Devices, systems, and methods for imparting uniaxial or biaxial mechanical strain to samples including biological samples

Abstract

A strain-inducing system to strain a sample deposited upon a substrate includes a cassette including substrate interface members spaced from each other to create a gap therebetween. One or more of the substrate interface members includes an abutment member extending therefrom. A base has a sample chamber configured to removably receive the cassette therein, and a strain-inducing interface is configured to be placed in connection with the base and with the cassette. A strain body of the strain inducing interface includes a strain path including a strain surface which extends in a predetermined path around an axis of the strain body. The extending strain surface is configured to be placed in abutting contact with the abutment member. Rotation of the strain body relative to the cassette about the axis of the strain body causes a change in a size of the gap and straining of the sample.

Description

Attorney Docket No. 25-003PCTDEVICES, SYSTEMS, AND METHODS FOR IMPARTING UNIAXIAL OR BIAXIAL MECHANICAL STRAIN TO SAMPLES INCLUDING BIOLOGICAL SAMPLESSTATEMENT OF GOVERNMENT INTEREST

[0001] This invention was made with government support under grant number #HD044750 awarded by the National Institutes of Health. The government has certain rights in the invention.CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims benefit of U.S. Provisional Patent Application SerialNo. 63 / 778,121, filed March 26, 2025, the disclosure of which is incorporated herein by reference.BACKGROUND ART

[0003] The following information is provided to assist the reader in understanding technologies disclosed below and the environment in which such technologies may typically be used. The terms used herein are not intended to be limited to any particular narrow interpretation unless clearly stated otherwise in this document. References set forth herein may facilitate understanding of the technologies or the background thereof. The disclosure of all references cited herein are incorporated by reference.

[0004] Physical mechanics plays a critical role in orchestrating tissue shape and subsequent function across a range of contexts including, for example, early embryonic development and later organogenesis, carcinogenesis, wound healing, and regeneration. Mechanical strain, induced both by the local microenvironment and external sources, can guide biological processes such as cell migration, proliferation, cell fate change, etc. For instance, externally- applied compressive forces can reduce cancer cell proliferation and induce apoptosis, and inAttorney Docket No. 25-003PCTother cases, compressive forces can guide growth cone migration and drive collective neural crest cell migration.

[0005] Lesions to mechanical processes may lead directly to birth defects via inappropriate strain and tissue malformation or by altering normal mechanically-triggered biological processes. These lesions can drive structural birth defects in organ formation, such as congenital heart defects, spina bifida, and ventral body wall closure. For instance, changes in embryo bulk mechanical properties can facilitate or delay the mesenchymal-to-epithelial transitions in Xenopus heart progenitor cells. These mechanical changes can cause cardiac defects; specifically, increased external mechanical tension is shown to induce 50% more mesenchymal- to-epithelial transition in heart progenitor cells, giving rise to cases of cardiac edema. Another study found that lack of tissue strain during gastrulation disrupted planar-cell-polarity in the ciliated epithelium of Xenopus embryos. That defect was rescued by applying exogenous strain similar to the normal gastrulation strain. Understanding how mechanics in general, and mechanical strain in particular, function in developmental processes is key to identifying underlying causes of diseases and birth defects.

[0006] High strain can also change tissue mechanical properties by fluidizing or solidifying tissues, facilitating transitions between so-called solid-like and fluid-like states by "unjamming" or "jamming" cells in the tissue. Such transitions may involve alterations in adhesive junctional complexes between cells to allow remodeling. Recent studies have described "jamming" and "unjamming" tissue behaviors during embryonic development, but it remains unknown whether or how mechanical strain alters cell-cell junctions enabling transitions between fluid- and solidlike states. Nonetheless, the capacity of a tissue to remodel is critical for its ability to dissipate strain energy, and the mechanical cues those strains encode.

[0007] Establishing causal relationships between mechanical cues and their effects on multicellular tissues requires tools capable of experimentally generating temporally and spatially defined strains (for example, externally controlled strain rates), which are compatible with high resolution live-cell imaging. Key cellular and extracellular features including the nucleus, cytoskeleton, cell adhesions, and extracellular matrix have all been implicated in establishing mechanical properties as well as in transducing strain cues into signal transduction pathways.Attorney Docket No. 25-003PCTHigh resolution live-cell microscopy combined with image analysis pipelines can quantify the distribution of polarity factors; the dynamics of the cytoskeleton, adhesion, and membrane remodeling; and how those processes are coupled to signal transduction. The ability to control tissue strain over minutes to hours akin to controlling gene activity has immediate applications in studying the influence of mechanical cues in remodeling both synthetic and native tissues within complex micromechanical microenvironments.

[0008] A number of strain-inducing or "stretcher" systems have been used to apply strains to living tissues in combination with microscopic analysis. Simple stretchers for suspended cell monolayers include wire cantilevers. More sophisticated uniaxial or biaxial stretchers use clamps or posts to bond tissues to motorized actuators. Clamp- and actuator-based systems are typically bulky and weigh considerably more than the 250 g mass limit of fast z-scanning stages used for high resolution confocal sectioning. Another technique to apply strain is to induce compression along one axis and thus generate tensile strain along the other two axes. Although this technique is easy to implement, the technique typically achieves only small strains and is limited to larger bulk tissue samples such as whole embryos. Indentation of an elastic substrate including seeded cells or tissues thereon is also commonly used to induce tensile strain. In these indentation devices, an elastic substrate is fixed on posts, and an indenter is used to press on the substrate to deform the substrate, generating strain on seeded cells. Indentation requires steric access for positioning and travel along the z-axis (see FIG. 2), which can limit access for high resolution optics.

[0009] A number of previously studied stretchers have also been designed to generate strain on cells or tissues by bonding or attaching them to an elastic substrate. The earliest efforts to stretch embryonic tissues used rubber substrates. A number of commercial systems use posts to fix the edges of an elastic substrate, with strain subsequently applied by a linear actuator along the edge of the device. Another commercial device uses a macro-scale indenter to induce strains up to 30%. Such systems have advantages and limitations, but none are well suited for high resolution confocal live-cell microscopy.

[0010] Thus, in various fields of biological study including various omics, it is desirable to induce a controlled amount or "dose" of mechanical strain (that is, at least one of stretching,Attorney Docket No. 25-003PCTcompressing and shearing strain) upon of living biological sample. To carry out a system analysis of the roles of mechanical strain of living biological samples in various mechanisms, requires a systematic analysis. Although it is desirable to develop devices, systems, and methods to induce controlled mechanical strain upon living tissue (for example, for high-resolution microscopic study of such tissue), there has been limited success in developing such devices, systems, and methods.

[0011] Recently a low-weight, strain-inducing system suitable to be seated upon a microscope stage of and to function with the optics thereof to microscopically study strain in a variety of materials including live tissue and cells has been developed. PCT International Patent Publication No. WO 2025 / 019,681 the disclosure of which is incorporated herein by reference. It remains desirable to further develop strain-inducing systems to facilitate, for example, the controlled application of biaxial strain for microscopic studied of living tissue, cells, and other substrates.SUMMARY OF THE INVENTION

[0012] A strain-inducing system for applying strain to a sample deposited upon a sample substrate, wherein the sample substrate is stretchable, includes a cassette including substrate interface members. The substrate interface members are spaced from each other to create a gap therebetween. Each of the substrate interface members is configured to be attached to a separate area of a surface of the sample substrate so that a portion of the sample substrate extends across the gap. One or more of the substrate interface members includes an abutment member extending therefrom. The strain-inducing system further includes a base having a sample chamber configured to removably receive the cassette therein and a strain-inducing interface configured to be placed in connection with the base and with the cassette. The strain-inducing interface includes a strain body including an extending strain path. The extending strain path includes an extending strain surface which extends in a predetermined path around an axis of the strain body. The extending strain surface is configured to be placed in abutting contact with the abutment member. Rotation of the strain body relative to the cassette about the axis of the strain body causes a change in a size of the gap when the abutment member is in contact with the extending strain surface. The change in size of the gap caused by rotation of the strainAttorney Docket No. 25-003PCTbody is determined by change in radial position of the extending strain surface as it extends around the axis of the strain body and the degree of angular rotation of the strain body about the axis of the strain body.

[0013] In a number of embodiments, the strain-inducing interface further includes a guide body. The guide body includes an extending guide path. The extending guide path includes an extending guide surface which extends in a predetermined path. The extending guide surface extends generally linearly. The extending guide surface is configured to be placed in abutting contact with the abutment member when the strain body and the guide body are placed in predetermined positions relative to each other and in connection with an assembly of the base and the cassette such that the strain body is rotatable relative to the guide body and to the cassette. The abutment member extends from the substrate interface member to first be placed in abutting contact with one of the extending strain surface and the extending guide surface, and then to be placed in abutting contact with the other of the extending strain surface and the extending guide surface. The extending guide surface constrains the abutment member to generally linear motion.

[0014] Each of a plurality of the substrate interface members may include an abutment member extending therefrom. The strain body may include a plurality of extending strain paths, wherein each of the plurality of extending strain paths includes an extending strain surface which extends in a predetermined path around the axis of the strain body. Each of the extending strain surfaces may be configured to be placed in abutting contact with an associated one of the abutment members. Rotation of the strain body relative to the cassette about the axis of the strain body causes a change in a size of the gap when each of the extending strain surfaces is in abutting contact with the associated one of the abutment members. The change in size of the gap caused by rotation of the strain body about the axis of the strain body is determined by change in radius of each of the plurality of extending strain surfaces as it extends around the axis of the strain body and the degree of angular rotation of the strain body about the axis of the strain body.

