WO2025019681A2

WO2025019681A2 - Devices, systems, and methods for imparting mechanical strain to samples including biological samples

Info

Publication number: WO2025019681A2
Application number: PCT/US2024/038560
Authority: WO
Inventors: Lance A. DAVIDSON; Emily Irene HEARTY; Jing Yang; Timothy N. WALTER; Yingli Wang
Original assignee: University Of Pittsburgh - Of The Commonwealth System Of Higher Education
Priority date: 2023-07-19
Filing date: 2024-07-18
Publication date: 2025-01-23
Also published as: WO2025019681A3

Abstract

A system includes a cassette including a. first end member spaced from a second end member and a sample substrate, which is stretchable, connected between the first end member and the second end member so that a. portion of the sample substrate extends across a gap between the first end member and the second end member. The sample substrate is adapted to have a sample deposited on the portion thereof which extends across the gap. The first end member includes a first cooperating abutment member. The system also includes an assembly including a body having a sample chamber to removably receive the cassette therein and a cassette interface. The cassette interface includes an abutment member extending to contact the first cooperating abutment member. The system further includes a first actuator in connection with the body to impart motion to the first cooperating abutment member.

Description

DEVICES, SYSTEMS, AND METHODS FOR IMPARTING MECHANICAL STRAIN TO SAMPLES INCLUDING BIOLOGICAL SAMPLES

GOVERNMENTAL INTEREST

[0001] This invention was made with government support under grant numbers HD044750, HL127711; and HL136566 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 Serial No. 63/514,464, filed July 19, 2023, the disclosure of which is incorporated herein by reference.

BACKGROUND

[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 micro environment 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 in other 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 rises to cases of cardiac edema. Another study found that lack of tissue strain during gastrulation disrupted planar-cellpolarity in the ciliated epithelium of Xenopus embryos; importantly, this 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 solid-like 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. High 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] Previously developed 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 consist of wire cantilevers. More sophisticated uniaxial or biaxial stretchers use clamps or posts to bond tissues to motorized actuators. Clamp- and actuatorbased 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 with seeded cells or tissues 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, which can limit access for high resolution optics.

[0009] 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, compressing 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, required a systematic analysis. Although it is desirable to develops devices, systems, and method 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. SUMMARY

[0011] In one aspect, a system for applying strain to a sample includes a cassette including a first end member spaced from a second end member and a sample substrate, which is stretchable, connected between the first end member and the second end member so that a portion of the sample substrate extends across a gap between the first end member and the second end member. The sample substrate is adapted to have the sample deposited on the portion thereof which extends across the gap. The first end member includes a first cooperating abutment member. The system also includes an assembly including a body having a sample chamber or volume to removably receive the cassette therein and a cassette interface. The cassette interface includes a first abutment member which extends to contact the first cooperating abutment member when the cassette is in the sample chamber. The system further includes a first actuator in connection with the body. The first actuator includes a first movable actuator arm configured to be placed in connection with the first abutment member to impart motion to the first abutment member and thereby impart motion to the first cooperating abutment member when the cassette is in the sample chamber to control a width of the gap and thereby a width of the portion of the sample substrate extending across the gap. In a number of embodiments, the cassette interface is removably connectible or operatively connectible to the body.

[0012] The second end member may include a second cooperating abutment member, and the cassette interface may include a second abutment member which extends to contact the second cooperating abutment member when the cassette is in the sample chamber. Likewise, the system may further include a second actuator in connection with the body. The second actuator includes a second movable actuator arm configured to be placed in connection with the second abutment member to impart motion to the second abutment member and thereby impart motion to the second cooperating abutment member when the cassette is in the sample chamber, and to control the width of the gap and thereby the width of the portion of the sample substrate extending across the gap.

[0013] In a number of embodiments, the sample chamber is adapted to contain a volume of a liquid therein. The sample chamber may, for example, includes a transparent bottom section. The body may be adapted to be placed in operative connection with a stage of a microscope so that an objective of the microscope is aligned with the sample deposited upon the sample substrate via the transparent bottom section.

[0014] The first end member of the cassette and the second end member may include at least one extendible member connected therebetween. The extendible member is configured to provide resistance to torsional motion of the first end member relative to the second end member.

[0015] In a number of embodiments, the first end member of the cassette includes a first upper member attached to a first lower member, and the second end member includes a second upper member attached to a second lower member. The first lower member of the first end member and the second lower member of the second end member may be connected via two spaced extendible members extending therebetween,. The two spaced extendible members providing resistance to torsional motion of the first end member relative to the second end member. In a number of embodiments, a first end of the sample substrate is positioned between the first upper member, the first lower member and a second end of the sample substrate is positioned between the second upper member and the second lower member, and the sample substrate extends across a gap or space between the two spaced extendible members. The first end of the sample substrate may optionally be attached to at least one of the first upper member and the first lower member via an adhesive. Similarly, the second end of the sample substrate may optionally be attached to at least one of the second upper member and the second lower member via an adhesive. The first upper member and the first lower member may optionally be attached via an adhesive, and the second upper member and the second lower member may optionally be attached via an adhesive. In a number of embodiments, the first lower member, the second lower member, and the spaced extendible members are formed monolithically. The second end member may include a second cooperating abutment member,. The first cooperating abutment member may, for example, extend upward from an upper surface of the first upper member, and the second cooperating abutment member may extend upward from an upper surface of the second upper member. In a number of embodiments, the first upper member and the first lower member of the first end member and second upper member and the second lower member of the second end member have a thickness in the range of 38 - 127 μm.

[0016] The cassette interface may, for example, include a first end, a second end, a first flexible crossbeam extending between the first end and the second end, and a second flexible crossbeam extending between the first end and the second end and spaced from the first flexible crossbeam. The first abutment member may extend downward from the first flexible crossbeam. The second abutment member, when present, may extend downward from the second flexible crossbeam. The first moveable actuator arm may be configured to contact the first crossbeam. The second moveable actuator arm, when present, may be configured to contact the second crossbeam. In a number of embodiments, the first actuator includes a first piezo linear actuator and the second actuator includes a second piezo linear actuator.

[0017] The system may further include a control system in communicative connection with the first actuator and in communicative connection with the second actuator, when present. The control system may include a processor system and a memory system in operative connection with the processor system. The memory system may include one or more algorithms stored therein which are executable by the processor system (to, for example, effect control of the first actuator and the second actuator).

[0018] In embodiments in which the body may be adapted to be placed in operative connection with a stage of a microscope, the microscope may, for example, be an inverted brightfield or confocal microscope. The system may further include a control system in communicative connection with the stage of the microscope. The control system may include a processor system and a memory system in operative connection with the processor system. The memory system may have one or more algorithms stored therein which are executable by the processor system to control a position of the stage of the microscope to align the objective with a determined portion of the sample during application of strain.

[0019] In general, the sample may be any composition or object that may be strained via strain of the sample substrate upon which the sample is deposited. The sample may a biological sample or a non-biological sample. A non-biological sample may be a natural sample or a synthetic sample. In the case of a biological sample, the sample may be a living sample (for example, live tissue or live cells).

[0020] The strain applied by the system may include at least one of extension strain, compressive strain, and shearing strain. Combinations of such strains may be applied in a controlled manner using the system hereof.

[0021] In another aspect, a method of stretching a sample includes providing a system as described herein. In that regard, the system may include a cassette including a first end member spaced from a second end member and a sample substrate, which is stretchable, connected between the first end member and the second end member so that a portion of the sample substrates extends across a gap between the first end member and the second end member. The sample substrate is adapted to have the sample deposited on the portion thereof which extends across the gap, the first end member including a first cooperating abutment member. The system also includes an assembly including a body having a sample chamber to removably receive the cassette therein, and a cassette interface. The cassette interface includes a first abutment member which extends to contact the first cooperating abutment member when the cassette is in the sample chamber. The system further includes a first actuator in connection with the body. The first actuator includes a first movable actuator arm configured to be placed in connection with the first abutment member to impart motion to the first abutment member and thereby impart motion to the first cooperating abutment member when the cassette is in the sample chamber to control a width of the gap, and thereby control a width of the portion of the sample substrate extending across the gap.

[0022] The method further includes inserting the cassette including the sample in the sample chamber, placing the first abutment member in operative connection with the first cooperating abutment member after the cassette is inserted in the chamber, and imparting motion to the first abutment member via the first actuator.

[0023] As described above, the second end member may include a second cooperating abutment member, and the cassette interface may include a second abutment member which extends to contact the second cooperating abutment member when the cassette is in the sample chamber. Likewise, the system may further include a second actuator in connection with the body. The second actuator includes a second movable actuator arm configured to be placed in connection with the second abutment member to impart motion to the second abutment member and thereby impart motion to the second cooperating abutment member when the cassette is in the sample chamber, and to control the width of the gap and thereby the width of the portion of the sample substrate extending across the gap.

[0024] In a number of embodiments, the sample chamber is adapted to contain a volume of a liquid therein. The sample chamber may, for example, includes a transparent bottom section. The body may be adapted to be placed in operative connection with a stage of a microscope so that an objective of the microscope is aligned with the sample deposited upon the sample substrate via the transparent bottom section. [0025] The first end member of the cassette and the second end member may include at least one extendible member connected therebetween. The extendible section is configured to provide resistance to torsional motion of the first end member relative to the second end member.

[0026] In a number of embodiments, the first end member of the cassette includes a first upper member attached to a first lower member, and the second end member includes a second upper member attached to a second lower member. The first lower member of the first end member and the second lower member of the second end member may be connected via two spaced extendible members extending therebetween,. The two spaced extendible members providing resistance to torsional motion of the first end member relative to the second end member. In a number of embodiments, a first end of the sample substrate is positioned between the first upper member, the first lower member and a second end of the sample substrate is positioned between the second upper member and the second lower member, and the sample substrate extends across a gap between the two spaced extendible members. The first end of the sample substrate may optionally be attached to at least one of the first upper member and the first lower member via an adhesive. Similarly, the second end of the sample substrate may optionally be attached to at least one of file second upper member and the second lower member via an adhesive. The first upper member and the first lower member may optionally be attached via an adhesive, and the second upper member and the second lower member may optionally be attached via an adhesive. In a number of embodiments, the first lower member, the second lower member, and the spaced extendible members are formed monolithically. The second end member may include a second cooperating abutment member,. The first cooperating abutment member may, for example, extend upward from an upper surface of the first upper member, and the second cooperating abutment member may extend upward from an upper surface of the second upper member. In a number of embodiments, the first upper member and the first lower member of the first end member and second upper member and the second lower member of the second end member have a thickness in the range of 38 - 127 μm.

