time-synchronous time markers 29, 30, and 31 are recorded by the measuring devices 7, 10, and 14 during the acquisition of the temporal developments, thus enabling a precise temporal correlation of the temporal developments of the force between the holding elements 5, the electric field potential in area 11, and the oscillating calcium ion concentration in area 16. This temporal correlation takes place in the correlation device 9.An end device 20 is used to display the correlated temporal developments and to input user instructions to the correlation unit 9.

[0060] Fig. 2 illustrates the measuring device 14, also referred to in this application as the first measuring device, with the fluorescence light microscope 15. The fluorescence light microscope 15, designed as an epifluorescence light microscope, is shown only schematically. However, its structure and function are known to those skilled in the art. It depicts a temporal development of the fluorescence of markers located in the area 16, which is measured by the Ca 2+- ion concentration at the respective location of area 16 is dependent. The fluorescence excitation light from the fluorescence light microscope 15 is directed through a transparent base 25 and the equally transparent culture medium 6 onto area 16 of the object 2 in the measuring cell 4, just as the fluorescence light 4 from area 16 is directed through the transparent medium 6 and the transparent base 25 of the measuring cell 4 to the REHBERG HÜPPE + PARTNER - 13 - Originally submitted version 22030PCT 22.09.2025

[0061] The fluorescence light microscope 15 is used. For selecting the area 16, the measuring device 14 has an xyz positioning unit 26. With the first measuring device 14, an initial temporal development of the oscillating calcium ion concentration at the respective location of the area 16 is recorded.

[0062] Fig. 3 shows the measuring device 10, which is also referred to here as the second measuring device. The measuring device 10 has a base unit 27, which includes the signal amplifier 8 and which can be positioned relative to the measuring cell 4 by means of an xyz positioning unit 28. The microelectrode array 12 is positioned in the area 11 of the object 3 by means of the xyz positioning unit 28. For this purpose, the microelectrode array 12 is released in the xy direction with a distance in the z direction above the area 11, so that it sinks in the culture medium 6 down to the object 2 and adheres to it. The microelectrode array 12 can specifically be a so-called MEA film, commercially available from Multi Channel Systems MCS GmbH, Reutlingen, Germany. The second measuring device 10 records a second temporal development of the electric field potential at the locations of the individual microelectrodes of the microelectrode array 12 in the area 11.

[0063] Fig. 4 separately illustrates the measuring device 7, also referred to here as the third measuring device, for recording the first temporal development of forces exerted by the object 2 on the holding elements 5 in the measuring cell 4. The holding elements 5 are attached to side walls 21 of the measuring cell 4, with at least one of the holding elements 5 being movably supported to enable the measurement of the contraction force exerted by the object 2 on the holding elements 5 from outside the measuring cell 4. This is indicated in Fig. 4 by a feedthrough 22 through the side wall 21 shown here on the left for the holding element 5 shown here on the left. Outside the measuring cell 4, this holding element 5 is connected to a force sensor 23, which is also designed as a pull actuator with an integrated displacement sensor 24. A suitable force sensor is commercially available from FlITEK Advanced Sensor Technology, Inc., California, USA.The third measuring device 14 records a third temporal development of the contraction force exerted by the measuring object 2 on the holding elements 5.

[0064] The measuring device 7 can not only measure the force exerted by the object 2 on the holding elements 5, but this force can also be measured as a function of the current distance between the holding elements 5 or a time-dependent change in this distance between the holding elements. REHBERG HÜPPE + PARTNER - 14 - Originally submitted version 22030PCT 22.09.2025

[0065] 5. Furthermore, the force on the retaining elements 5 can be actively influenced by varying the distance between the retaining elements 5. As an alternative to mounting the retaining elements 5 on or in the side walls 21, the retaining elements 5 can also project into the measuring cell 4 from below or above. In principle, the object being measured 2 can also extend vertically or at an angle within the measuring cell 4. However, as a rule, the object being measured 2 will be aligned horizontally between the retaining elements 5, as shown in Fig. 4.