[0015] In a number of embodiments, the strain-inducing interface further includes a guide body, and the guide body includes a plurality of extending guide paths. Each of the plurality ofAttorney Docket No. 25-003PCTextending guide paths includes an extending guide surface which extends in a predetermined path. The extending guide surfaces extend generally linearly. Each of the extending guide surface is configured to be placed in abutting contact with an associated one of the abutment members when the strain body and the guide body are placed in predetermined positions relative to each other and in connection with an assembly of the base and the cassette such that the strain body is rotatable relative to the guide body and to the cassette. Each of the abutment members extends from one of the substrate interface member to first be placed in abutting contact with one of the extending strain surfaces or one of the extending guide surfaces, and then to be placed in abutting contact with the other of one of the extending strain surfaces and one of the extending guide surfaces. Each of the extending guide surface constrains the associated one of the abutment member to generally linear motion.

[0016] Each of the plurality of extending strain paths may further include another extending strain surface which extends generally parallel to the extending strain surface to form an extending strain slot. Each of the plurality of extending guide members may include another extending guide surface which extends generally parallel to the extending guide surface to form an extending guide slot. In such an embodiment, the strain body includes a plurality of extending strain slots, and the guide body includes a plurality of extending guide slots.

[0017] In a number of embodiments, the guide body is placed adjacent to the cassette and between the cassette and the strain body so that each of the abutment members extends through one of the plurality of extending guide slots and into one of the plurality of extending strain slots.

[0018] The base may be configured to be placed in connection with the guide body and with the strain body. In a number of embodiments, the base includes a seating into which the guide body is positioned over the cassette such that each of the abutment members extends through a different one of the extending guide slots. The seating prevents the guide body from rotating relative to the base. The base may further include a seating into which the strain body is rotatably positioned over the guide body such that each of the abutment members extends into a different one of the plurality of extending strain slots.Attorney Docket No. 25-003PCT

[0019] In a number of embodiments, the cassette includes four substrate interfaces, wherein each of the substrate interfaces has one of the abutment members extending therefrom. In such an embodiment, the guide body may include four extending guide slots, and the strain body may include four extending strain slots. Each of the substrate interfaces may extend to form a cross shape. Each of the substrate interfaces may include an inner section to which the sample substrate is attached and an outer section from which one of the abutment members extends. Each outer section of each substrate interface may be connected to each adjacent outer section by an extendable member. The extendable member providing resistance to flexure of the substrate interfaces out of a plane defined by the original configuration thereof.

[0020] In a number of embodiments, the system further includes an actuator body in connection with the strain body to impart rotational motion to the strain body. The actuator body may be a manual actuator, or the actuator may be a powered actuator. The strain system may further include a motor to power the actuator body and a system configured to determine position of the actuator body. The strain system may, for example, further include a motor, and the actuator body may include gear teeth which are configured to be placed in connection with a first gear which is driven by the motor. The strain system may further include a second gear which is in connection with the gear teeth of the actuator body and in connection with a system configured to determine the position of the actuator body. The system configured to determine the position of the actuator body may, for example, include an encoder. The system may further include electronic circuitry in connection with the motor and with the encoder to control the position of the actuator body over time.

[0021] In a number of embodiments, the plurality of extending strain slots is configured to create one of isotropic and anisotropic biaxial strain in the sample substrate. The plurality of extending strain slots may alternatively be configured to create uniaxial strain.

[0022] In a number of embodiments, the system is configured to be placed in connection with a stage of a microscope so that an objective of the microscope is aligned with the sample, and the sample is viewable via the objective during straining. The sample chamber may be adapted to contain a volume of a liquid therein. The sample may, for example, be a biological sample.Attorney Docket No. 25-003PCT

[0023] A method of inducing strain in a sample includes providing a strain-inducing system as described herein, attaching the sample to the sample substrate, attaching the sample substrate to the substrate interface member of the cassette, placing the cassette in the sample chamber of the base, placing the strain body in connection with the cassette, and rotating the strain body of the system relative to the base and to the cassette. In a number of embodiments, the system is configured to be placed in connection with a stage of a microscope so that an objective of the microscope is aligned with the sample, and the sample is viewable via the objective during straining. The sample chamber may be adapted to contain a volume of a liquid therein. The sample may, for example, be a biological sample.

[0024] A cassette for use in a system in which a substrate is strained includes four substrate interfaces, each of the substrate interfaces having an abutment member extending therefrom. Each of the four substrate interface members is spaced from each other to create a gap therebetween. Each of the substrate interface members is configured to be attached to a separate area of a surface of the substrate so that a portion of the substrate extends across the gap. Each of the four substrate interface member is independently movably relative to the other substrate interface members via application of force to the abutment member extending therefrom to strain the portion of the substrate extending across the gap.

[0025] The present devices, systems, and methods, along with the attributes and attendant advantages thereof, will best be appreciated and understood in view of the following detailed description taken in conjunction with the accompanying drawings.BRIEF DESCRIPTION OF DRAWINGS

[0026] FIG. 1 illustrates an isometric, exploded view of an embodiment of a strain-inducing system hereof, wherein the strain interface (that is, the strain body and guide body) of the strain-inducing system is illustrated as transparent.

[0027] FIG. 2 illustrates an isometric view of the strain-inducing system of FIG. 1 in an assembled state, wherein the strain interface of the strain-inducing system is illustrated as transparent.

[0028] FIG. 3 illustrates a top view of the strain-inducing system of FIG. 1 in an assembled state, wherein the strain interface of the strain-inducing system is illustrated as transparent.Attorney Docket No. 25-003PCT

[0029] FIG. 4 illustrates a side view of the strain-inducing system of FIG. 1 in an assembled state.

[0030] FIG. 5A illustrates a strain body for use in connection with the strain-inducing system of FIG. 1, which functions to provide first, predetermined strain profile.

[0031] FIG. 5B illustrates another strain body for use in connection with the strain-inducing system of FIG. 1, which functions to provide a second strain profile, different from the first strain profile.

[0032] FIG. 5C illustrates another strain body for use in connection with the strain-inducing system of FIG. 1, which functions to provide a third strain profile, different from the first and second strain profiles.

[0033] FIG. 5D is a photograph illustrating the generally symmetrical biaxial strain (in the x and y directions) induced in a sample substrate using the strain body of FIG. 5A, wherein each of the strain paths (in the form of slots in the illustrated embodiment) functions as an eccentric cam surface for the associated abutment member.

[0034] FIG. 5E is a photograph illustrating the asymmetrical strain (in the x and y directions) induced in a sample substrate using the strain body of FIG. 5B and demonstrating the movement of each of the extending abutment members, contact members or pins of the cassette along the associated guide path (in the form of a slot in the illustrated embodiment) and strain path (in the form of a slot in the illustrated embodiment) upon rotation of the strain body, wherein each of the strain paths functions as an eccentric cam surface for the associated abutment member.

[0035] FIG. 6 illustrates a top view of the actuator of the strain-inducing system of FIG. 1.

[0036] FIG. 7 illustrates a top view of the strain body of the strain-inducing system of FIG. 1 and FIG. 5A.

[0037] FIG. 8 illustrates a top view of the guide body (which may function as a microscope stage interface) of the strain-inducing system of FIG. 1.

[0038] FIG. 9 illustrates the base or body of the strain-inducing system of FIG. 1.

[0039] FIG. 10 illustrates a side view of the cassette of the strain-inducing system of FIG. 1.

[0040] FIG. 11 illustrates an isometric view of the cassette of the strain-inducing system of FIG. 1.Attorney Docket No. 25-003PCT

[0041] FIG. 12 illustrates an isometric, exploded view of the cassette of the strain-inducing system of FIG. 1.

[0042] FIG. 13A illustrates another embodiment of a strain body hereof in which the strain paths include an extending arced (cam) surface that abutment members of the cassette contact and follow during induction of movement thereof.

[0043] FIG. 13B illustrates another embodiment of a guide body hereof in which the guide paths include a generally linearly extending surface that abutment members of the cassette contact and follow during induction of movement thereof.

[0044] FIG. 13C illustrates another embodiment of a guide body hereof in which the orientation or angle of the generally linear guide paths (in the form of slots) are different than the embodiment illustrated in, for example, FIGS. 1 and 8.

[0045] FIG. 13D illustrates another embodiment of a strain body hereof in which the strain paths (in the form of slots) extend in a generally linear path and have the same beginning and ending radii (distance from axis A) as the strain paths illustrated in, for example, FIGS. 1 and 5A.

[0046] FIG. 13E illustrates another embodiment of a strain body hereof in which the strain paths (in the form of arced slots) have the same radius or radial position ri (distance from axis A) at each end of the strain path and the distance from the axis varies along the length of the strain path to a maximum of r4 at the midpoint of the strain path.

[0047] FIG. 13F illustrates pre-stretching or pre-straining of a cassette hereof wherein the upper portion of the drawing illustrates a top view of the cassette in a relaxed or unstrained state (in broken lines) and in a pre-stretched or pre-strained state (in solid lines) with a jig in connection with the cassette in the pre-stretched or pre-strained state, and the lower portion of the drawing illustrates a side view of the cassette in the pre-stretched or pre-strained state with the jig in connection with the cassette.

[0048] FIG. 13G illustrates a top view (left), a side view (center) and an isometric view (right) of the jig for use in pre-stretching or pre-straining cassettes hereof.

[0049] FIG. 14 illustrates schematically the strain-inducing system of FIG. 1 positioned on the stage of a confocal inverted microscope.Attorney Docket No. 25-003PCT

[0050] FIG. 15A illustrates a flow chart that sets forth a representative embodiment control methodology that was used in a number of studies of an embodiment of powered strain system hereof.

[0051] FIG. 15B illustrates an isometric, exploded or disassembled view of a representative embodiment of a powered strain system hereof.

[0052] FIG. 15C illustrates a top view of the powered strain system of FIG. 15A in an assembled state.

[0053] FIG. 15D illustrates an end view of the powered strain system of FIG. 15A in an assembled state.

[0054] FIG. 15E illustrates a side view of the powered strain system of FIG. 15A in an assembled state.