[0027] The cassette interface may, for example, include a first end, a second end, a first flexible crossbeam extending between the first end and the second end, and a second flexible crossbeam extending between the first end and the second end and spaced from the first flexible crossbeam. The first abutment member may extend downward from the first flexible crossbeam. The second abutment member, when present, may extend downward from the second flexible crossbeam. The first moveable actuator arm may be configured to contact the first crossbeam. The second moveable actuator arm, when present, may be configured to contact the second crossbeam. In a number of embodiments, the first actuator includes a first piezo linear actuator and the second actuator includes a second piezo linear actuator.

[0028] The system may further include a control system in communicative connection with the first actuator and in communicative connection with the second actuator, when present. The control system may include a processor system and a memory system in operative connection with the processor system. The memory system may include one or more algorithms stored therein which are executable by the processor system (to, for example, effect control of the first actuator and the second actuator).

[0029] In embodiments in which the body may be adapted to be placed in operative connection with a stage of a microscope, the microscope may, for example, be an inverted brightfield or confocal microscope. The system may further include a control system in communicative connection with the stage of the microscope. The control system may include a processor system and a memory system in operative connection with the processor system. The memory system may have one or more algorithms stored therein which are executable by the processor system to control a position of the stage of the microscope to align the objective with a determined portion of the sample during application of strain.

[0030] As described above, the sample may be any composition or object that may be strained via strain of the sample substrate upon which the sample is deposited. The sample may a biological sample or a non-biological sample. A non-biological sample may be a natural sample or a synthetic sample. In the case of a biological sample, the sample may be a living sample (for example, live tissue or live cells). Further, the strain applied by the system may include at least one of extension strain, compressive strain, and shearing strain. Combinations of such strains may be applied in a controlled manner using the system hereof.

[0031] In another aspect, a microscope system includes a microscope including a stage and an objective. The microscope system further includes a strain system for applying strain to a sample. The strain system includes a cassette including a first end member spaced from a second end member and a sample substrate, which is stretchable, connected between the first end member and the second end member so that a portion of the sample substrates extends across a gap between the first end member and the second end member. The sample substrate is adapted to have the sample deposited on the portion thereof which extends across the gap, the first end member including a first cooperating abutment member. The system also includes an assembly including a body having a sample chamber to removably receive the cassette therein, and a cassette interface. The cassette interface includes a first abutment member which extends to contact the first cooperating abutment member when the cassette is in the sample chamber. The system further includes a first actuator in connection with the body. The first actuator includes a first movable actuator arm configured to be placed in connection with the first abutment member to impart motion to the first abutment member and thereby impart motion to the first cooperating abutment member when the cassette is in the sample chamber to control a width of the gap, and thereby control a width of the portion of the sample substrate extending across the gap.

[0032] The second end member may include a second cooperating abutment member, and the cassette interface may include a second abutment member which extends to contact the second cooperating abutment member when the cassette is in the sample chamber. Likewise, the system may further include a second actuator in connection with the body. The second actuator includes a second movable actuator arm configured to be placed in connection with the second abutment member to impart motion to the second abutment member and thereby impart motion to the second cooperating abutment member when the cassette is in the sample chamber, and to control the width of the gap and thereby the width of the portion of the sample substrate extending across the gap.

[0033] In a number of embodiments, the sample chamber is adapted to contain a volume of a liquid therein. The sample chamber may, for example, includes a transparent bottom section. The body may be adapted to be placed in operative connection with the stage of a microscope so that the objective of the microscope is aligned with the sample deposited upon the sample substrate via the transparent bottom section.

[0034] The microscope may, for example, be an inverted brightfield or confocal microscope. The strain system may be further characterized as described herein.

[0035] In a further aspect, a cassette for use in applying strain to a sample includes a first end member spaced from a second end member. The first end member includes a first upper member attached to a first lower member, and the second end member includes a second upper member attached to a second lower member. The first lower member of the first end member and the second lower member of the second end member are connected via two spaced extendible members extending therebetween. The two spaced extendible members provides resistance to torsional motion of the first end member relative to the second end member. The cassette further includes a sample substrate, which is stretchable. The sample substrate is connected to the first end member, between the first upper member and the first lower member, at a first end of the sample substrate, and is connected to the second end member, between the second upper member and the second lower member, at a second end of the sample substrate so that a portion of the sample substrate extends across a gap between the first end member and the second end member. The sample substrate is adapted to have the sample deposited on the portion thereof which extends across the gap.

[0036] In a number of embodiments, at least one of the first upper member and the second upper member includes a cooperating abutment member via which force can be applied to the cassette. The first upper member may, for example, include a first cooperating abutment member extending therefrom, and the second upper member may include a second cooperating abutment member extending therefrom, via which force can be applied to the cassette. In a number of embodiments, the at least one of the first upper member, the first lower member, and the first end of the sample substrate therebetween are attached via an adhesive, and at least one of the second upper member, the second lower member, and the second end of the sample therebetween, are attached via an adhesive.

[0037] The first lower member, the second lower member, and the spaced extendible members may, for example, be formed monolithically. The first upper member and the first lower member of the first end member, and second upper member and the second lower member of the second end member may, for example, have a thickness in the range of 38 - 127 μm.

[0038] In a number of embodiments, the first end of the sample substrate, which is connected between the first upper member and the first lower member, is laterally wider that the portion of the sample substrate which extends across the gap, and the second end of the sample substrate, which is connected between the second upper member and the second lower member, is laterally wider that the portion of the sample substrate which extends across the gap.

[0039] 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 THE DRAWINGS

[0040] FIG. 1A illustrates schematically an embodiment of a strain system hereof positioned in operative connection with an inverted microscope.

[0041] FIG. 1 B illustrates an isometric view of the strain system of FIG. 1A for use with a microscope stage assembly.

[0042] FIG. 1C illustrates an isometric exploded view of the strain system of FIG. 1A.

[0043] FIG. 1D illustrates a side exploded view of the strain system of FIG. 1A and an embodiment of a cassette hereof.

[0044] FIG. 1E illustrates a top view of the strain system of FIG. 1A.

[0045] FIG. 1F illustrates a bottom view of the strain system of FIG. 1A.

[0046] FIG. 1G illustrates a side view of the strain system of FIG. 1A.

[0047] FIG. 1H illustrates a front view of the strain system of FIG. 1A.

[0048] FIG. 1I illustrates: panel (a) an enlarged top view photograph of the strain system of FIG. 1A inserted into or onto a microscope stage of a confocal microscope; panel (b) a side perspective view photograph of the strain system inserted into or onto a microscope stage of a confocal microscope; and panel (c) a front perspective view photograph of the strain system inserted into or onto a microscope stage of a confocal microscope.

[0049] FIG. 2A illustrates a top isometric view of a top component of the stage assembly of the strain system of FIG. 1A.

[0050] FIG. 2B illustrates a bottom isometric view of a top component of the stage assembly.

[0051] FIG. 2C illustrates a top view of a top component of the stage assembly.

[0052] FIG. 2D illustrates a front view of a top component of the stage assembly.

[0053] FIG. 3A illustrates a top isometric view of a bottom component of the stage assembly of the strain system of FIG. 1A. [0054] FIG. 3B illustrates a top view of a bottom component of the stage assembly of the strain system of FIG. 1A.

[0055] FIG. 3C illustrates a bottom, hidden line view of a bottom component of the stage assembly.

[0056] FIG. 3D illustrates a front, hidden line view of a bottom component of the stage assembly.

[0057] FIG. 3E illustrates a side, hidden line view of a bottom component of the stage assembly.

[0058] FIG. 4A illustrates an isometric view of a cassette interface of the strain system of FIG. 1A (having the form of and sometimes referred to herein as an H-bridge).

[0059] FIG. 4B illustrates a hidden line, isometric view of the cassette interface.

[0060] FIG. 4C illustrates a hidden line, front view of the cassette interface.

[0061] FIG. 4D illustrates a top view of the cassette interface.

[0062] FIG. 4E illustrates a bottom view of the cassette interface.

[0063] FIG. 4F illustrates a side view of the cassette interface.

[0064] FIG. 5A illustrates: a top disassembled view of an embodiment of a cassette hereof illustrates the abutment blocks, a bottom component or shim which may, for example, be cut from a polymer such as a polyester or PES, a polymeric (for example, polydimethylsiloxane PDMS) sheet used to attach/seed samples such as biological samples, and a top component or shim which may, for example, be cut from a polymer such as a polyester or PES.

[0065] FIG. 5B illustrates an isometric view of the abutment blocks.

[0066] FIG. 5C illustrates a front view of the abutment blocks.

[0067] FIG. 5D illustrates a top view of the assembled cassette.

[0068] FIG. 5E illustrates a bottom view of the assembled cassette.

[0069] FIG. 5F illustrates a top isometric view of the assembled cassette. [0070] FIG. 5G illustrates a front view of the assembled cassette.

[0071] FIG. 5H illustrates a side view of the assembled cassette.

[0072] FIG. 6 illustrates a flowchart for an embodiment of an AutoCenter plugin hereof.

[0073] FIG. 7 illustrates schematically an embodiment of a method of achieving sample stretching using a cassette hereof.

[0074] FIG. 8 illustrates: panel (a) a top-view photograph of the cassette in a relaxed state; panel (b) a bottom-view photograph of the cassette in a relaxed state; panel (c) a top-view photograph of the cassette in a stretched state; and panel (d) a bottom-view photograph of the cassette in a stretched state, wherein the cassette is positioned within the liquid- filled chamber of a strain system hereof in operative connection with a microscope stage.

[0075] FIG. 9A illustrates a method of using an embodiment of explant jig for sample (for example, tissues or cells) attachment.

[0076] FIG. 9B illustrates a side, hidden-line view of the explant jig of FIG. 9A.

[0077] FIG. 10 illustrates an embodiment of a procedure for using a strain system hereof to effect compression of a sample.

[0078] FIG. 11 illustrates the use of an embodiment of a pre-stretch stabilizer hereof to prestretch a cassette hereof.