[0066] Fig. 5 shows, over three contraction events, the temporal evolution 33 of the electric field potential measured with measuring device 10 at the top, the temporal evolution 32 of the oscillating calcium ion concentration measured with measuring device 14 in the middle, and the temporal evolution 34 of the contraction force of the object 2 on the holding elements 5, measured with measuring device 7 at the bottom. These forces were recorded at corresponding points on an exemplary object 2. Using the time markers 29 to 31 provided by the correlation device 9, the temporal evolutions 32 to 34 of the three parameters describing the dynamic state of the object 2—electric field potential, oscillating calcium ion concentration, and contraction force—are synchronized with sub-millisecond accuracy. The same events, i.e., contractions, are thus observed synchronously in all three dimensions.The time markers themselves are not shown in Fig. 5 because they are each registered in a separate channel of the multi-channel signals.

[0067] Fig. 6 shows the superimposed temporal developments 32 to 34 of the three parameters electric field potential, oscillating calcium ion concentration, and contraction force for the middle of the three contraction events according to Fig. 5.

[0068] REHBERG HÜPPE + PARTNER - 15 - Originally submitted version 22030PCT 22.09.2025

[0069] REFERENCE MARK LIST

[0070] 1 Device

[0071] 2. Measuring object

[0072] 3 contractile biological tissue

[0073] 4 measuring cells

[0074] 5 retaining element

[0075] 6 Cultural medium

[0076] 7 (third) measuring device

[0077] 8 signal amplifiers

[0078] 9 Correlation device

[0079] 10 (second) measuring device

[0080] 11 (second) area

[0081] 12 microelectrode array

[0082] 13 signal amplifiers

[0083] 14 (first) measuring device

[0084] 15 Fluorescence light microscope

[0085] 16 (first) area

[0086] 17 Management

[0087] 18 Management

[0088] 19 Management

[0089] 20 terminal devices

[0090] 21 Side wall

[0091] 22 Implementation

[0092] 23 Force sensor

[0093] 24 Actuator with displacement sensor

[0094] 25 transparent floor

[0095] 26 xyz positioning unit

[0096] 27 Base unit

[0097] 28 xyz positioning unit

[0098] 29 Time stamp

[0099] 30 Time stamp

[0100] 31 Time stamp

[0101] 32 First temporal development REHBERG HÜPPE + PARTNER - 16 - Originally submitted version 22030PCT 22.09.2025

[0102] 32 second temporal development

[0103] 33 third temporal development

Claims

REHBERG HÜPPE + PARTNER - 17 - Originally submitted version 22030PCT 22.09.2025 PATENT CLAIMS 1. Method for measuring several parameters of a measurement object (2) from a contractile biological tissue (3) that describe a current dynamic state of the measurement object (2), wherein the measurement object (2) is suspended from at least two spaced-apart holding elements (5) movable in the direction of their spacing relative to each other, wherein a first temporal development (32) is recorded by time-resolved recording of a first distribution of a chemical potential over the first region (16) of the measurement object (2) located between the holding elements by means of fluorescence microscopy, wherein a second temporal development (33) is recorded,by using a microelectrode array (12) attached to a second area (11) of the object (2) suspended from the holding elements to detect a second distribution of an electric field potential over the second area (11) in a time-resolved manner, a third temporal development (34) is detected by detecting and / or changing the distance between the holding elements (5) in a time-resolved manner and / or by detecting a force acting in the direction of the distance between the holding elements (5) in a time-resolved manner, and the first temporal development (32), the second temporal development (33) and the third temporal development (34) are correlated with each other in time, time markers (29 to 31) are set during the detection of the different temporal developments (32 to 34) and are recorded together with the respective temporal development (32, 33 and 34).

2. Method according to claim 1, wherein the same or time-synchronous time markers (29 to 31) are set and registered as parts of the three temporal developments (32 to 34) or each in a separate channel of a multi-channel signal.

3. Method according to claim 1 or 2, wherein the first temporal evolution (32), the second temporal evolution (33) and the third temporal evolution (34) are analyzed relative to the time markers (29 to 31) in order to capture the current dynamic state of the object being measured (2) with time resolution. REHBERG HÜPPE + PARTNER - 18 - Originally submitted version 22030PCT 22.09.2025 4. Method according to one of the preceding claims, wherein the second region (11), over which the distribution of the electric field potential is detected with time resolution, at least partially overlaps with the first region (16), over which the first distribution of the chemical potential is detected with time resolution, or with a projection of the first region (16) in a principal direction of the fluorescence microscopic imaging.