[0055] FIG. 15F illustrates an isometric view of the powered strain system of FIG. 15A in an assembled state.

[0056] FIG. 15G illustrates isometric view of a representative embodiment of a removable base hereof with a cover glass in connection therewith (left) and with a cover glass in position to be placed in connection therewith (right).

[0057] FIG. 15H illustrates a representative embodiment of a strain guide hereof configured to provide a constant rate of radial displacement per rotational displacement of 0.1 mm per degree of rotation in the illustrated embodiment.

[0058] FIGS. 16A illustrates a graph of a simulated or ideal strain with a linear relationship to displacement and measured data for studies of an embodiment of a strain system hereof for the first principal strain as a function of abutment member displacement.

[0059] FIGS. 16B illustrates a graph a simulated or ideal strain with a linear relationship to displacement and measured data for studies of an embodiment of a strain system hereof for XX strain as a function of abutment member displacement.

[0060] FIGS. 16C illustrates a graph a simulated or ideal strain with a linear relationship to displacement and measured data for studies of an embodiment of a strain system hereof for YY strain as a function of abutment member displacement.Attorney Docket No. 25-003PCT

[0061] FIGS. 16D illustrates a graph a simulated or ideal strain with a linear relationship to displacement and measured data for studies of an embodiment of a strain system hereof for XY strain as a function of abutment member displacement.

[0062] FIG. 16E sets forth the coordinate system used in defining strain relative to the cassette in FIGS. 16A through 16D.

[0063] FIG. 17 illustrates Table 1 which sets forth terminology used in connection with strain. DESCRIPTION

[0064] It will be readily understood that the components of the embodiments, as generally described and illustrated in the figures herein, may be arranged and designed in a wide variety of different configurations in addition to the described representative embodiments. Thus, the following more detailed description of the representative embodiments, as illustrated in the figures, is not intended to limit the scope of the embodiments, as claimed, but is merely illustrative of representative embodiments.

[0065] Reference throughout this specification to "one embodiment" or "an embodiment" (or the like) means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearance of the phrases "in one embodiment" or "in an embodiment" or the like in various places throughout this specification are not necessarily all referring to the same embodiment.

[0066] Furthermore, described features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. In the following description, numerous specific details are provided to give a thorough understanding of embodiments. One skilled in the relevant art will recognize, however, that the various embodiments can be practiced without one or more of the specific details, or with other methods, components, materials, et cetera. In other instances, well known structures, materials, or operations are not shown or described in detail to avoid obfuscation.

[0067] As used herein and in the appended claims, the singular forms "a," "an,” and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "an actuator" includes a plurality of such actuators and equivalents thereof known to those skilled in the art, and so forth, and reference to "the actuator" is a reference to one orAttorney Docket No. 25-003PCTmore such actuators and equivalents thereof known to those skilled in the art, and so forth. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each separate value, as well as intermediate ranges, are incorporated into the specification as if individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contraindicated by the text.

[0068] The terms "electronic circuitry," "circuitry" or "circuit," as used herein include, but is not limited to, hardware, firmware, software, or combinations of each to perform a function(s) or an action(s). For example, based on a desired feature or need. a circuit may include a software-controlled microprocessor, discrete logic such as an application specific integrated circuit (ASIC), or other programmed logic device. A circuit may also be fully embodied as software. As used herein, "circuit" is considered synonymous with "logic." The term "logic," as used herein includes, but is not limited to, hardware, firmware, software, or combinations of each to perform a function(s) or an action(s), or to cause a function or action from another component. For example, based on a desired application or need, logic may include a software-controlled microprocessor, discrete logic such as an application specific integrated circuit (ASIC), or other programmed logic device. Logic may also be fully embodied as software.

[0069] The term "processor," as used herein includes, but is not limited to, one or more of virtually any number of processor systems or stand-alone processors, such as microprocessors, microcontrollers, central processing units (CPUs), and digital signal processors (DSPs), in any combination. The processor may be associated with various other circuits that support operation of the processor, such as random-access memory (RAM), read-only memory (ROM), programmable read-only memory (PROM), erasable programmable read only memory (EPROM), clocks, decoders, memory controllers, or interrupt controllers, etc. These support circuits may be internal or external to the processor or its associated electronic packaging. The support circuits are in operative communication with the processor. The support circuits are not necessarily shown separately from the processor in block diagrams or other drawings.Attorney Docket No. 25-003PCT

[0070] The term "controller," as used herein includes, but is not limited to, any circuit or device that coordinates and controls the operation of one or more input and / or output devices. A controller may, for example, include a device having one or more processors, microprocessors, or central processing units capable of being programmed to perform functions.

[0071] The term "software," as used herein includes, but is not limited to, one or more computer readable or executable instructions that cause a computer or other electronic device to perform functions, actions, or behave in a desired manner. The instructions may be embodied in various forms such as routines, algorithms, modules, or programs including separate applications or code from dynamically linked libraries. Software may also be implemented in various forms such as a stand-alone program, a function call, a servlet, an applet, instructions stored in a memory, part of an operating system or other type of executable instructions. It will be appreciated by one of ordinary skill in the art that the form of software is dependent on, for example, requirements of a desired application, the environment it runs on, or the desires of a designer / programmer or the like.

[0072] As used herein, the term "approximately" when used in connection with a value means within 5%, within 2%, or within 1% of the value unless otherwise indicated herein or otherwise clearly contraindicated by the text. As used herein the term "and / or" means one of or both of an entity. Thus, A and / or B means A or B, or both A and B.

[0073] As used herein, relative positional terms such as above, top, upper, below, bottom, lower and the like refer to the orientation illustrated in, for example, FIG. 1 and relative to the orientation when systems hereof are used in connection with microscope 10 as illustrated in FIG. 13, where in the base of the system is positioned at the lowermost or bottom position.

[0074] To test the physiological roles of strain, it is desirable that a strain-inducing system or strain system usable with a sample such as tissue be able to apply relatively large strains (as, for example, observed during embryonic morphogenesis or during cycles of physiological function) to tissue samples cultured ex vivo. During the most rapid phases of embryonic morphogenesis, for example, tissues experience large strain, ranging from 50% to greater than 500%. For example, tissues undergoing convergent extension exhibit more than 2-fold changes in length, greater than 200% strain, during zebrafish and Xenopus gastrulation and neurulation. ToAttorney Docket No. 25-003PCTreplicate those high in vivo levels of strain, a number of embodiments of strain systems hereof are capable of reaching 100% strain or more. Furthermore, simultaneous observation of intracellular cytoskeletal and adhesion dynamics requires high numerical aperture oil immersion objective lenses that typically have small working distances that require tissues to be less than 200 pm from the coverslip. To acquire high-resolution imaging sequences while applying strain, stable tissue mounts should minimize out-of-plane torsion that would otherwise drive samples out of plane beyond the objective's working distance. Finally, the total mass of the strain system should be compatible with piezo or galvo-driven z stages that are commonly used in rapid confocal sectioning in live cell imaging systems (for example, Leica, Zeiss, Nikon, and Thorlabs).

[0075] The strain inducing systems, stretcher systems, or strain systems hereof are capable of inducing high strain (that is mechanical strain or deformation, which may, for example, be uniaxial, biaxial or shearing strain) on, for example, living tissues, cells, and other materials. The strain can be readily controlled in direction, amplitude, rate etc. The strain-inducing systems hereof also enable imaging at high-resolution on a microscope such as an inverted confocal microscope. In a number of embodiments of strain systems hereof, the system includes a number of subsystems or components including a strain-inducing interface, a readily interchangeable cassette, a base (which may function a microscope stage assembly or insert which is readily designed or customizable for integration with a particular microscope), and an actuator or actuators (which may be manual or powered). The modular design of strain systems hereof enables integration with, for example, an inverted compound microscope equipped for high-resolution confocal imaging. The cassette-based design allows simple exchange of samples for technical and biological replicates. Furthermore, the cassette design allows one to image samples directly through a simple cover glass, instead of through support substrates, such as elastic substrates, which are not optimized for high-resolution imaging. Additionally, the cassette design hereof is easily modified to accommodate diverse experimental models. The interchangeable cassettes hereof may alternatively be referred to as interchangeable cartridges, carriers, or supports.

[0076] As described above, strain systems hereof may be used to apply mechanical strain to living tissues, which can be live imaged using high-resolution confocal microscopy. FIGS. 1Attorney Docket No. 25-003PCTthrough 12 illustrate an embodiment of a strain-inducing system or strain system 100 hereof and various subsystems or components thereof. FIGS. 13A through 13E illustrates alternative embodiments of strain bodies and guide bodies for use in the system of FIG. 1 (or another system hereof). FIG. 14 illustrates schematically the strain system 100 of FIG. 1 upon a stage 11 of an inverted confocal microscope 10. As, for example, illustrated in FIG. 1, strain system 100 includes a body or base 200, a cassette 300, a strain-inducing or strain interface or strain inducing interface 400 (including a guide body 500 and a strain body 600, in the illustrated embodiment), and an actuator 700, which is connectible or operatively connectible to strain body 600 to induce rotation therein. A used herein, the term "actuator" refers to a component that produces or transfers force, torque, or displacement when an electrical, pneumatic, hydraulic, or manual input is supplied to the actuator.