[0079] FIG. 12 illustrates characterization of the strain profile of a sample sheet or substrate hereof, wherein: panel (a) illustrates a schematic of the sample or substrate sheet coated with fluorescent beads, and a center region and an edge region are indicated on the schematic; panel (b) illustrates fluorescent beads traced at the edge; panel (b’) illustrates fluorescent beads traced at the center locations; wherein arrows show position changes between two beads, and the scale bars = 100 μm; panel (c) illustrates έ_XX strain rate map of the edge; panel (c’) illustrates έ_XX strain rate map of the center of the PDMS substrate, wherein a phase lookup table is applied, and darkened areas (negative numbers) indicate contraction, while lightened areas (positive number) indicate elongation of the material along x-axis; and panel (d) illustrates quantification of έ_XX of edge and center over 0-15, 15-30, 30-45 minutes, wherein the error bars, standard deviation. [0080] FIG. 13 illustrates calculation of the velocity of the edges of the cassette wherein panel (a) illustrates a reslice of the timelapse video of one of the cassette edges, showing a kymograph-like trajectory of the edge traveled in distance (horizontal axis) and time (vertical axis), wherein the edge appeared to travel in a straight line, indicating constant velocity during stretching; panel (b) illustrates a right triangle with an angle θ which was used to calculate the velocity, with the horizontal leg as the distance d and the vertical leg as the time t, wherein the length of the hypotenuse was represented by I; and panel (c) illustrates velocity of one side of the cassette edge which was calculated by dividing distance by time, wherein velocity = 52 ± 3.5 μm/minute, and n = 10.

[0081] FIG 14A illustrates quantification of tissue and cellular strain in Xenopus laevis organotypic explants in a Stage 13 animal cap organotypic explant labeled with membrane- mNeonGreen at relaxed state (left) and stretched state (right), wherein dashed line outlined the region-of-interests, where the same region was traced and imaged through 8 stretch steps. Scale bar = 35 μm. * indicated the same cell before and after stretching.

[0082] FIG. 14B illustrates (top images) a έ_XXT strain rate map overlayed with cell outlines represented continuous tissue strain rate over two consecutive stretch steps, (middle images) a έ_XXC strain rate mapped using individual cellular strain, which strain was calculated based on individual cell shape, with no variation of strain within the single cell. N, 81 cells; and (bottom images) a concordance/ discordance map which was represented by the differences between έ_XXC and έ_XXT, wherein a magenta-cyan phase lookup table was applied, where both magenta and cyan indicated discordance behaviors between tissue and the individual cell, and white represented concordance, and wherein * indicated the same cell across the frames, and D represented cells that divided throughout stretching.

[0083] FIG. 14C illustrates quantification of both tissue and cellular strain rate έ_XX between each two consecutive stretch steps, wherein error bars, standard deviation.

[0084] FIG. 14D illustrates quantification of cumulative tissue and cellular strain ε_XX across the 8 stretch steps, wherein error bars, standard deviation.

[0085] FIG. 14E illustrates tissue-cell concordance/ discordance by plotting differences between έ_XXC and έ_XXT against absolute values of their sum, wherein the bottom region represented insignificant movement of both tissue and the cell, the center region represented tissue and the cell strained concordantly, the right region represented discordance that the cell strained larger than the tissue, and the left region represented discordance that the tissue strained larger than the cell, wherein T = tissue; C = cell. σ_C+T = 0.081 , standard deviation of |έ_XXC| + |έ_xxT|. σ = 0.057, and standard deviation of έ_XXC — έ_XXT.

[0086] FIG. 15A illustrates high-resolution live imaging studies of a Stage 11 animal cap organotypic explant labeled with mem -mN eon Green and keratinS-mCherry at relaxed state (top) and stretched state (bottom), wherein * indicates the same cell across frames, and the last column shows keratin filaments in a single cell, a white broken line outlined the single cell, a long arrow highlights the filament that was tortuous at relaxed state but straightened after stretching, a short arrow highlights the separation between the filaments and the cell junction, and scale bar = 10 μm.

[0087] FIG. 15B illustrates schematic of seeding cardiomyocytes or HUVECs on fibronectin- or collagen-coated cassette, wherein a droplet of suspended cardiomyocytes or HUVECs was pipetted onto the PDMS substrate of the cassette with 500 μL of respective medium at the bottom of the petri dish; after cells attach to the PDMS substrate, 12 mL of medium was added to fully submerge the cassette; and the cassette was flipped back and put into microscope stage insert before imaging.

[0088] FIG. 15C illustrates high-resolution live imaging studies of cardiomyocytes labeled with membrane and nucleus markers were stretched and imaged for 6 stretch steps, wherein white arrows indicate straining of the cell membrane, and scale bar = 10 μm.

[0089] FIG. 15D illustrates a έ_XXCM strain rate map of cardiomyocytes.

[0090] FIG. 15E illustrates high-resolution live imaging studies of HUVECs labeled with membrane and in which nucleus markers were stretched and imaged for 6 stretch steps, wherein white arrows highlight tearing of cell-cell contacts and the scale bar = 20 μm.

[0091] FIG. 15F illustrates high-resolution live imaging studies of a single HUVEC at relaxed (left) and stretched (right) state, wherein short white arrows highlight the fenestrated holes in the cell membrane, and long white arrows highlight the detachment of cell membrane to the PDMS substrate, and wherein scale bar = 10 μm. [0092] FIG. 16A illustrates a cassette design for different a pure shear strain profile, wherein the broken-lined boxes indicate positions for substrate attachment .

[0093] FIG. 16B illustrates a cassette design for unilateral strain, wherein the broken-lined box indicate a positions for substrate attachment.

[0094] FIG. 16C illustrates a design for biaxial stretch and shearing, wherein the broken-lined boxes indicate positions for substrate attachment.

[0095] FIG. 17A illustrates a top view of an embodiment of a cassette assembly jig hereof.

[0096] FIG. 17B illustrates an isometric view of the cassette assembly jig of FIG. 17A.

[0097] FIG. 17C illustrates photographs demonstrating use of the assembly jig of FIG. 17A in an embodiment of a method of assembling a cassette using the assembly jig.

DETAILED DESCRIPTION

[0098] 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.

[0099] 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.

[00100] 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.

[00101] 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 or more 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.

[00102] 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 functions) 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.

[00103] 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 separate from the processor in block diagrams or other drawings.

[00104] 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.

[00105] 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.

[00106] To test the physiological roles of strain, it is desirable that a tissue straininducing system or strain system be able to apply relatively large strains (as, for example, observed during embryonic morphogenesis) 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 100%. For example, tissues undergoing convergent extension exhibit more than 2-fold changes in length, greater than 100% strain, during zebrafish and Xenopus gastrulation and neurulation. To replicate those high in vivo levels of strain, embodiment 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 μm 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).

[00107] To overcome the limitations of previous stretcher designs, the strain inducing systems or strain systems hereof are capable of inducing high strain (that is mechanical strain or deformation, which may be stretching, compressing or shearing strain) on, for example, living tissues and provide for 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 three subsystems or components including a readily interchangeable cassette, one or more motorized actuators, and a microscope stage assembly or insert which is readily designed or customizable for integration with a particular microscope. 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.

[00108] In a number of studies, use of a strain system hereof was demonstrated with organotypic explants from Xenopus laevis embryos. Stretching tissues with the strain systems hereof allows, for example, quantification of cell strain heterogeneity across the tissue and visualization of remodeling of intracellular keratin filaments under tension. The broader applicability of the strain systems hereof was demonstrated by modifying cassettes hereof for use with human umbilical endothelial cells (HUVECs) and mouse neonatal cardiomyocytes, permitting observations of cell morphological changes under large strain. In various embodiments, the strain systems and methods hereof thus enable high-resolution confocal imaging of living tissues under high strain (stretching, compressing, or shearing strain) from diverse animal models. The flexible and customizable systems hereof provide powerful tools for gaining insight into how mechanical cues function in remodeling tissues. Although the cassettes and strain systems hereof are particularly well suited for use in connection with biological samples such as tissues and cells, they may be used to induce strain in samples other than biological samples. Although representative embodiment of cassettes and strain systems hereof are discussed in connection with biological samples, one skilled in the art will appreciate that such cassettes and strain systems may be used in connection with non-biological samples, including, for example, synthetic materials or natural materials.

[00109] A representative embodiment of a strain inducing system or strain system 10 hereof is illustrated, in FIGS. 1A through 1I. FIG. 1A illustrates schematically strain system 10 in operative connection with a stage 110 of an inverted confocal microscope or microscope system 100. As known in the microscope arts, components of microscope system 100 that increase the overall system magnification include objective 120 and eyepiece 124. Light is provided by a light source 130. Objective 120, which is located closest to the sample, relays a real image of the sample to eyepiece 124 and to an appropriate detector 140 as known in the microscope arts.

[00110] FIG. 1I illustrate in panel (a) an enlarged top view photograph of the strain system of FIG. 1A inserted into or onto a microscope stage of a confocal microscope; panel (b) a side perspective view photograph of the strain system inserted into or onto a microscope stage of a confocal microscope; and panel (c) a front perspective view photograph of the strain system inserted into or onto a microscope stage of a confocal microscope. FIG. 1B illustrates a top isometric view of the representative embodiment of strain system 10 illustrated in FIG. 1A while FIGS. 1C and ID illustrate isometric and exploded view, respectively of strain system 1A. In the illustrated embodiment, strain system 10 include an assembly or stage assembly 20, which may, for example, interface with microscope stage 110. Assembly 20 incudes a body 22, which function as a stage insert or stage interface in the illustrated representative embodiment. Hereafter, body 22 is referenced a stage interface 22.

[00111] Body or stage interface 22 include stage top or top section 22a (further illustrated in FIG. 2A through 2D) and a stage bottom or bottom section 22b (further illustrated in FIGS. 3 A through 3E). Stage interface 22, which may, for example, be formed from a polymeric material (for example, via 3D-printing), may be readily designed or customized to fit various commercial inverted brightfield or confocal microscopes. Stage bottom 22b of stage interface 22 is designed and formed, for example, to sit securely or tightly on the platform of microscope stage 110 to provide stability and prevent shifting during, for example, live imaging. A sample volume or chamber 24 is formed in stage bottom 22b to receive a sample via a sample cassette as described below. In a number of embodiments, sample chamber 24 may include a transparent bottom 26 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 of light. In a number of studied embodiments, stage bottom included a 45 x 50 mm solution-fillable sample chamber 24. In the illustrated embodiment, stage bottom 22b further included two mounts 28 for securely mounting actuators 40 (for example, motorized linear actuators such as piezo actuators) via passages 28a therein.