5. Method according to one of the preceding claims, wherein the time markers (29 to 31) are triggered by the first temporal development (32) and / or the second temporal development (33) and / or the third temporal development (34), wherein optionally the first temporal development (32), the second temporal development (33) and the third temporal development (34) are correlated only for a limited partial period defined in relation to the time markers (29 to 31).

6. Method according to one of the preceding claims, wherein the distance between the holding elements (5) and / or the force acting in the direction of the distance between the holding elements (5) and / or an external voltage applied to an electrode and electrically stimulating the object being measured (2) is repeatedly actively changed with a fourth temporal development, wherein the fourth temporal development is defined relative to the time markers (29 to 31) and / or relative to the first temporal development (32) and / or the second temporal development (33) and / or the third temporal development (34).

7. Method according to claim 6, wherein the fourth temporal development is defined in such a way as to impose or train a dynamic state of interest on the object of measurement (2) in relation to the time markers (29 to 31) and / or in relation to the first temporal development (32) and / or the second temporal development (33) and / or the third temporal development (34).

8. Method according to claim 7, wherein the object of measurement (2) is exposed to an active substance in the imposed or trained dynamic state of interest in order to detect an effect of the active substance on the dynamic state of interest of the object of measurement (2). REHBERG HÜPPE + PARTNER - 19 - Originally submitted version 22030PCT 22.09.2025 9. Method according to claim 7 or 8, wherein the same dynamic state of interest is set for a plurality of identical measuring objects (2).

10. Method according to any of the preceding claims, wherein the tissue (3) is cardiac muscle tissue.

11. Device (1) for measuring several parameters of a measurement object (2) made of a contractile biological tissue (3) that describe a current dynamic state of the measurement object (2), in particular according to the method according to one of the preceding claims, comprising at least two spaced-apart holding elements (5) movable relative to each other in the direction of their spaced-apart position, which are configured to suspend the measurement object (2) thereon, comprising a first measuring device (14) comprising a fluorescence light microscope (15), which is configured to detect a first temporal development (32) by detecting a first distribution of a chemical potential over the first region (16) of the measurement object (2) suspended on the holding elements with time resolution by fluorescence microscopy of a first region (16) located between the holding elements, comprising a second measuring device (10) comprising a microelectrode array (12),which is designed to detect a second temporal development (33) by detecting a second distribution of an electric field potential over the second area (11) of the measuring object (2) suspended from the holding elements in a second area (11) located between the holding elements with the microelectrode array (12) attached to the measuring object (2), with a third measuring device (7) designed to detect a third temporal development (34) by detecting and / or changing the distance between the holding elements (5) in a time-resolved manner and / or detecting a force acting in the direction of the distance between the holding elements (5) in a time-resolved manner, and with a correlation device (9) designed to correlate the first temporal development (32), the second temporal development (33) and the third temporal development (34) with each other in a time-resolved manner,where, during the recording of the various temporal developments (32 to 34), it sets time markers (29 to 31) that start from the first, REHBERG HÜPPE + PARTNER - 20 - Originally submitted version 22030PCT 22.09.2025 The measuring device (14), the second measuring device (10) and the third measuring device (7) together with the respective temporal development (32, 33 and 34 respectively) are recorded.

12. Device (1) according to claim 11, comprising a measuring chamber, wherein the holding elements (5) are arranged on opposite side walls of the measuring chamber, wherein the fluorescence light microscope (15) of the first measuring device (14) is arranged to image the measuring object (2) suspended on the holding elements (5) through a transparent bottom of the measuring chamber, and wherein the microelectrode array (12) of the second measuring device (10) is connected to attach itself from above to the measuring object (2) suspended on the holding elements (5).

13. Device (1) according to claim 11, comprising a positioning device for positioning the microelectrode array (12) of the second measuring device (10) relative to the holding elements (5) and, optionally, for releasing the positioned microelectrode array (12) in a release position above a target position on the measuring object (2) suspended on the holding elements.

14. Device (1) according to one of claims 11 to 13, comprising an actuator device configured to actively change the distance between the holding elements (5) and / or the force acting in the direction of the distance between the holding elements (5), and / or an electrode device comprising an electrode configured to actively apply an external voltage to the electrode that electrically stimulates the object being measured (2).

Images