[0077] Base 200 may function as a stage interface in embodiments in which strain system 100 is to be used in connection with a microscope such as microscope 10 of FIG. 14. Base 200 may, for example, be formed from a polymeric material (for example, via 3D-printing), and may be readily designed or customized to fit various microscopes such as commercial inverted brightfield or confocal microscopes. In a number of embodiments, base or stage interface 200 may be 3D-printed out of a polymer such as Polylactic Acid (PLA) using a Fused Deposition Modeling (FDM) printer and can be easily modified to fit a wide variety of microscope stages. Base 200 may be readily designed and formed, for example, to sit securely or tightly on the platform of microscope stage 11 (see FIG. 14) to provide stability and prevent shifting during, for example, live imaging. A sample volume or chamber 210 is formed in base 200 to receive a sample via sample cassette or cassette 300 as described below. In a number of embodiments, sample chamber 210 may include a transparent bottom and may be suitable to hold a liquid (for example, an aqueous medium) that is formulated to maintain a live biological sample. Certain samples (for example, samples which are not living biological samples) may not require a liquid medium. As used herein, the term "transparent" refers to a physical property of allowing light to pass through the material without appreciable scattering or fluorescent absorbance of light. In a number of embodiments, a bottom of base 200 includes an opening 220 which cooperates with a coverslip (not shown) to form the transparent bottom of chamber 210, allowing direct andAttorney Docket No. 25-003PCTunimpeded microscopy of the sample. In the illustrated embodiment, base 200 includes a rectangular area or seating 222 formed by the inner wall of chamber 210 to seat a transparent cover slip (not shown) over opening 220.

[0078] In the illustrated embodiment of cassette 300 (see, for example, FIGS. 1 through 3 and 10 through 12), cassette 300 includes a plurality of substrate interface members 310, which are movable relative to each other. Substrate interface members 310 are spaced from each other to create a gap 320 therebetween. With reference to FIGS. 10 through 12, each of the substrate interface members 310 includes a centrally or radially inward positioned first section 310a which is adapted or configured to be attached to a separate area of a surface of a sample substrate 800 so that a portion of sample substrate 800 extends across gap 320. As illustrated in FIG. 11, a sample S is, for example, positioned at the center of sample substrate 800 in a generally square-shaped or square-shaped gap formed in the vicinity of the inner ends of first sections 310a of substrate interface members 310. One skilled in the art will appreciate that other cassette and gap geometries are possible. At least one of the substrate interface members (for example, a single substrate interface member used to induce a uniaxial strain in conjunction with a stationary substrate interface member through movement thereof relative to the other substrate interface members). Each of the plurality of substrate interface members 310 may further include an abutment member, contact member, or pin 330 extending therefrom (from a top surface therefrom in the illustrated embodiment). In the illustrated embodiment, abutment members 330 are attached to an outer or radially outer second section 310b of substrate interface members 310 (for example, via an adhesive).

[0079] In the illustrated embodiment (as best illustrated in, for example, FIGS. 11 and 12), cassette 300, when assembled, includes four substrate interface members 310, forming an X or cross-shaped cassettes wherein each abutment member 330 is positioned generally at the corner of a square (in the vicinity of the outer end of each substrate interface member 310). As, for example, illustrated in FIG. 12 (and described in PCT International Patent Publication No. WO 2025 / 019,681 for another, different cassette), in a number of embodiments, cassette 300 may be formed from an upper or top sheet or shim 302a and a lower or bottom sheet or shim 302b of, for example, a polymeric material such as a polyester (PES) polymeric material.Attorney Docket No. 25-003PCTThe polymeric material may be cut to form upper section or layer 300a and a lowersection 300b or layer of cassette 300. In FIG. 12, upper shim 302a and lower shim 302b are illustrated in broken lines and the cutout section(s) thereof used to form upper section 300a and lower section 300b of cassette 300 are illustrated in solid lines. Upper shim 302a was cut to include or form four separate or unconnected upper sections of substrate interface members 310 in a number of studied embodiments. Each upper section includes an opening or seating 312, in which a lower, base section of abutment members 330 may be seated during assembly (for example, via a UV-curable optical adhesive) to cooperate with straininterface 400. Similar to upper sheet or shim 302a, lower shim 302b was cut to include lower sections of substrate interface members 310. The upper sections and lower sections are aligned (or stacked; see, for example, FIG. 12) during assembly such that the upper sections are positioned over the lower sections and then connected to form an assembly (using, for example, a UV-curable optical adhesive). Through induced movement of abutment members 330, each substrate interface member 310 is independently movable relative to other substrate interface members 310.

[0080] In a number of embodiments, shims 302a and 302b hereof were 38 to 127 pm, or 38 to 76 pm, in thickness to facilitate high-resolution imaging by maintaining the sample within the working distance of high-numerical aperture objectives. The thickness of shims 302a and 302b of the cassette shim assembly can be adjusted according to the biological sample, (for example, tissue or cell types). Due to the nature of plasticity of a polymeric shim assembly such as a PES shim, the deformation substrate interface members 310 formed therefrom is negligible under the forces experienced during normal use of systems hereof. Deformation is transmitted to the elastic or stretchable sheet or substrate 800 (positioned between and attached to the upper and bottom sections of substrate interface members 310) and to extendable sections, spring sections or cutout sections 340 of cassette 300, which are formed in lower section 300b thereof. Extendable (or deformable) sections 340 are formed in lower shim 302b in the illustrated embodiment and function to reduce or minimize out-of-plane torsion (that is, movement in the z direction, out of the original planar conformation), which is important to maintain focus during microscopy, and to stabilize a tissue sample attached to substrate 800 during stretching. ToAttorney Docket No. 25-003PCTsecurely mount stretchable sheet or sample substrate 800 onto upper and lower PES sections 300a and 300b hereof, a "sandwich" design was used in which sample substrate 800 was glued between the centrally or radially inward sections of upper section 300a and centrally or radially inward sections of lower section 300b (which form sections 310a of substrate interface members 310 when assembled). As illustrated in FIG. 12, sample substrate 800 may be formed in an X or cross shape to increase or maximize area for adhesive contact.

[0081] In the illustrated embodiment, base 200 provides a support for strain interface 400 and actuator 700 which form a strain assembly or mechanism to impart strain to cassette 300. In that regard, after cassette 300 is placed within chamber 210, strain interface 400 is placed in connection therewith. In the illustrated embodiment, strain interface 400 includes a guide body 500 (in the form of a first plate) and a strain body 600 (in the form of a second plate). In the illustrated embodiment, each of guide body 500 and strain body 600 are illustrated as being transparent. In a number of studied embodiments, each of guide body 500 and strain body 600 was formed from a polymer such as acrylic polymer. In the illustrated embodiment, guide body 500 is placed over cassette 300 (after cassette 300 is seated in chamber 210 of base 200). Opposite ends of guide body 500 are seated in generally rectangular seating 230 formed as indents or relief areas on opposite ends of base 200. A lower surface of the opposite ends of guide body 500 rest upon lower ledges 232 of such indents. Seating 230 prevents rotation of guide body 500 relative to base 200 and cassette 300 when guide member is positioned in therein and over cassette 300. In the illustrated embodiment, a plurality (that is, two, three, four or more; wherein four are included in the illustrated embodiment) of extending abutment members, extending pins, or path follower 330 of cassette 300 extend to contact or abut guide paths formed in guide member 500. Each guide path provides an extending surface which is contacted by abutment members 330 to guide or direct the movement of that abutment member 330 along the extending surface during movement of that abutment member 330 induced by rotation of strain body 600 about axis A thereof. In the illustrated embodiment, such guide paths extend linearly or generally linearly and are formed as guide slots or guide passages 510 formed in guide body 500. Each abutment member 330 extends independently through a difference passage 510. In a number of other embodiments, a single abutmentAttorney Docket No. 25-003PCTmember may be provided to, for example, produce a uniaxial strain. Guide body 500 further include a passage 520 positioned inward or radially inward of guide slots 510. Passage 520 may facilitate imaging as well as access to cassette 300. In the illustrated embodiment, each guide slot 510 extends linearly or generally linearly, as described above, and is spaced or oriented (or are approximately spaced or oriented) 90 degrees from adjacent guide slots 510.

[0082] After seating of guide body 500 as described above, strain body 600 is placed thereover such that a portion of the perimeter of strain body 600 rests on surface or ledges 242 of arced seatings 240 of base 200. Abutment members 330 extend to contact or abut strain paths formed in strain member 500. Each strain path provides an extending surface which is contacted by abutment members 330 to guide or direct the movement of that abutment member 330 along the extending surface. In the illustrated embodiment the strain paths are formed as strain slots or strain passages 610 (having parallel arced surfaces). Each abutment member 330 extends at least partially into one of strain slots 610. In general, the radius (the distance from axis A of strain body 600 to the extending contact surface) of the strain paths hereof varies over the length thereof (which extends around axis A with a defined orientation) such that motion is induced in an associated abutment member 330 upon rotation of strain body 600 about axis A (relative to cassette 300). In the illustrated embodiment of FIGS. 1 through 5E and 7, strain slots 610 extend in a predetermined arc to induce strain in sample substrate 800 via in interaction of one of abutment members 310 independently with one of extending strain slots 610 upon rotation of strain body 600 about axis A (see FIG. 2) relative to the assembly of base 200, cassette 300 and guide body 500. In that regard, each strain slots 610 may be eccentrically arced relative to axis A to interact with one of abutment members 310 in a modified manner of a follower face cam mechanism (that is, a modified four-follower face cam mechanism in the illustrated embodiment). In the illustrated embodiment of FIGS. 1 through 5E and 7, the arced contact surface or cam contact surface is designed to provide a constant rate of motion of abutment members 330 for a constant rate of rotation of strain body 600. Similar to passage 520 of guide body 500, passage 620 of strain body 600 may facilitate imaging as well as access to cassette 300.Attorney Docket No. 25-003PCT

[0083] In the illustrated embodiment, upon rotation of strain body 600, the combination of slots 510 and 610 of guide member 500 and strain body 600, respectively, changes rotational motion of strain body 600 into linear motion independently for each abutment member 310. Guide member 500 is prevented from rotating relative to base 200 by seating 230, and linearly extending guide slots 510 constrain abutment members 310 to generally linear or linear motion along the length of guide slots 510. As described further below, by changing the manner in which the radius of the contact surface(s) of the strain paths vary over the length thereof, the motion induced in the abutment members can be varied or adjusted using different strain bodies in connection with individual cassettes 300 which are manufactured in the same or like manner. Moreover, the motion of each abutment member 330 can be controlled independently. In the case of the arced strain slots 610, the radius increases along the length thereof and the difference between the beginning radius, and the end radius (or end distance from axis A) of each individual arced strain slot 610 determines the degree or distance of linear motion of the associated abutment member 330. As described above, the motion induced in abutment members 330 can be adjusted independently for each abutment member 310, thereby controlling the strain profiles (uniaxial, biaxial, isotropic or symmetrical, anisotropic or asymmetrical, shear, etc.) applied to sample substrate 800 and to the sample positioned thereof. In that regard, rotation of strain body 600 relative to cassette 300 causes a change in a size of gap 320. The change in size of the gap hereof is determined by variables including the number and configuration of abutment members of the cassette, the configuration of each of the extending strain paths, the direction or orientation of each of the extending guide paths, the amount or degree of rotation of strain body 600, and the direction of rotation of strain body 600.