[00112] Assembly or stage assembly 20 further includes a cassette interface 60, which has the form or shape of an H-shaped bridge or “H-bridge” in the illustrated embodiment. Cassette interface 60 (which is further illustrated in FIGS. 4A through 4F) cooperates with a removable cassette 80 (see, for example, FIGS. 4D and 5A through 5H) and actuators 40 to impart stretch to the biological sample. As described further below, removable cassette 60, which interfaces with the tissue sample and imparts strain/ stretch thereto, is placed or positioned within the liquid-filled sample chamber 24 to be placed in operative connection with cassette interface 60.

[00113] As illustrated in, for example, FIGS. IB and 1C, the cassette interface 60 is removably connectable to stage interface 22 via a cassette interface rest, mount, or seating 28, which may, for example, function to ensure a conforming or tight fit, and a connected opening 30. Referring, for example, to FIGS. 4A through 4F, cassette interface 40 includes one or more downward-extending (relative to the orientation of the microscope FIG. 1A and generally having the orientation of the gravitational vector in the figure as well as in typical use) abutment members 62 configured to contact and form an operative connection with cassette 80. In the illustrated embodiment, such extending abutment members 62 are cantilevered members which extend downward from transvers beams or crossbeams 66 of cassette interface 60. In that regard, extending abutment members 62 extend downward from moveable, flexible, and/or resilient extending elements 68, which form flexible crossbeams 66 of cassette interface 60. In the illustrated embodiment, two, generally parallel, extending elements 68 are positioned on each lateral side of cassette interface 60. The opposite ends of extending elements 68 of crossbeams 66 are attached to and extend between two opposing end members 70 of cassette interface 60. As, for example, illustrated in FIGS. IB, 1C and ID, end members 70 cooperate with seating 30 to place cassette interface 60 in cooperative engagement with stage interface 22. Fasteners, as known in the art, may securely connect cassette interface 60 to seating 30 of stage top 22a via aligned passages in each element.

[00114] In the illustrated embodiment, each extending abutment member (or cantilever member) 62 is connected or anchored between extending elements 68 of one of the generally parallel pairs of extending elements 68, and extends downward therefrom. The function of extending elements 68 of cassette interface 60 as a flexible crossbeam is described further below. In a number of embodiments, crossbeams 66 of cassette interface 60 (and thereby cassette 80) may be aligned with and mechanically coupled to one or more actuators 40 through simple linear abutment or contact therewith as, for example, illustrated in FIG. 1B.

[00115] Referring, for example, to FIG. 1D and FIGS. 5 A through 5H, the relative position of abutment members 82 (or the distance therebetween) of cassette 80 is controlled by the movement of one or more actuators arms 42 of actuators 40 (two, in the illustrated embodiment) which reciprocally extend from actuators 40 to abut laterally outer extending element 68 of crossbeams 66 of cassette interface 60. In the illustrated embodiments, abutment member 82 are extending members, but such abutment members may alternatively be formed as depressions or seating in an upper surface of cassette 80. As used herein, an actuator refers to a component with converts energy to motion. In a number of studied embodiments, two NEW FOCUS™ PICOMETER™ piezo linear actuators (available from Newport Corporation of Irvine, California) were used.

[00116] Strain (for example, extension or compression) occurs as crossbeams 66 are displaced, transmitting displacement to abutment members 82 on either side of operatively connected cassette 80. In preparation for uniaxial extension stretching, for example, two linear actuators arms 42 are extended to deflect crossbeams 66 inward (that is, toward each other). Next, cassette 80 is placed in a relaxed state into the culture-media filled sample chamber, with the coverslip serving as bottom 26 of the chamber 24, allowing direct and unimpeded microscopy of the sample. Abutments 82 of cassette 80 are aligned with, but not yet contacting, the lower portion of extending members 62 of cassette interface 60. At this point, relaxed cassette 60 may be placed in connection with crossbeams 66 by retracting actuator arms 42. As crossbeams 66 relax, they contact cassette abutments 82. A small, initial uniaxial bilateral stretch of cassette 80 may be used to immobilize the sample within the field of view. [00117] Actuators 40, cassette interface 60, and cassette 80 (with a resilient or stretchable sample substrate 90 — see, for example, FIGS. 5A and 5D through 5F), may be rigidly integrated. As further discussed below, large scale strain may, for example, be applied in steps as cassette extending members 62 are moved apart (in extension studies) and sample substrates 90 (formed, for example, from PDMS) stretches. In a number of representative embodiments, exposed sample substrate 90 within cassette 80 could be stretched to a maximum of 6 mm, generating up to 233% strain. Movement of cassette extending member 62 (via control of linear actuators arms 42) may, for example, be controlled by a computer-based control system (as illustrated in FIG. 1C) (using, for example, LabView) or be manually controlled via a joystick. The motion/position of actuator arms 42 may also be controlled manually (for example, via adjustment knobs on actuators 4G- not shown), semiautomatically, and/or automatically. Powered actuators 40 may, for example, be placed in communicative connection with a computerized control system as illustrated in FIG. 1C, which includes a processor system and a memory system in communicative connection therewith to control the energy supplied to actuator 40 and thereby the motion imparted to extending actuator arms 42. The memory system may include one or more software algorithms stored therein and executable by the processor system to control motion/position of the extending actuator arms 42.

[00118] In representative studies, image acquisitions were carried out with microscope automation software (^Manager 2.0). ^Manager is an open-source, cross-platform application via which one may control motorized microscopes, stages, illuminators, scientific cameras, stages, and a number of other microscope accessories. See, for example, Edelstein, A. D. et al. Advanced methods of microscope control using ^Manager software. Journal of biological methods 1 (2014).

[00119] In a number of embodiments, all components of stage assembly 20 were formed from polymeric materials via 3D-printing using, for example, Fused Deposition Modeling (FDM). In a number of studied representative embodiments, polylactic acid (PLA), a thermoplastic polymer, was used in FDM printing the components of stage assembly 20. Many polymers other than PLA, metals, and other materials, may be used in fabricating the components of stage assembly 20. 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 stage top 22a and stage bottom 22b of stage assembly 20 and/or other components hereof are readily adjustable to function with a particular microscope/microscope stage and for a range of predetermined uses.

[00120] FIGS. 5A through 5H illustrates further details of a representative embodiment of removable/disposable cassette 80 hereof. FIG. 5A illustrates a top, disassembled view a representative embodiment of cassette 80 hereof, which was similar in size or dimensions to a US quarter coin. FIGS. 5B and 5C illustrated isometric and side views of abutment members respectively. Assembled cassette 80 is illustrated in FIGS. 5D through 5H. In the illustrated embodiment, cassette 80, when assembled, includes a first cassette end member 80a and a second cassette end member 80b (see FIGS. 5D through 5H). In a number of embodiments, cassette 80 was formed from an upper or top sheet or shim 84 and a lower or bottom sheet or shim 86 of, for example, a polymeric material such as a polyester (PES) polymeric material. Upper shim 84 was cut to include or form two end members 84a and 84b, each of which includes an opening or seating 85a and 85b, respectively, in which abutment members 82 are seated during assembly. Cooperating members, abutment members or stretcher blocks 82 were, for example 3D-printed using stereolithography (SLA), and attached using a UV-curable optical adhesive to and extend upward from lateral end members 84a and 84b of upper shim 84 to cooperate with the extending abutment members or cantilever members 62 of cassette interface 60. Similar to upper shim 84, lower shim 86 was cut to include two end members 86a and 86b. Upper end member 84a and 84b as well as lower end members 86a and 86b are aligned (or stacked; see, for example, FIG. 5G) such that upper end member 84a is positioned over lower end member 86a and upper end member 84b is position over lower end member 86b and then connected to form an assembly (using, for example, a UV-curable optical adhesive).

[00121] In the illustrated embodiment, lower shim 86 was cut to include two extendable sections 88, wherein one extendable section 88 extending between lower end members 86a and 86b and positioned on each side of a gap 89. Such extendable sections or member 88 assist in stabilizing cassette end member 80a and 80b by resisting torsional or out of plane (that is, in the z-direction, referring to FIG. 1C) movement between cassette end members 80a and 80b. The sample thus remains flat or in plane (that is, generally parallel to the plane of stage 110 of the microscopelOO). During stretching or extension of the sample, extending sections 88 elongate as described further below. [00122] A flexible/stretchable polymeric film or sheet 90 which is desirably biocompatible (for example, a polydimethylsil oxane or PDMS sheet) extends across gap 89 formed between lateral end members 80a and 80b of the fully assembled shim assembly of cassette 80 (see, for example, FIGS. 5D through 5F). In a number of embodiments, a dumbbellshape PDMS sheet or sample substrate 90 was used to attach/seed biological samples. The larger surface area of the lateral ends of the dumbbell-shaped sample substrate 90 mechanically stabilize the attachment to lateral end members 80a and 80b of cassette 80 during stretching via the increased surface (contact) area thereof compared to the central section (between lateral ends of dumbbell- shaped sample substrate 90) which extends in gap 89 and is used to attach/seed biological samples. Sample substrate 90 may, for example, be securely attached to cassette end member 80a and 80b using a UV-curable optical adhesive as described above, during forming of the assembly of cassette 80. Cassettes 80 of different thickness may be manufactured for use with biological samples or animal models having different thicknesses. Cassettes 80 of different thicknesses may, for example, be readily identified via color-coding or other identifying features.