[0084] Multiple strain bodies and / or guide bodies (have various strain and / or guide path designs) may be formed (for example, cut) and may be easily and readily swapped to create different strain trajectories (for example, uniaxial, biaxial strain, etc.) on a sample substrate 800 and upon a sample attached thereto. In a number of studied embodiments, each of the guide bodies and strain bodies were acrylic plates which were laser cut pieces in a customized manner to generate different strain profiles. For example, FIGS. 5A and 5C illustrate examples of strainAttorney Docket No. 25-003PCTbodies 600 and 600" to effect isotropic or symmetrical biaxial strain in the x and y directions. (1:1, 2:2) on sample substrate 800, while FIG. 5B illustrates an example of a strain body 600' to effect anisotropic or asymmetrical strain (1:2), etc.), resulting in shear, on sample substrate 800. In the case of strain body 600 of FIG. 5A, the strain in gap 320 results is 3 mm (as determined by the difference between end radii or distances from axis A, r₁ and r₂, of strain slots 610) in each of the x and y directions (as defined by the orientation of guide slots 510, which are shown in broken lines in FIG. 5A) over an angular rotation of 70 degrees (designated 3×3×70 on strain body 600). A photograph of such a strain profile, wherein abutment members 330 are at their widest strain positions, is provided in FIG. 5D. In the case of strain body 600' of FIG. 5B, the strain in gap 320 is 3 mm in the x direction (as determined by the difference between r₁ and r₂ or strain slots 610a') and 6 mm in the y direction (as determined by the difference between r₁ and r₃ of strain slots 610b') over an angular rotation of 70 degrees (designated 3×6×70 on strain body 600'). A photograph of such a strain profile, wherein abutment members 330 are near their widest strain positions, is provided in FIG. 5D. In the case of strain body 600” of FIG. 5C, the strain in gap 320 is 6 mm (as determined by the difference between r₁ and r₃) in each of the x and y directions over an angular rotation of 70 degrees (designated 6×6×70 on strain body 600). In the case of a strain guide having a radius or distance from axis A that is constant over the length thereof (wherein r₁ and r₂ are equal), the change in position of associated abutment member330 would be zero over the range of angular rotation. Once again, strain bodies (for example, acrylic plates) as well as guide bodies hereof can be readily and independently swapped between experiments or studies to enable the user to perform many different experiments (that is, with different strain profiles) without extensive changes to the systems hereof. Likewise, the number of abutment members and initial positions thereof may be varied.

[0085] In the illustrated embodiment of, for example, FIG. 1, strain body 600 is positioned over guide body 500. However, the relative positions of strain body 600 and guide body 500 may be reversed. By positioning strain body 600 above guide body 500, operative connection of strain body 600 to an actuator such as actuator 700 may be facilitated. Although angular force may be applied directly to strain body 600 to cause rotation thereof, such rotation is facilitated using an actuator such as actuator 700.Attorney Docket No. 25-003PCT

[0086] In the embodiment of the photographs of FIG. 5D and 5E, actuator 700 is rotated via manual power (operating actuator 700 in the manner of a hand wheel) to rotate strain bodies 600 and 600", respectively. Similar to passage 520 of guide body 500 and passage 620 of strain body 600, passage 720 of actuator 700 facilitates imaging as well as access to cassette 300. In that regard, each of passages 520, 620, and 720 align upon assembly of system 100 as, for example, illustrated in FIGS. 2 and 3. In the illustrated embodiment, actuator 700 includes projections or projecting members 730 (see, for example, FIG. 1) extending from a lower surface thereof which are seated within openings or passages 630 of strain body 600 during assembly of system 100 to transfer rotational motion resulting from application of angular force to actuator 700 to strain body 600. Actuator 700 may be a powered actuator (for example, electrically, hydraulically or pneumatically powered) via a powering system 900 (illustrated schematically and in broken lines in FIG. 2) which may, for example, include a motorized worm gear as known in the art to cooperate with a gear-like strain body. Strain body 600 may, for example, be formed to include gear teeth around the perimeter thereof which cooperate with a worm gear.

[0087] As, for example, illustrated in FIG. 2, system 100 may further include electronic circuitry or a control system in communicative connection with powered actuator system 900, which is illustrated schematically in FIG. 2. The control system may, for example, include a processor system and a memory system in operative connection with the processor system. The memory system may include one or more software algorithms stored therein which are executable by the processor system to, for example, effect control of powered actuator system 900, and thereby rotation of actuator 700.

[0088] FIGS. 13A through 13B illustrate representative examples of various manners in which guide bodies and strain bodies hereof may be modified. FIG. 13A illustrates an embodiment of a strain body 600a hereof which includes strain paths in the form of extending arced (cam) strain surfaces 610a that abutment members of a cassette such as cassette 300 may abut and follow during induction of strain by rotation of strain body 600a. In the illustrated embodiment, extending strain surfaces 610a are the same (that is, in arc or curvature and orientation) as theAttorney Docket No. 25-003PCTradially inner surfaces of guide slots 610 of strain body 610. Strain body 600a will provide essentially the same strain profile as discussed above for strain body 600.

[0089] FIG. 13B illustrates another embodiment of a guide body 500a hereof in which the guide paths are in the form of generally linearly extending or linearly extending guide surfaces 512a of extending members 510a. An abutment member of a cassette such as cassette 300 can abut and follow one of extending guide surfaces 512a during induction of strain via rotation of a strain body hereof. Guide surfaces 512a extend in an essentially identical manner (that is, have essentially the same orientation and length) as the guide surfaces of guide slots 510 of guide body 500.

[0090] A combination of guide body 500a and strain body 600a in a strain assembly hereof will provide essentially the same strain profile as provided by the combination of guide body 500 and strain body 600 in strain assembly 400. Use of slots such as guide slots 510 and strain slots 610 may provide advantages in certain embodiments over the use of a single strain surface as provided by guide surface 512a and strain surface 610a. For example, the generally parallel surface of guide slots 510 and the generally parallel or offset arcs of strain slots 610 may provide an advantage in the case of initial misalignments in the position of one or more of the abutment members and in preventing misalignment during induced motion thereof. By forming such slots so that the distance between the generally parallel or offset surfaces is only slightly larger than the corresponding dimension of the associated abutment member (which may, for example, be cylindrical in shape), the associated abutment member may move readily along the length of the slots while being well constrained thereby.

[0091] FIG. 13C illustrates another embodiment of a guide body 500b hereof in which the orientation or angle of extension of the generally linearly (or linearly) extending guide paths (in the form of guide slots 510) are different than the embodiment of guide body 500 illustrated in, for example, FIGS. 1 and 8, to provide a different strain profile therefrom.

[0092] FIG. 13D illustrates another embodiment of a strain body 600b hereof in which the strain paths (in the form of strain slots 610b) extend in a generally linearly extending or linearly extending path and have the same beginning and ending radii (that is, the distance from axis A', r₁ and r₂, respectively) as the strain paths of strain slots 610 illustrated in, for example, FIGS. 1Attorney Docket No. 25-003PCTand 5A. While use of strain body 600b in connection with guide body 500 will provide the same overall strain as use of strain body 600 over a 70 degree angular rotation, unlike the case of strain body 600, the rate of strain will not be constant for a constant rate of angular rotation of strain body 600b. However, one may readily vary the rate of rotation of strain body 600b in a controlled manner to achieve a constant rate of strain using strain body 600b. That result may, for example, be readily achieved under control of electronic circuitry and a powered or powering system 780 as described in connection with FIG. 2.

[0093] FIG. 13E illustrates another embodiment of a strain body 600c hereof in which the strain paths (in the form of arced strain slots 610c) have the same initial and ending radii (ri; distance from axis A) and the radius or distance from axis A varies along the length of strain slots 610c to a maximum of at the midpoint of each strain slot 610c. Such strain paths would result in extension strain during travel of an abutment member along the first half of the strain path (traveling from ri to r and compression strain during travel of the abutment member in the second half of the strain path (traveling from r4 to ri).

[0094] Compression can be induced in embodiments of strain bodies such as strain bodies 600, 600’, 600", 600a and 600b by rotation of the strain body in a counterclockwise direction (in the illustrated orientation of, for example, FIG. 1). One may, for example, perform a cycling strain study in which the representative example of strain body 600 is rotated 70 degrees in the clockwise direction to induce 3 mm or strain in sample substrate 800 and then rotated 70 degrees in the counterclockwise direction (inducing compression) to return sample substrate 800 to the zero or initial position. Such cycling can be repeated multiple times.Further, a sample substrate can be pre-strained or pre-stretched before placement in connection with cassette 300 such that the initial position of the abutment member 330 is at the largest radius end of strain slots 610 and the strain body can be rotated in a counterclockwise direction to induce compressive strain in sample substrate 800 and in the attached sample S. Cycling studies and pre-stretching for compressive strain studied are discussed in PCT International Patent Publication No. WO 2025 / 019,681. In cycling or compression studies, it is desirable that substrate 800 is not permanently deformed during stretching such that it will notAttorney Docket No. 25-003PCTreadily return to its original configuration when compressive (that is, gap-reducing) strain is induced.

[0095] As described above, compression studies on the biaxial tissue strain systems hereof may be conducted in a similar manner to that disclosed in PCT International Patent Publication No. WO 2025 / 019,681 for a uniaxial design. As described in connection with FIGS. 13F and 13G, one may integrate an indent or notch 332 near the vicinity of a lower (in the orientation of the illustrated embodiment of FIG. 13F), base section of abutment members 330, near the point where the base section of each abutment member 330 is attached to one of substrate interface members 310 of cassette 300. A jig 350 dimensioned in a predetermined manner for a given amount of pre-stretch or pre-strain, which may be constructed of a polymeric material using a plastic jig, and which includes notches which may conform to and cooperate with notches 332, may be used to maintain cassette 300 in a pre-stretched or pre-strained state before a biological sample or other sample is mounted as described above. Once the sample is mounted and cassette 300 is mounted in a strain system hereof, jig 350 may be removed, and then the strain system hereof may be operated to relax (compress) cassette 300 to generate compression on the mounted biological sample or other sample.

[0096] FIG. 14 illustrates schematically strain-inducing system 100 in operative connection with a stage 11 of an inverted confocal microscope or microscope system 10. As known in the microscope arts, components of microscope system 10 that increase the overall system magnification include objective 12 and eyepiece 13. Light is provided by a light source 14.Objective 12, which is located closest to the sample, relays a real image of the sample to eyepiece 13 and to an appropriate detector 15 as known in the microscope arts. Electronic circuitry or a control system (such as illustrated in FIG. 2 or a separate electronic circuitry or control system) may be placed in communicative connection with stage 11 of microscope 10. As described above, the control system may, for example, include a processor system and a memory system in operative connection with the processor system. The memory system may have one or more software algorithms stored therein which are executable by the processor system to, for example, control a position of stage 11 of microscope 10 to align the objective with a determined portion of the sample during application of strain (as, for example, describedAttorney Docket No. 25-003PCTin PCT International Patent Publication No. WO 2025 / 019,681). Sample preparation and strain (as well as compression) studies may, for example, be conducted using system 100 hereof in a manner as described in PCT International Patent Publication No. WO 2025 / 019,681 as modified, when desirable, for use with system 100.

[0097] FIG. 15A illustrates a flow chart that sets forth a control methodology that was used in a number of studies of strain system 1100 hereof as illustrated in FIGS. 15B through 15H. Strain system 1100, which was very similar in design and function to strain system 100, was controlled via an external control box including electronic circuitry as described in connection with FIG. 2. Elements of strain system 1100 are numbered similarly to like element of strain system 100 with addition of 1000 to the reference number of the like element of strain system 100. In the studied embodiments, the electronic circuitry included software that ran on an Arduino UNO and was configured to interface with an automated open-source microscope control software (Micro-Manager) using serial communication.

[0098] In a number of studies, and referring, for example, to FIGS. 15B through 15F, the electronic circuitry ran a powered or driven actuator system 1900 including a simple stepper motor 1910 (for example, a 28BYJ-48 stepper motor available from various vendor including Amazon) that drove a 5:1 bevel gear train. A first driven gear 1920 rotated an actuator body 1700 of strain system 1100. Actuator body 1700 functioned in the manner of a wheel gear. Actuator body 1700 included gear teeth 1702 on a lower surface thereof (in the illustrated orientation) which interact with first drive gear 1920. First driven gear 1920 was a bevel gear, the gear teeth of which is operatively connected to gear teeth of actuator body 1700 via a 5:1 gear ratio as described above. First driven gear 1920 is connected to motor drive shaft (not shown) via a press fit. The motor drive shaft passed through a bearing 1924 (for example, a 608 ball bearing) positioned in a drive housing 1926, which was mounted (via connectors such as nuts and bolts, screws, etc. as known in the mechanical connector arts) to biaxial stage insert 1130 of strain system 1100. Stage inserts hereof can be made for a specific microscope stage or be formed to universally fit to multiple microscopes stages. Drive housing 1926 secured stepper motor 1910, first driven gear 1920, and bearing 1924. Biaxial stage 1130 included or supported a base 1200 which functioned similarly to base 200 of strain system 100. As described above forAttorney Docket No. 25-003PCTbase 200, base 1200 includes an area or seating 1222, which may be rectangular, formed by the inner wall of a chamber 1210. Seating 1222 may, for example, seat a transparent cover slip 1150 over an opening 1220

[0099] In a number of embodiments, base or tray 1200 was designed as a disposable component to localize media and prevent contamination of biaxial stage insert 1130 while allowing strain experiments to continue. As, for example, illustrated in FIG. 15G, base 1200 was fabricated by gluing cover glass 1150 to polymeric base 1200 and then using the assembly in place of a single cover glass. An experiment using multiple drugs in connection with a biological sample would, for example, require a unique interchangeable base or tray 1200 for each unique combination of drugs to prevent contamination between mixtures.

[0100] A second driven gear 1920' was also a bevel gear which connected to gear teeth 1701 of actuator body 1700 via a 5:1 gear ratio. Second driven gear 1920' was connected to a rotary encoder 1930 (for example, the AMT103-V-8mm encoder available from Same Sky Devices of Lake Oswego, Oregon USA). Rotary encoder 1930 was configured to track the position of the gear train to create a closed loop system. Second driven gear 1910' is connected to an inner wheel 1932 of rotary encoder 1930 via a press fit. An encoder housing 1936 was mounted (via connectors as known in the mechanical connector arts) to biaxial stage 1130. Encoder housing 1936 secured encoder 1930 and second driven gear 1920'.

[0101] As, for example, illustrated in FIG. 15E, in the embodiment of strain system 1100, actuator body 1700 included extending section 1710, which extended downward (in the illustrated orientation), passing between first driven gear 1920 and second driven gear 1920'. Extending section had a generally frustoconical shape in the illustrated embodiments. Pegs, projections or projecting members 1730 (see FIG. 15B) extended downward from a lower surface of extending section 1710 to cooperate with passages 1630 in strain body 1600 of a strain interface or strain-inducing interface 1400 of strain system 1100. Strain interface 1400 further included a guide body which operated in a similar manner to guide body 500. A housing or cover section 1950, including an opening 1960 therein, was used to apply compressive force to actuator body 1700 to retain actuator body 1700 in operative connection with first and second driven gears 1920 and 1920 and with strain body 1600. In the illustrated embodiment,Attorney Docket No. 25-003PCTcover section 1950 included extending legs 1970 having passages or openings therein for connection to cooperating passages in biaxial stage 1130 via connectors as known in the mechanical connector arts. Passage or opening 1960 of cover section 1950, passage or opening 1620 of strain body 1600, passage or opening 1520 of guide body 1500 align upon assembly of strain system 1100 to facilitate imaging as well as access to cassette 1300. See, for example, FIGS. 15B and 15C.

[0102] In operating strain system 1100, actuator 1700 was connected to strain body 1600 via projecting members 1730 in a manner similar to which actuator 700 is placed in connection with strain body 600. By driving first driven gear 1920, actuator 1700 and strain body 1600 are driven, and cassette 1300 (including a sample mounted thereof) is strained as described herein. Cassette 1300 may be essentially identical to cassette 300. Use of stepper motor 1910 with a 5:1 gear reduction allowed accurate control over the rotation of actuator 1700. Using gear reduction increases the torque while decreasing the speed of rotation of actuator 1700, thereby enabling more precise movement. Second drive gear 1920' and rotary encoder 1930 enable tracking of the position of actuator 1700. Motor 1910 and rotary encoder 1930 were both controlled via the Arduino UNO, and custom programming which ran on a loop that first checked to see if there is an input source. The program then checked the current position of rotary encoder 1930. The source of input could, for example, be from a controller such as a wired joystick, but it is more effective to use serial commands to set a target position. When a target position was identified, the program activated a subprogram that ran the motor while checking position data to stop the motor once the target position had been met within a tolerance of 0.1 degrees. The program then reset and wait for the next source of input. To integrate the strain system hereof with a microscopes, a custom MicroManager script that sends serial commands and then captures an image once the target position has been reached was used.

[0103] As described above, by rotating strain body 1600, one changes the position of the cassette pins and applies a specific amount of strain to the sample at the center of cassette 1300. A number of design embodiments of strain slot hereof included arcs that connected the start and end points but were not linear along the full trajectory as the strain guide was rotated.Attorney Docket No. 25-003PCTIn a number of studied embodiments, strain slot design (for example, the design of strain slots 1610 in of strain body 1600 and strain slots 1610' of strain body 1600' illustrated in FIG. 15H) to accurately control the angular position along the full trajectory as the strain body is rotated between the start point and the end point. Rotary encoder 1930, mounted into the gear train of the powered or driven actuator system 1900, provides real-time position data. Creating strain slots into strain bodies hereof may be based on the movement of a stepper motor coupled into the gear train. Such strain slots may, for example, provide a constant rate of radial displacement per rotational displacement (for example, 0.1 mm per degree of rotation in the embodiment of FIG. 15H). That type of arc geometry is also known as an " Archimedean spiral." Custom strain bodies hereof may readily implement a range of different types of strain including uniaxial, equibiaxial, anisotropic biaxial, or specific shear strains based on the independent radial displacement of each of the four cassette pins. By using, for example, rotary encoder 930, stepper motor 1910 and a customized strain body, strain systems 1100 and other strain systems hereof can create a specific cumulative strain at any point along the trajectory as the strain body is rotated. Such a design provides the user with increased capabilities using fewer strain bodies. Experimental studies may be designed that image samples at variable levels of strain or strain rates without the need to swap strain bodies.