[00123] The material properties of the extending sections 88 and stretchable sheet/substrate 90 (for deposition of the biological sample) may, for example, be readily selected to provide for fully elastic extension/stretchmg. In that regard, cassette gap 89 and sample substrate 90 (extending across gap 89) may return to their original length upon removal of a stretching force therefrom (that is, to a “zero” or original state). In a number of studies hereof, cassette 90 returned to the zero state over a range of strains studied. Returning to a zero state may be important if the nature of the studies being conducted require it. Moreover, it may be desirable to repeat the stretching and relaxation process multiple times (for example, over a range frequencies) in a number of studies. The materials used in the representative embodiments hereof were suitable to stretch and return to a zero state for, for example, one or a few uses. However, materials (for example, other polymers, metals, and/or composite materials) may be used to provide multiple cycles of stretching and relaxation. In other embodiments, it may be suitable to use materials that undergo nonrecoverable stretching in a single use if there is no need to conduct studies after returning to a zero state. Suitable materials for a particular study are readily chosen by those skilled in the art using known engineering principles and constraints (for example, moduli, weight limitations, etc.). [00124] In a number of embodiments, shims 84 and 86 hereof were 38 — 127 μm in thickness to facilitate high-resolution imaging by maintaining the sample within the working distance of high-numerical aperture objectives. As described above, thickness of shims 84 and 86 of the shim assembly can be adjusted according to 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 in the lateral end members 84a, 84b, 86a, 86b (and cassette end members 80a and 80b formed therefrom) is negligible under the forces experienced during normal use. Deformation is transmitted to the elastic or stretchable sheet or substrate 90 and to the extendable sections 88. As described above, the extendable (or deformable) sections or cutout sections 88 of cassette 80 minimizes out-of-plane torsion and stabilizes tissue sample during stretching. To securely mount stretchable sheet or sample substrate 90 onto the PES shims hereof, a “sandwich” design was used in which stretchable sheet was glued between the lateral end members 84a, 84b of the upper shim 84 and lateral end members 86a, 86b of lower shim 86 of cassettes 80 in studied embodiments. In such studied embodiments, the two 3D-printed (photopolymer resin), cooperating abutment members or stretch blocks 82 were glued onto the upper surface of upper end members 84a and 86a of cassette 80 to enable a cooperating connection to abutment members 62 of the cassette interface 60 of stage assembly 20. Samples attached to sample substrate 90 are thereby physically coupled to cassette 80. Thus, as end members 80a and 80b of cassette 80 are moved and substrate sheet 90 is strained, samples are deformed. In a number of embodiments, after assembly of strain system 10 hereof, exposed PDMS sample substrate 90 (and, correspondingly, gap 89 between the end members 80a and 80b of cassette 80) was approximately 1.8 mm wide. As described above, stretchable sample substrate or sheet 90 was stretched to strains in excess of 200% in a number of studies.

[00125] Since large displacements can generate apparent drift of the image across the field of view, a custom microscope control AutoCenter software routine or algorithm was developed to recenter the image. The AutoCenter routine was based on the built-in autofocus plugin of the microscope automation software (μManager 2.0). In brief, the AutoCenter routine runs between actuator movements and adjusts the position of the XY-plane stage to counter drift of the sample. The plugin module was executed at the beginning of each specified acquisition timepoint with two user inputs: (1) the channel to use for the adjustment, and (2) a search range along the Z-axis (usually equal to the Z-stack range set at the beginning of the acquisition). Using the user-input search range, the AutoCenter routine will find the Z position that exhibits the greatest sharpness and capture a reference image. The module will then calculate the xy displacement between the new reference image (from the current time point) and the previous reference image (from the last time point) using conjugate multiplication of the Fourier transforms of the two images, inverse transforming the result, and then finding the Ax, Ay deviation of the brightest pixel from the center of the image. Ax and Ay are used to set the stage to the new position in register with the earlier time point

[00126] FIG. 6 illustrates a flowchart for an embodiment of an AutoCenter plugin hereof. Between stretches of the motor/actuator 40, the xy stage is adjusted to account for drifting of the tissue that occurs during stretching. This is implemented with a custom autofocus plugin for Micro Manager, which handles image acquisition through Image! (an open source software for processing and analyzing scientific images). The plugin uses the core autofocus functions, making it appear in the autofocus menu. The plugin module to adjust the xy stage to account for drift is executed at the beginning of each specified timepoint of image acquisition. The user supplies two inputs: the channel in which to focus and the μm range to use for z focus at the beginning of each timestep. First, the acquisition window is checked for an image. If no image exists, the plugin will do nothing and wait for the next round of images. If there is an image in the acquisition window, the Oughtafocus routine will be run from the middle of the stack plus and minus half the search range. The sharpest z is used to set the z position of the stage. Two images are needed to calculate the displacement. The first is taken from the middle z of the stack for the previous timepoint. The second is snapped at the newly focused z and stored within the plugin so it does not interfere with the acquisition. As described above, the displacement between these two images is calculated by conjugate multiplication of the Fourier Transforms of the images, inverse transforming the result, and then finding the deviation of the brightest pixel from the center of the image. This calculated dx and dy is then multiplied by the pixel size and used to set the new stage position.

[00127] FIG. 7 illustrates a schematic of cassette 80 at a relaxed state with linear actuators 40 pushing inward (toward a center line of cassette 80) to deform crossbeams 66 (top left) inward. As arms 42 of actuators 40 retracted, resilient crossbeams 66 become straight and stretch cassette 80 (top right). Schematic illustrations of a tissue sample attached to substrate 90 of cassette 80 at a relaxed state (middle left; gap width x₁) and at a maximum stretched state (middle right; gap width x₂) are provided. Side views of cassette 80 being stretched and imaged on an inverted scope (wherein sample chamber bottom 26 is formed by a glass coverslip), with the tissue sample facing towards the objective lens are provided at the bottom of FIG. 7. The H-bridge cantilevers may snap onto the stretcher blocks and push outward as the actuator arms retract. FIG. 8 illustrates photographs of a cassette in a relaxed state and a stretched state as described in connection with FIG. 7 while strain system 10 hereof is in operative connection with microscope stage 110.

[00128] In the normal, undeformed, or relaxed state of crossbeams 66 of cassette interface 60, each crossbeam 66 (including a pair of extending elements 68 as described above) is generally parallel to other crossbeam 66 and extends in a generally straight line between ends 70 of cassette interface 60. The terms “relaxed” and “stretched” as used herein refer to state of cassette 80, associated sample substrate 90, and the sample. When crossbeams 66 (and extending abutment members/cantilever members 62 thereof) are pushed inward or toward each other, the sample-loaded cassette 80, which has been immersed within a liquid of chamber 24 of stage bottom 22b, may be maneuvered (for example, using forceps) such that the abutment members/cantilever members 62 are placed in operative connection with the cooperative abutment member or stretch blocks 42 of cassette 80 without exerting tension or stretching force on cassette 80. In a number of embodiments, extending abutment members or cantilever members 62 of crossbeams 66 may, for example, snap onto cooperating abutment members or stretch blocks 42 of cassette 80. During imaging, the biological sample (tissue) desirably face downward (on a lower surface of stretchable sample sheet 90 of cassette 80), that is, oriented toward the objective of microscope 100. During stretching, upon controlled retraction of actuators arms 42, crossbeams 66 of cassette interface 60 become less deformed and move toward their relaxed position, thereby placing tension upon and stretching cassette 80 via the interaction between abutment members 62 and cooperating abutment members 82. In other words, cooperating abutment members or stretch blocks 82 of cassette 80 are pushed outward by abutment members 62 of cassette interface 60 as actuator arms 42 retract. The reciprocal (extending/retracting) motion of actuator arms 42, the motion of the abutment members 62, the motion of cooperating abutment members 82, and the stretching motion of the assembly of the cassette 80, sample sheet 90, and the sample is generally linear motion along the x axis in the xy plane.

[00129] FIG. 9A illustrates a method of using an embodiment of explant jig 200 for sample (for example, tissues or cells) attachment. Studied embodiments of explant-mounting jig 200 were 3D-printed using stereolithography with Formlabs clear resin. A purpose or function of explant-mounting jig 200 is to provide a flat surface and direct access for tissue/cell attachment at the bottom of the polydimethylsiloxane sample substrate 90.

[00130] As illustrated in FIG. 9A, a cassette 80 hereof may, for example, be coated with a desired extracellular matrix for tissue/cell (sample S) attachment before the procedure. Cassette 80 is flipped or inverted (with cooperating abutment members or stretcher blocks 82 facing downward) and seated onto explant-mounting jig 200. Explant-mounting jig 100 (FIG. 9A and 9B) includes two cutouts or passages 210 to seat or accommodate cooperating abutment members 82. The height of jig 200 may be slightly higher than cooperating abutment members 82, allowing top shim 84 to sit flat on the surface of the jig 200. Next, cassette 80 with the jig 200 are placed into, for example, a 60mm Petri dish filled with desired tissue/cell culture medium. Organotypic tissue or cells (sample S) are placed onto sample substrate 90 of cassette 80, allowing attachment to the surface of sample substrate 90 for hours to days (wherein the time depends on tissue/cell types). Once the tissue or cells (sample S) are securely attached to the surface of sample substrate 90, cassette 80 is removed from explant-mounting jig 200 and transferred to sample chamber 24 in bottom section 22b of stage assembly 20. Cassette 80 is positioned upright within sample chamber 24, with cooperating abutment members 82 facing upward. Abutment members 62 of cassette interface 60 contact the inner face of cooperating abutment members for expansion/ stretching as described above. During imaging, tissue/cell sample S faces downward toward the microscope objective.