[0104] Characterization with a strain-predictive finite element model (see Experimental section) demonstrated that an ideal cassette generates strain with a linear relationship to displacement. Using seven datapoints obtained from the finite element model, a linear curve was fit to represent strain as a function of peg displacement as illustrated in FIGS. 16A through 16D. FIG. 16E sets forth the coordinate system used in defining strain relative to cassette 1300. Terminology and equations used in determining, for example, cumulative strain and strain rate are set forth in FIG. 17.

[0105] To further validate the strain systems hereof, nine experiments were performed with bead-coated cassettes. In that regard, fluorescent beads bound to the PDMS surface of the cassette were tracked. The resulting images were processed using Strain MapperJ, a custom macro, which can track the fluorescent bead positions over time and calculate the 2D strain field, e.g., a strain map was used. StrainMapperJ (available via GitHub) is a software toolAttorney Docket No. 25-003PCTdesigned for visualizing and quantifying the mechanics of deforming tissues, which is often used in biological research to analyze kinematic features such as strain. Each cassette was displaced from an original distance of 28.25 mm up to 32.75 mm measured axially between each opposed pair of cassette pegs. This displacement achieved a maximum first principal strain of 0.83 at the sample region of the PDMS.

[0106] The linear curves defined by the finite element model were compared with the experimental data using root mean squared error (RMSE) as illustrated in FIGS. 16A through 16D. See H. Kamble et al., " An electromagnetically actuated double-sided cell-stretching device for mechanobiology research," Micromachines, vol. 8, no. 8, p. 256 (2017). It was found that the RMSE value of the first principal strain was 0.15. The strain along the x and y axis yielded RMSE values of 0.27 and 0.29, respectively. Shear strain had the lowest RMSE value of 0.089.

[0107] The data set forth in FIGS. 16A through 16D illustrates four types of strain and a biaxial cassette with X and Y vectors to define the coordinate system (see FIG. 16E). Each of FIGS. 16A through 16D illustrates a different type of cumulative strain. XX and YY are the cumulative strain measured along the X and Y axes, respectively. Those strains could also be referred to as "normal strain" because the XX strain occurs normal to the YZ plane and the YY strain occurs normal to the XZ plane. Alternatively, the XY strain represents "shear strain" and is the cumulative strain measured parallel to the XY plane. Another way of understanding how shear and normal strain relate to each other is using the strain tensor.εxxεxyεxzεxxεxy0ε = εyxεyyεyzε = εyxεyy0εzxεzyεzz0 0 0

[0108] One can simplify the strain tensor to two dimensions by setting the third row and column to zero because the deformation along the z-axis is assumed to be negligible. The principal strain of FIG. 16A, is calculated using the strain tensor and represents a theoretical maximum strain that occurs during the loading condition.ε1, ε2Attorney Docket No. 25-003PCT

[0109] The equation above can be used to calculate the first and second principal strains where the first principal strain represents a maximum strain and the second principal strain represents a minimum strain. In both cases, the equation is derived by setting the shear strain to zero using a transformation matrix. The first principal strain represents the ideal equibiaxial strain where the sample is only subjected to equal amounts of cumulative strain in the X and Y directions.

[0110] The experimental validation of strain systems hereof indicates that the strain generated at the center of each cassette is similar to the simulation. As described above, RMSE was used to quantitatively determine the fitness of the data to the model. A goal for strain systems hereof is to have an RMSE value of less than 0.10 for each of the strain types. While similar, the RMSE values were higher than expected in the nonoptimized systems which were studied. Moreover, the respective error in the XX and YY strains is likely due to misalignment of the cassette axis to the image axis, causing a lower measured strain. This analysis does not apply to the first principal strain, which accumulates all the loading conditions into one maximum strain. Error in the first principal strain is potentially occurring because of overly broad assumptions in the finite element model.

[0111] The design of cassettes hereof was demonstrated to withstand large deformation. The strain systems hereof are compatible with both high resolution confocal and stereo microscopes. The validation methods described above were proven to be a useful measure of system parameters and will continue to be used to optimize performance, experimental conditions, and models.

[0112] Living tissues can readily be attached to the cassettes hereof, which can be swapped in and out with ease. Actuators can be easily assembled to rotate the gear and stretch the cassette with the tissues attached. In several studied embodiments, the acrylic plates were laser cut pieces which can be customized to generate different strain profiles (for example, isotropic biaxial strain (FIGS. 5A and 5C; 1:1,1:2), anisotropic biaxial strain (shear strain; FIG. 5B 1:2), etc.) on the sample. In addition, the acrylic plates can be easily swapped to enable the user to perform a wide variety of different experiments without extensive changes to the systems hereof. The modular design of the strain systems hereof enables, for example, one toAttorney Docket No. 25-003PCTincorporate high-resolution confocal microscopy with live imaging of multiple samples, while keeping the cost of fabrication low.

[0113] Polymer, metals, and other materials may be used in fabricating the components of systems hereof. Use of polymers, however, assists in achieving low weight and relatively low fabrication cost. Use of 3-D printing of polymeric components further facilitates relatively low fabrication cost and flexibility in production. The dimensions and shapes of the base of straininducing systems or strain systems hereof and / or other components hereof are readily adjustable to function with, for example, a particular microscope / microscope stage and for a range of predetermined uses. In a number of embodiments, representative strain system 100 weight less than 200 g. Such a light weight allows systems hereof to be used with a precise z- stage controller (piezo or galvo) on an inverted brightfield or confocal light microscope. The modular design, including exchangeable cassettes, allows rapid sample exchange. The uniaxial or biaxial stretching cassette is integrated with a modifiable strain interface (for example, include exchangeable 3D cut acrylic plate as guide bodies and strain bodies) which encodes a defined trajectory through a rotational mechanism to create, for example, symmetric or asymmetric biaxial stretching. The system hereof enable manual or computer controlled biaxial tissue stretching to 100% strain, 200% strain, and beyond while keeping the tissue sample within the 150 micrometer working distance of the microscope objective.

[0114] Experimental

[0115] Strain system fabrication of studied embodiments. A number of studied embodiments of strain systems hereof included five main components: a cross-shaped cassette (see, for example, Fig. 1A), a motorized drive and gear system, a customized microscope stage, and strain-programmed cams or strain interfaces (that is, strain bodies and guide bodies) (see, for example, Fig. IB). The system functions using an interchangeable strain-programmed four- follower face cam mechanism to deform an easily swapped sample-carrying, elastic cassette.

[0116] Cassette fabrication. Cassettes hereof were designed as flexure mounts for samples including cell or tissue samples. In the studied embodiments, each cassette was a solid component assembled from 5 pieces of polyester (PES) shim, a single piece of polydimethylsiloxane (PDMS), and 4 extending abutment members or pegs for interconnectionAttorney Docket No. 25-003PCTwith a strain interface including a strain body and a strain guide. Shim and PDMS were custom cut on a vinyl cutter. The abutment member assemblies were fabricated by a 3D resin printer. Cut shim and PDMS pieces were cleaned with acetone and ethanol before being bonded by ultraviolet (UV) activated glue. The glue was also used to attach the abutment member assemblies to the cassette. After assembly, the entire cassette was cured under UV light and then cleaned again with acetone and ethanol. Upon assembly, each single-use cassette included a clean PDMS membrane sandwiched between two layers of PES for stability and four extending member glued to the top face. A cross-shape section of PDMS was exposed at the center of the cassette where samples may be mounted after additional preparation.

[0117] Application of strain to cassettes. To apply strain to the cassettes, the abutment members of the cassettes were positioned within the strain slots of the strain body and strain guide. The strain body and strain guide function as two strain-programmed cams which define the motion of each peg, the linear cam (strain guide), and the strain cam (strain body). The strain cam or strain body was designed with four parametrically curved strain slots, while the linear cam was designed to have four straight perpendicular guide slots. After aligning and projecting the four abutment members of the cassette into different slots in the two, cams which are stacked on top of each other, the strain cam or strain body is rotated to move the cassette abutment member at a rate defined by the curve of the strain slot. The linear cam or strain guide remains stationary and defines a linear path that constrains the motion of each extending abutment member. By stacking the strain guide and strain body of the strain interface and positioning the cassette abutment member to fit through each of the slots, the rotation of the strain guide will determine the velocity of each abutment member, and the linear strain guide will determine the direction in which each extending member moves. The strain guide and the strain body were cut out of an acrylic sheet using a CO2laser and aligned within a 3D printed housing.

[0118] Finite element analysis. A strain-predictive finite element model of the strain system hereof was developed using Autodesk Fusion, which is a cloud-based, product development platform used for 3D design, engineering, and manufacturing, integrating CAD, CAM, CAE, and ECAD tools, which is available from Autodesk of San Francisco, California. A goal of the modelAttorney Docket No. 25-003PCTwas to develop a baseline against which one can test the performance of experiments and identify any required improvements in, for example, system design and / or experimental setup. In was assumed that the extending abutment members mounted to the cassette follow the linear trajectory defined by the strain guide. In addition, it was assumed that out of plane motion is limited and that all pieces of the cassette are perfectly bonded together. Because the model is used as a perfect representation of the cassette function with live samples, it was anticipated that optimal strain fields would be centered on the PDMS. Thus, simulated finite element model performance at the center point of the cassette was analyzed.

[0119] Fluorescent bead validation. To validate the linearity of the strain field, displacement of fluorescent beads bound to the PDMS surface of the cassette was tracked. To prepare bead- coated PDMS cassettes, the surface was first activated with oxygen plasma by placing cassettes into an oxygen plasma cleaner for 2 minutes. Immediately after removing the cassettes, a desired or predetermined coating was added. In the case of fluorescent beads, 5 pL of a slurry made of one drop 10 pm fluorescent beads (Duke Scientific) and 1 ml ethanol was applied. The bead-coated cassettes were dried in a dust free chamber.

[0120] To evaluate the performance of the cassettes and strain system, bead-coated regions on the cassette were stretched to radial displacements defined by the strain body and imaged at regular intervals. A goal was to stretch each cassette to a maximum displacement of 4.5 mm, which was measured axially between each opposed pair of cassette abutment members. To achieve that displacement, a strain guide that encoded a rotation of a 0.1 mm / degree strain over a 45 degree rotation was constructed (see FIG. 15H). The motor was paused every 3 degrees so that images could be collected with a fluorescence stereomicroscope. To automate the rotation and capture images, a custom Micro-Manager script was used to communicate with an Arduino Uno. Micro-Manager is a software package for control of automated microscopes (micro-manager.org). The custom script was written to automate both microscope and stretcher operation. The script oversees communication between the stretcher, the Arduino Uno microcontroller, and the Micro-Manager software.

[0121] Image analysis. The images collected from the fluorescent bead validation were analyzed using ImageJ. Image] (available via GitHub) is a free, open-source Java-based softwareAttorney Docket No. 25-003PCTdesigned for scientific image processing and analysis, which was developed by the National Institutes of Health (NIH). Digital correlation microscopy using StrainMapperJ. Before processing with StrainMapperJ, the images were cropped to a region of interest that represented a central region on the PDMS at which it was anticipated biological samples would be tracked. The images were then averaged and plotted to understand the relationship between cassette displacement and the sample strain.

[0122] The foregoing description and accompanying drawings set forth a number of representative embodiments at the present time. Various modifications, additions and alternative designs will, of course, become apparent to those skilled in the art in light of the foregoing teachings without departing from the scope hereof, which is indicated by the following claims rather than by the foregoing description. All changes and variations that fall within the meaning and range of equivalency of the claims are to be embraced within their scope.

Claims

Attorney Docket No. 25-003PCTClaims1. A strain-inducing system for applying strain to a sample deposited upon a sample substrate, the sample substrate being stretchable, comprising:a cassette comprising substrate interface members, the substrate interface members being spaced from each other to create a gap therebetween, each of the substrate interface members being configured to be attached to a separate area of a surface of the sample substrate so that a portion of the sample substrate extends across the gap, one or more of the substrate interface members comprising an abutment member extending therefrom;a base having a sample chamber configured to removably receive the cassette therein, a strain-inducing interface configured to be placed in connection with the base and with the cassette, the strain-inducing interface comprising a strain body comprising an extending strain path, the extending strain path including an extending strain surface which extends in a predetermined path around an axis of the strain body, the extending strain surface being configured to be placed in abutting contact with the abutment member, wherein rotation of the strain body relative to the cassette about the axis of the strain body causes a change in a size of the gap when the abutment member is in contact with the extending strain surface, wherein the change in size of the gap caused by rotation of the strain body is determined by change in radial position of the extending strain surface as it extends around the axis of the strain body and the degree of angular rotation of the strain body about the axis of the strain body.

2. The system of claim 1 wherein the strain-inducing interface further comprises a guide body, the guide body comprising an extending guide path, the extending guide path include an extending guide surface which extends in a predetermined path, the extending guide surface extending generally linearly, the extending guide surface being configured to be placed in abutting contact with the abutment member when the strain body and the guide body are placed in predetermined positions relative to each other and in connection with an assembly of the base and the cassette such that the strain body is rotatable relative to the guide body and to the cassette, wherein the abutment member extends from the substrate interface member to firstAttorney Docket No. 25-003PCTbe placed in abuting contact with one of the extending strain surface and the extending guide surface, and then to be placed in abutting contact with the other of the extending strain surface and the extending guide surface, the extending guide surface constraining the abutment member to generally linear motion.

3. The system of claim 2 wherein each of a plurality of the substrate interface members comprises an abutment member extending therefrom and the strain body comprises a plurality of extending strain paths, each of the plurality of extending strain paths including an extending strain surface which extends in a predetermined path around the axis of the strain body, each of the extending strain surfaces being configured to be placed in abutting contact with an associated one of the abutment members, wherein rotation of the strain body relative to the cassette about the axis of the strain body causes a change in a size of the gap when each of the of extending strain surfaces is in abutting contact with the associated one of the abutment members, wherein the change in size of the gap caused by rotation of the strain body about the axis of the strain body is determined by change in radius of each of the plurality of extending strain surfaces as it extends around the axis of the strain body and the degree of angular rotation of the strain body about the axis of the strain body.

4. The system of claim 3 wherein the strain-inducing interface further comprises a guide body, and the guide body comprises a plurality of extending guide paths, each of the plurality of extending guide paths comprising an extending guide surface which extends in a predetermined path, the extending guide surfaces extending generally linearly, each of the extending guide surface being configured to be placed in abutting contact with an associated one of the abutment members when the strain body and the guide body are placed in predetermined positions relative to each other and in connection with an assembly of the base and the cassette such that the strain body is rotatable relative to the guide body and to the cassette, wherein each of the abutment members extends from one of the substrate interface member to first be placed in abutting contact with one of the extending strain surfaces or one the extending guide surfaces, and then to be placed in abutting contact with the other of one of the extending strain surfaces and one of the extending guide surfaces, each of the extending guide surface constraining the associated one of the abutment member to generally linear motion.Attorney Docket No. 25-003PCT5. The system of claim 4 wherein each of the plurality of extending strain paths further comprises another extending strain surface which extends generally parallel to the extending strain surface to form an extending strain slot, and each of the plurality of extending guide members comprises another extending guide surface which extends generally parallel to the extending guide surface to form an extending guide slot, such that the strain body comprises a plurality of extending strain slots and the guide body comprises a plurality of extending guide slots.

6. The system of claim 5 wherein the guide body is placed adjacent to the cassette and between the cassette and the strain body, so that each of the abutment members extends through one of the plurality of extending guide slots and into one of the plurality of extending strain slots.

7. The system of claim 6 wherein the base is configured to be placed in connection with the guide body and with the strain body.

8. The system of claim 7 wherein the base comprises a guide body seating into which the guide body is positioned over the cassette such that each of the abutment members extends through a different one of the extending guide slots, the guide body seating preventing the guide body from rotating relative to the base.

9. The system of claim 8 wherein the base comprises a strain guide seating into which the strain body is rotatably positioned over the guide body such that each of the abutment members extends into a different one of the plurality of extending strain slots.

10. The system of any one of claims 5 through 9 wherein the cassette comprises four substrate interfaces, each of the substrate interfaces having one of the abutment members extending therefrom, the guide body comprises four extending guide slots, and the strain body comprises four extending strain slots.

11. The system of claim 10 wherein each of the substrate interfaces extends to form a cross shape, each of the substrate interfaces comprising an inner section to which the sample substrate is attached and an outer section from which one of the abutment members extends.

12. The system of claim 11 wherein each outer section of each substrate interface is connected to each adjacent outer section by an extendable member, the extendable member providing resistance to flexure of the substrate interfaces out of a plane defined by an original configuration thereof.Attorney Docket No. 25-003PCT13. The system of claim 10 further comprising an actuator body in connection with the strain body to impart rotational motion to the strain body.

14. The system of claim 13 wherein the actuator body is a manually actuated.

15. The system of claim 13 wherein the actuator body is actuated in a powered manner.

16. The system of claims 15 wherein the strain system further comprises a motor to power the actuator body and a system configured to determine position of the actuator body.

17. The system of claim 15 wherein the strain system further comprises a motor, and the actuator body comprises gear teeth which are configured to be placed in connection with a first gear which is driven by the motor.

18. The system of claim 16 wherein the strain system further comprises a second gear which is in connection with the gear teeth of the actuator body and in connection with a system configured to determine the position of the actuator body, wherein optionally the system configured to determine the position of the actuator body comprises an encoder19. The system of claim 18 further comprising electronic circuitry in connection with the motor and with the encoder to control the position of the actuator body over time.

20. The system of claim 10 wherein the plurality of extending strain slots is configured to create one of isotropic and anisotropic biaxial strain in the sample substrate.

21. The system of any one of claims 1 through 20 wherein the system is configured to be placed in connection with a stage of a microscope so that an objective of the microscope is aligned with the sample, and the sample is viewable via the objective during straining.

22. The system of any one of claims 1 through 20 wherein the sample chamber is adapted to contain a volume of a liquid therein.

23. The system of claim 22 wherein the system is configured to be placed in connection with a stage of a microscope so that an objective of the microscope is aligned with the sample, which is a biological sample, and the sample is viewable via the objective during straining.

24. A method of inducing strain in a sample, comprising:providing a strain-inducing system as described in any one of claims 1 through 20, attaching the sample to the sample substrate,Attorney Docket No. 25-003PCTattaching the sample substrate to the substrate interface member of the cassette,placing the cassette in the sample chamber of the base,placing the strain body in connection with the cassette, androtating the strain body of the system relative to the base and to the cassette.

25. The method of claim 24 wherein the system is configured to be placed in connection with a stage of a microscope so that an objective of the microscope is aligned with the sample, and the sample is viewable via the objective during straining.

26. The method of claim 24 wherein the sample chamber is adapted to contain a volume of a liquid therein.

27. The method of claim 26 wherein the system is configured to be placed in connection with a stage of a microscope so that an objective of the microscope is aligned with the sample, which is a biological sample, and the sample is viewable via the objective during straining.

28. A cassette for use in a system in which a substrate is strained, comprising: four substrate interfaces, each of the substrate interfaces having an abutment member extending therefrom, each of the four substrate interface members being spaced from each other to create a gap therebetween, each of the substrate interface members being configured to be attached to a separate area of a surface of the substrate so that a portion of the substrate extends across the gap, wherein each of the four substrate interface member is independently movably relative to the other substrate interface members via application of force to the abutment member extending therefrom to strain the portion of the substrate extending across the gap.

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