[00131] Various modifications may be readily made in the operation of system 10 to enable unilateral compression of a tissue or a cell sample instead of expansion or stretching. In that regard, as illustrated in FIG. 10, cassette 80 may be pre-stretched (see left, “pre-stretched” side of FIG. 10) before deposition of sample S thereon. For example, one may use a pre-stretch stabilizer 300 hereof (see FIG. 11) before tissue/cell attachment. Once the tissue or cells (sample S) are attached onto sample substrate 90 of pre-stretched cassette 80, cassette 80 is transferred to sample chamber 24. As illustrated in FIG. 10, crossbeams 66 are at a straight configuration with extending abutment members 62 extended downward, contacting the inner surfaces of cooperating abutment members 82 of cassette 80, maintaining cassette 80 in a stretched state with the sample relaxed. To compress sample S, arms 42 of linear actuators 40 push inward on crossbeams 66 to deform crossbeams 22 to a curved configuration. Cassette 80 is thereby relaxed, compressing sample S (see right, “relaxed” side of FIG. 10). [00132] In a number of studied embodiments, pre-stretch stabilizer 300 was cut out from a 380 μm thick polyester shim using a 2D-cutter. A purpose or function of pre-stretch stabilizer 300 is to keep cassette 80 at a stretched configuration before tissue/cell (sample S) attachment. As illustrated in FIG. 1l a modified explant-mounting jig 200a is modified (as compared to explant-mounting jig 200) in a manner to cooperate with pre-stretched cassette 80 with a stabilizer 300. A cassette 80 is coated with desired extracellular matrix for sample S attachment before the procedure. Pre-stretch stabilizer 300 is used to stretch cassette 80. Cooperating abutment members 82 may, for example, have a trapezoid-shape groove 83, whereby a side of pre-stretch stabilizer 300 can snap into groove 83 to keep cassette 80 in a stretched configuration (see FIG. 11). Stretched cassette 80 is then flipped or inverted (with cooperating abutment members 82 facing down) and placed onto modified explant-mounting jig 200a. Modified jig 200a includes two seating, passages, or cutouts 210a and an indented surface 220a around the center to accommodate two cooperating abutment members 82 and stabilizer 300, respectively. The height of jig 200a may be slightly higher than cooperating abutment members 82, allowing upper surfaces of lateral end member 84a and 84b of shim 84 to sit flat on the surface of jig 200a, while pre-stretch stabilizer is seated within indented or lowered surface 220a. Pre-stretched cassette 80 with jig 200a is then placed into a 60mm Petri dish filled with desired tissue/cell culture medium. A sample S such as organotypic tissue or cells are then, for example, placed onto sample substrate 90 of pre-stretched cassette 80, allowing the tissue or cells (sample S) to attach to sample substrate 90 for hours to days (wherein the time depends on tissue/cell (sample) types). Once sample S is securely attached to the lower surface of sample substrate 90, cassette 80, along with pre-stretch stabilizer 300, is removed from jig 200a and transferred to sample chamber 24 in stage assembly 20. Prestretched cassette 80 is positioned upright, with cooperating abutment members 82 facing up and sample S facing down. Cassette interface 60 may, for example, be inserted into seating 32 so that abutment members 62 of cassette interface 60 are approximately halfway through the height of cooperating abutment members 82. Then, stabilizer 300 is removed by bending one side of stabilizer 300 (for example, using forceps). After removing stabilizer 300, cassette interface 60 is inserted further until abutment members 62 reach top shim 84 of cassette 80.

[00133] Substrate strain uniformity is important to uniform deformation across attached tissues. To validate strain uniformity using the devices, systems and methods hereof, sample sheet or substrate 90 in cassette 80 was coated with 5 μm-diameter green fluorescing polymer beads (see FIG. 12). Linear actuators 40 were controlled by the LabView program to stretch cassette 80 at a constant velocity of 52 ± 3.5 μm per minute for 45 minutes (see FIG. 13) and cassette 80 was imaged every 30 seconds. To verify the strain profile across sample substrate 90, two locations were selected: (1) near the site where sample sheet or sample substrate 90 connects with end member 84b and 86b forming end 80b of cassette 80 and (2) at the center of PDMS sample substrate 90 where it was intended to track tissue strain (panel (a) of FIG. 12). The fluorescent beads were traced every 15 minutes during stretch to investigate the strain uniformity across PDMS sample substrate 90 (panel (b) of FIG. 12).

[00134] To quantify strain uniformity on the PDMS substrate, a custom macro for digital image correlation was used to analyze strain between image pairs across regions of interests (StrainMapper). Boyle, J. J,, et al. 'Simple and accurate methods for quantifying deformation, disruption, and development in biological tissues', JR Soc Interface 11(100): 20140685 (2014) and Stepien, T. L., Lynch, H. E., Yancey, S. X., Dempsey, L, & Davidson, L. A. Using a continuum model to decipher the mechanics of embryonic tissue spreading from time-lapse image sequences: An approximate Bayesian computation approach. PLoS One 14, 460774, doi:10.1371/joumal.pone.0218021 (2019). StrainMapper assumes the image represents a continuous field and carries out a warping transformation allowing the calculation of principal strain rates έ_XX, έ_YY, and έ_XY. Here strain rate έ_XX along the x-axis (e.g., the stretch axis) between the edge and was compared to the center of the PDMS substrate since the stretcher system is a uniaxial tension system. Elongation along x-axis results in positive strain (darkened areas) whereas shortening results in negative strain (lightened areas) in the strain rate έ_XX colormap, respectively (panel (c) of FIG. 12). During the initial 30-minute interval, the edge of the PDMS sample substrate 90 where it is bonded to PES shims 84 and 86 of cassette 80, exhibited heterogeneous strain with large variance (σ² = 0.013, 6.2 x 10^-3 for 0 — 15 min, and 15 - 30 min, respectively) across the field of view. That period was marked by localized contraction proximal to the border of the frame with relatively high elongation at the center of the frame. However, the subsequent span of 30 to 45 minutes after the start of stretching shows a uniform strain distribution (σ² = 6.5 x 10^-5) across the edge region of stretched sample substrate 90 (panels (c) and (d) of FIG. 12). In contrast to strains at the edge of sample substrate 90, the variation of strain was consistently small at the center of sample substrate 90 (σ² = 5.4 x 10^-5, 7.6 x 10^-3, 3.7 x 10^-3 for 0 to 15 min, 15 to 30 min, and 30 to 45 min, respectively) (panels (c’) and (d) of FIG. 12). The uniaxial strain έ_XX at the center of PDMS sample substrate 90 was homogeneous. Thus, tissue samples attached to center will experience consistently homogeneous substrate strain during stretching. The remainder of the studies therefore focused on tissues and cells within the center of PDMS sample substrate 90.

[00135] To test strain system 10 on live tissues, preliminary studies of stretching Xenopus laevis organotypic explants were conducted and imaged with an inverted brightfield microscope. Then, to visualize cells and test the system with a confocal microscope, membrane-mNeonGreen mRNAs were expressed in the Xenopus embryos, which were cultured to the early to mid-gastrula stage. Regions of ectoderm expressing mNeonGreen were micro surgically dissected from the embryos. To observe apical cell surfaces during imaging, explants were cultured on the prospective undersurface of fibronectin-coated PDMS sample substrate 80 bound in a cassette 80 for at least an hour at room temperature. To attach explants to the bottom of cassette 80, fibronectin-coated PDMS-cassette 80 was flipped and placed onto a 3D-printed explant-mounting jig 100 as described above, stabilizing cassette 80 horizontally for secure mounting of the explant to the undersurface of sample substrate 90 (see FIGS. 9A and 9B). Once the tissue sample adhered to sample substrate 90, cassette 80 was inverted so that the apical face of the organotypic explant sample faced the objective, to allow tracking of the same group of cells during stretch (see FIG. 14A wherein boxes surround areas of tissue, and a cell in indicated by asterisks).

[00136] To better mimic in vivo tissue strain such as those during early development, actuators 40 generated up to 150% strain within cassette 80. The term stretch step (“S#”) refers to a single stretch step. To minimize image blurring that occurred during displacement, cell groups were tracked and imaged over 8 additive stretch steps, e.g. S1 to S3 (FIG. 14B). In that case, one stretch step displaces cassette ends 80a and 80b by 375 μm in total; the initial relaxed state is indicated as S0 and the maximum stretched state, after 8 stretch steps, as S8 (3 mm total grip-to-grip displacement).

[00137] To compare tissue and individual cellular mechanical behaviors under tension, two independent analysis pipelines were used to quantify tissue strains and individual cellular strains along the x-axis, roughly along the axis of stretch and represented by subscript xx. As custom image processing macro (StrainMapper) was used to calculate tissue strain rate έ_XXT between two consecutive stretch steps. Variations in strain were found within the tissue in each stretch step (σ² ~ 0.005) (see FIGS. 14B (top images) and 14C) were greater than variations in PDMS sample substrate 90 strain determined above (σ² ~ 1x10^-5) (see panels (c’) and (d) of FIG. 13). This suggests that the mechanical heterogeneity of the tissues under tension reflects sample variation rather than substrate heterogeneity. From that observation, it was suspected that individual cells also experienced heterogenous strain. To measure strain on a cell-by-cell basis, individual cells were segmented and registered across stretch steps with a segmentation software (Seedwater Segmenter, a graphical Python program to interactively segment image stacks of cells in tissue with edge-labels and available on GitHub). Mashbum, D. N., Lynch, H. E., Ma, X. & Hutson, M. S. Enabling user-guided segmentation and tracking of surface- labeled cells in time-lapse image sets of living tissues. Cytometry A 81, 409-418, doi:10.1002/cyto.a.22034 (2012). A custom image processing pipeline (FIJI and MATLAB) was used to quantify shape and position information from segmented cells. From cell shape changes the individual cellular strain rate έ_XXC was calculated between two stretch steps of cells that stayed in the imaging frame through all 8 stretch steps. The analysis excluded cells that divided during stretch, since those may not accurately reflect changes in strain that is solely due to the stretching. Similar to the findings from the bulk analysis pipeline, heterogeneous cellular strain (σ² varied from 0.002 to 0.007) was observed across the tissue, with both positively and negatively strained cells (FIG. 14B middle images).

[00138] It was next desired to investigate how consistently the tissue and individual cells react to tension. Comparing έ_XXT and έ_XXC at each stretch step (S0 to S1, S1 to S2, etc.), no significant differences was found (FIG. 14C). Cumulative tissue and cellular strains ε_XX were also compared by calculating strain between the relaxed state (S0) and each stretch step (S1, S2, etc.). Again, no significant differences were found between cumulative tissue and cellular strain at each stretch step (FIG. 14D). To investigate cell-to-cell variation between tissue and cell strain, differences between the two analytic pipelines were mapped across the tissue reporting whether tissue and cell strains were in concordance or discordance (FIG. 14B bottom images). The cell and tissue strains are in concordance if the difference between tissue and cellular strain is within one standard deviation. If the difference is greater than one standard deviation, the cellular and tissue strain are in discordance. It was observed that most tissue-cell strains were concordant (FIG. 14E, center regions of graph), suggesting consistent tissue and cell behaviors under tension. Also observed were cases wherein (1) tissues and cells only displayed small fluctuations in strain (FIG. 14E, bottom region), (2) individual cells strained more than the tissue, resulting in discordant strain (FIG. 14E, right region), and (3) individual cell strains that were less than the tissue or even contracted while the tissue was stretched (FIG. 14E, left region). Thus, it was determined that strain is heterogeneously distributed through Xenopus organotypic animal cap explant when under tension, with individual cells showing either concordant, or discordant strains within the tissue. Combining strain system 10 with image analysis pipelines thus reveals more complex behaviors of cells to strain than previously observed.

[00139] Building on the above studies, it was next sought to use strain system 10 with high resolution confocal fluorescent live-imaging, testing the compatibility of cassette 80 with a high numerical-aperture oil-immersion objective lens. Xenopus organotypic explants were stretched that express an intermediate filament reporter, keratin8-mCherry, and a membrane marker, membrane-mN eon Green. It was possible to observe and track keratin filament changes at a single filament level using a high numerical-aperture oil-immersion objective lens (FIG. 15 A). Prior to stretch, many curved keratin filaments were observed, suggesting a relaxed state. However, filaments straightened as stretch increased, suggesting keratin filaments bear more load after stretching (FIG. 15A, long arrows). In was also observed that in some cases where keratin filaments initially associated with the cell-cell junction would, after stretching, detach from junctions (FIG. 14A, short arrows). Both cases of keratin network remodeling support their role in carrying loads under tension.

[00140] Two other live cell model systems were also tested in strain system 10: mouse neonatal cardiomyocytes and human umbilical vein endothelial cells (HUVECs). To seed the cells on cassette 80, as in previous studies, ECM -coated cassettes 80 were prepared before the experiment by adsorbing either fibronectin (HUVECs) or collagen I (cardiomyocytes) onto PDMS sample substrates 90. After coating, cassette 80 was inverted and cells were loaded onto PDMS sample substrate 90 in 30 uL of medium containing either cardiomyocytes or HUVECs. Cells were incubated on cassette 80 until they attached to the surface of PDMS sample substrate 90. Vital dyes for nuclei and plasma membrane were added to the medium 30 minutes before imaging. As with tissue explants, cassette 80 was inverted, and placed in the strain system (see FIG. 15B and the Experimental section hereof). Strain was applied and images collected over 6 stretch steps.

[00141] Cardiomyocytes appeared to maintain cell-cell contacts while nuclei shape remained unchanged as they were stretched (FIG. 15C). A consistent field of cells throughout the stretch steps allowed one to map tissue strain between each consecutive stretch steps έ_XXCM using StrainMapper (FIG. 15D). Cardiomyocytes under high strain remain connected to each other and continue to beat. [00142] Since HUVECs are considerably larger than Xenopus embryonic cells or cardiomyocytes, a 25x objective was initially used to image confluent layers to include more cells in the imaging frame. In contrast to the Xenopus embryonic epithelium and mouse cardiomyocytes studies, applied strain disrupted HUVECs cell-cell adhesions, and caused cell membranes to detach from PDMS sample substrate 90 (FIG. 15E). Since stretching caused HUVEC membranes to retract and expose gaps between cells, it was not possible to perform a tissue strain analysis. Next, a higher numerical aperture objective was used and single isolated cells under high substrate strain were observed. The seeding density was lowered, allowing one to strain single HUVECs (FIG. 15F). While not obvious at rest, single cells under high strain exhibited multiple holes, similar to fenestrations that have been described in other endothelial tissues. At increased strains, cell membranes appeared to rupture or tear where fenestrated holes had appeared at lower strains (FIG. 15F, short arrows). Furthermore, cell-substrate adhesion was also disrupted after stretching, leaving only streaks of membrane attached to the substrate (FIG. 15F, long arrows). Comparing those two cell types, the preliminary observations indicated cardiomyocytes can remain attached to sample substrate 90 and maintain cell-cell contacts under strain. By contrast, highly strained confluent layers of HUVECs lost cell-cell adhesions and single cells ruptured at sites where fenestrations were observed, and both single, and confluent HUVECs lost cell-substrate adhesions.

[00143] Strain system 10 thus enables an effective mechanical stimulation of live samples while achieving high resolution live imaging. In the studied embodiment, strain system 10 made use of affordable 2D cutters and 3D printers to fabricate components that may, for example, be readily integrated with low-cost linear actuators 40. An exchangeable, 2D-cut cassette 80 that keeps the sample within the working distance of a high numerical-aperture objective lens. In the studied embodiments, cassette 80 and two linear actuators 40 were assembled into a custom designed microscope stage assembly 20 for simultaneous mechanical manipulation and visualization. Strain system 10 enables acquiring of high-resolution live images of organotypic explants during stretching and analysis of tissue and cellular scale engineering strain. In addition, it was possible to visualize intracellular intermediate filaments under strain at a single filament level. Representative cardiomyocytes isolated from neonatal mice and endothelial cells from human umbilical vein were also studied. Those model tissues demonstrated the utility of removable/exchangeable cassettes 80 and strain system 10 for visualizing and measuring the effects of strain on diverse cell and tissue types under strain with high-resolution confocal imaging. Although three animal models were studied herein, strain system 10 is compatible with most cultured cells, and organotypic explants that adhere to extracellular matrix. Furthermore, the low-cost, lightweight, and modular features of strain system 10 facilitate extensive customization for specific applications.

[00144] As described above, stage assembly 20 may be formed from a polymeric material and may, for example, be 3D-printed using, for example, an FDM printer. The weight of the stretching systems hereof may be readily maintained quite low (for example, below 200 grams). A relatively low weight is important in a number of embodiments to, for example, prevent interference with the operation of a z-direction motor of, for example, a z-axis- motorized xyz stage of a microscope. In that regard, such light weight allows strain systems 10 hereof to be used with a precise z-stage controller (piezo or galvo) on an inverted brightfield or confocal microscope. The overall design of stage assembly 20 and cassettes 80 operatively connectible thereto enable the biological sample to be positioned very close to the microscope objective (for example, within 200 μm or within 150 μm). As described above, living biological samples/tissues can be attached to cassettes 80 hereof, which can be swapped in and out with ease. Actuators 40 can be easily and readily be assembled into stage assembly 20 to provide the force to strain cassette 80 with the sample(s) attached on sample substrate 90 thereof. The modular design of the strain systems hereof enables one to incorporate high- resolution confocal microscopy with live imaging of multiple samples, while keeping the cost of fabrication low.

[00145] As described above, cassettes 80 hereof may be designed to provide stability along z-plane (that is, out of the xy plane or the plane of the stage) during stretching while permitting bilateral elastic deformation along at least the x-axis. Stretchable or deformable sample sheets or sample substrates 90 of cassettes 80 hereof, to which the biological (or other) sample is attached or seeded, helps to ensure that tension is efficiently transmitted and maximized in the sample during stretching. PDMS is a representative, biologically compatible and stretchable material (polymer) for use as the stretchable sheet or substrate for tissue attachment/ seeding.

[00146] In general, assembly or stage assembly 20 hereof is readily customizable to fit, for example, any inverted bright-field or confocal microscopes. In combination with a microscope, stage assembly 20, cassettes 80 and actuators 40 achieve an optimizable combination of high-resolution live imaging and effective mechanical manipulation of biological samples. The bilateral stretch of cassette 80 achieved in strain systems 10 hereof is suitable to keep the sample within the same imaging frame during strain.

[00147] Various adaptations can be readily integrated to broaden the applications of strain system 10. For example, cassette 80 may be readily adapted to use other biocompatible adhesives to attach tissue samples to sample substrates 90 such as cyanoacrylate, Cell-Tak (available from Coming), or poly-L-lysine. Furthermore, cassette 80 may be modified by changing grip positions or spring connections to provide different strain profiles (that is, shear strain, biaxial strain) as illustrated in FIGS. 16A through 16C, to mimic other dynamically changing microenvironments. The cassette in FIG. 16A is designed to provide pure shear strain to the tissue sample (illustrated in broken lines). By applying force along the indicated direction (anew F), central portion c of a first end section remains stationary, and two sides s of a second end section of the cassette move in the direction of the applied force F. The relevant movement induces shear strain to the attached sample, as depicted in the enlarged shear illustration at the bottom of FIG. 16 A. The cassette of FIG. 16B is designed to provide uniaxial stretch. By positioning the substrate (shown in broken lines) farther from the extendable (spring) sections, the cassette design further minimizes the effect of torsional movement of the extendable sections and the cassette. The cassette of FIG. 16C is designed to provide uniaxial extension/ stretch with shear. By applying forces in both the longitudinal and latitudinal directions as indicated by force arrows F, the substrate/sample is extended stretched laterally/horizontally and sheared longitudinally/vertically (see bottom of the figure).

[00148] Furthermore, the size of cassette sample substrate 90 can be increased to include larger numbers of tissue/cell samples for fixation. While the current design of strain system 10 was adapted to track and visualize live cellular and intracellular dynamics during stretching, other systems with multiple coupled strain systems may achieve high-throughput analysis of mechano-responses.

[00149] Strain system 10 enables, for example, testing of putative mechanosensors and mechanotransducers in living cells and provide insights into the signaling pathways and gene regulatory networks that respond to mechanical stimulation. In particular, the ability to generate and sustain high strain rates make this a unique tool to investigate the plastic behaviors of growing multicellular tissues and how mechanical cues play a role in development and disease. As described above, the strain systems hereof are capable of applying greater than 100% or greater than 200% mechanical strain on living biological samples such as living tissues. The samples may, for example, be live imaged using high-resolution confocal microscopy and provide dynamic, high resolution imaging during the straining process.

[00150] Although representative embodiments of the strain devices, systems, and methods hereof have been discussed for use in connection with microscopes, and in connection with biological samples, one skilled in the art will appreciate that the devices, systems, and methods may be used for any purpose in which it is desirable to controllably strain a sample (including biological and non-biological samples).

[00151] Experimental

[00152] Preparation and fabrication of the strain systems hereof. The top and bottom microscope stage insert, and the H-bridge or cassette interface were printed with Polylactic Acid (PLA; Prusa Research of Prague, Czech Republic) filaments in Fused Deposition Modeling 3d-printer (Prusa i3 MK3S; Prusa Research). Polyester (PES) sheets were purchased from Precision Brand of Grove, Illinois. Poly(dimethysiloxane) (PDMS) sheets (0.005”, 40D, Gloss finish) were purchased from Specialty Manufacturing, Inc. of Saginaw, Michigan. Top and bottom PES sheets of the cassette shims, and the dumbbell-shape PDMS sheets of the sample substrate were cut using Roland CAMM-1 GS-24 Vinyl Cutter (Roland DGA) with a 25 degree/.125 offset blade (USA-C125; Roland DGA of Irvine, California) to provide clean, smooth cutting edges. Then, PES and PDMS sheets were washed with 100% acetone, followed by extensive rinse of 100% ethanol and double deionized water for 24 hours. Rinsed products were dried between wax paper to prevent dust accumulation. Stretcher blocks or cooperating abutment members were 3D-printed using stereolithography (Form 2; Formlabs of Somerville, Massachusetts) with photocurable clear resin (Formlabs), followed by a 20-minute 100% isopropanol wash (Form Wash; Formlabs) to remove excess resin, and then cured in UV-light (405 nm) chamber (Form Cure; Formlabs) for 2 hours at 60°C.

[00153] The bottom shims (cut from PES sheets) were placed in a 3D-printed jig 400 to facilitate assembly (FIGS. 17A through 17C). Referring to FIG. 17C, a dumbbell-shape PDMS sample sheet or sample substrate 90 was placed onto bottom shim 86 with the dumbbell parts aligned with the protruding parts of the bottom shim 86. A thin layer of UV-curable optical adhesive (Norland Optical Adhesive 63; Edmund Optics) was applied to PDMS sample substrate 90 and to bottom shim 86 (excluding spring-like structures 88). Then, top shim 84 was placed onto bottom shim 86 using jig 400. Two cooperating abutment members or stretcher blocks 82 were bonded into the two cut-out holes 85a on top shims 84. Once assembled, cassette 80 was placed into UV-light (350nm) chamber to cure for 2 hours. Cassettes 80 were stored at room temperature before use and discarded after experiments.

[00154] Fluorescent bead coating. 30 μL of green fluorescent polymer microspheres (5.0 μm diameter; 1% solids; Duke Scientific Corporation of Fremont, California) was diluted in 420 μL of double deionized water. A cassette 80 was flipped (cooperating abutment members 82 at the bottom) and 100 μL of the diluted fluorescent beads solution was added onto PDMS sample substrate 90 of cassette 80. Cassette 90 was air-dried, covered with aluminum foil, until the liquid evaporated.

[00155] Microinjection of Xenopus laevis embryos and organotypic explant mounting. All Xenopus laevis work was approved by the University of Pittsburgh Division of Laboratory Animal Resources. Xenopus laevis embryos were obtained by the standard procedure. See Kay, B. K. Xenopus laevis: Practical uses in cell and molecular biology. Injections of oocytes and embryos. Methods Cell Biol 36, 663-669 (1991), the disclosure of which is incorporated herein by reference. 50 pg of membrane-tagged mNeonGreen mRNA was microinjected into 4-cell embryos. To visualize keratin 8 filaments, 100 pg of keratin8-mCherry mRNA was microinjected into 4-cell embryos. Injected and uninjected embryos were cultured in l/3x modified Barth Solution (MBS) to desired stages.

[00156] Cassettes 80 were flipped (cooperating abutment members 82 at the bottom) and put into an oxygen plasma cleaner (Harrick Plasma) for 2 minutes to activate surface of the sample substrates 90. 0.025 μg/μl of fibronectin (Chem Cruz) was added onto cassette 80 immediately after plasma cleaning and incubated at room temperature for 1 hour. Cassette 80 was then transferred into a 60 mm petti dish with mounting jig 200 in Danilchik’s For Amy medium with antibiotic and antimycotic (Sigma-Aldrich of S. Louis, Missouri) (FIG. 9 A). See, for example, Sive, H. L., Grainger, R. M. & Harland, R. M. Early development of Xenopus laevis : a laboratory manual. (Cold Spring Harbor Laboratory Press, 2000), the disclosure of which is incorporated herein by reference. An organotypic animal cap explant was microsurgically removed at early gastrula stage (Stage 10) and immediately transferred and positioned at the center of the PDMS substrate of the cassette (FIG. 9A). See Joshi, S. D. & Davidson, L. A. Live-cell imaging and quantitative analysis of embryonic epithelial cells in Xenopus laevis. J Vis Exp, doi: 10.3791/1949 (2010) and Nieuwkoop, P. D. & Faber, J. Normal tables of Xenopus laevis (Daudin), Amsterdam: Elsevier North-Holland Biomedical Press, (1967), the disclosures of which are incorporated herein by reference, A 1.5 mm x 12 mm glass coverslip bridge with high vacuum grease (DuPont) on both ends was gently pressed down to immobilize the explant and incubated at 14°C or room temperature to the desired stage. The glass bridge was removed before cassette 80 was transferred to stage assembly 20 strain system 10.

[00157] Human umbilical vein endothelial cell (HUVECs) culture. Pooled human umbilical vein endothelial cells (HUVECs; Promocell) were cultured in a sterile humidified incubator in complete endothelial cell growth medium (EC Growth Medium 2/EGM2, containing 2% FBS; Promocell) and 1 × antibiotic-antimycotic (Thermo Fisher Scientific) at 37 °C under 5% CO2. Upon confluency, cells were rinsed with HEPES BSS (Detach Kit, Promocell) and treated with 0.04% trypsin/0.03% EDTA for 5-7 minutes at room temperature until cells detached. After adding trypsin neutralization solution, cells were centrifuged at 220 × g for 3 minutes and gently resuspended in fresh EGM2. Cells were maintained in EGM2 for a maximum of six passages.

[00158] Mouse neonatal cardiomyocyte isolation and culture. All mouse work was approved by the University of Pittsburgh Division of Laboratory Animal Resources. Outbred Swiss Webster mice were used to generate cardiomyocytes for stretcher experiments. Neonatal mouse cardiomyocytes were isolated as described in Ehler, E., Moore-Morris, T. & Lange, S. Isolation and culture of neonatal mouse cardiomyocytes. J Vis Exp, doi: 10.3791/50154 (2013), the disclosure of which in incorporated herein by reference. Briefly, mouse pups were sacrificed at P2 and the hearts were removed, cleaned, minced, and digested overnight at 4°C in 20 mM BDM (2,3-butanedione monoxime) and 0.0125% trypsin in Hank's balanced salt solution. The next day, heart tissue was digested further in 15 mg/ml Collagenase/Dispase (Roche) in Leibovitz medium with 20 mM BDM to create a single-cell suspension. Cells were preplated for 1.5—2 h in plating medium (65% high glucose DMEM, 19% M-199, 10% horse serum, 5% fetal bovine serum, and 1% penicillin-streptomycin) to remove fibroblasts and endothelial cells. After preplating, cardiomyocytes were counted manually on a hemocytometer and cell density was adjusted to 3,000,000 cells/mL to seed onto the stretcher.

[00159] HUVECs and mouse cardiomyocytes seeding and live-labeling. Cassettes 80 were flipped (cooperating abutment members 82 at the bottom) and two 20 mm x 10 mm PDMS sample substrates 90 were placed onto shims 84, 86 of cassette 80, exposing PDMS sample substrate 90. This procedure ensured that only PDMS sample substrate 90 was surface activated in the oxygen plasma cleaner (Harrick Plasma) and shims 84, 86 remained hydrophobic. Cassettes 80 were surface activated in the oxygen plasma cleaner for 2 minutes. 0.025 μg/μl of fibronectin (Chem Cruz) or 0.25 μg/μl of rat tail Type I collagen (Millipore) were added onto cassette 80 immediately after plasma cleaning, followed by 1 -hour incubation at room temperature. The resulting fibronectin-coated cassettes 80 were then stored at 4°C until use. Collagen Type I was aspirated out from the surface of cassette 80, followed by 1-hour UV curing. Then the collagen-coated cassettes 80 were washed using phosphate-buffered saline (PBS; Sigma) and stored dry, covered with aluminum foil, at room temperature until use.

[00160] The 20 mm x 10 mm PDMS sample substrates 90 were removed from cassette 80 before seeding. 30 μL of HUVECs (250,000 cells/mL) or mouse cardiomyocytes (3,000,000 cells/mL) were placed onto PDMS sample substrate 90 of fibronectin-coated or collagen I-coated cassette 80, respectively, to form a droplet on top of PDMS sample substrate 90 (FIG. 15B).

[00161] Cassettes 80 with HUVECs droplets were cultured at 37°C for 2 hours to allow cell attachment, and then 8 mL of complete growth medium (Endothelial Cell Basal Medium- 2 C-22211, with Endothelial Cell Growth Medium 2 Supplement Pack C-39211, Promocell) was added into the petti dish to fully submerge the cassette. Submerged cassettes 80 were incubated for 16 hours at 37°C before imaging.

[00162] Cassettes 80 with cardiomyocytes were cultured at 37°C for 4 hours to allow cell attachment. Due to the long incubation time, 500 μL of cardiomyocyte plating medium was added to the bottom of the petri-dish to limit evaporation during incubation. After 4 hours, 12 mL of cardiomyocyte plating medium was added to the dish to fully submerge cassette 80. 16 hours post-plating, the plating medium was exchanged for cardiomyocyte maintenance medium (78% high glucose DMEM, 17% M- 199, 4% horse serum, 1 % penicillin/ streptomycin, 1 μm Ara-C, and 1 μm isoproterenol) and incubated at 37°C for 72 hours before imaging.

[00163] To label cells for live cell imaging, cassettes 80 were flipped back (cooperating abutment member 82 at the top) and Hoechst 33342 (nuclear stain; 2 μg/mL; Thermo Fisher Scientific) and CellMask Green (plasma membrane stain, lx working solution; Thermo Fisher Scientific) were added to the medium. Cells were incubated at 37°C in 5% CO₂ for 30 minutes in stain prior to imaging. [00164] Stretcher system setup and Imaging. Cassette 80 was transferred and positioned at the center of sample chamber 24 of stage bottom 22b with 4 mL of respective medium. Then, stage top 22a was placed onto stage bottom 22b. Two picomotor piezo linear actuators 40 (830 INF; Newport) with fully extended arms 42 were securely mounted at the motor mount 28. Cassette interface 60 was then inserted until the two ends fully contacted the cassette interface seating or rest 30 of stage top 22a (FIGS. 1B through 1H). The assembled microscope stage insert was placed onto an inverted microscope (see FIGS. 1A and II).

[00165] Picomotor piezo linear actuators 40 can, for example, be controlled by a LabView program with customizable velocity settings or manually operated by a joystick. Images at relaxed state and at the end of each stretch step were acquired using an inverted compound microscope (Leica) with a 63x/1.40NA oil immersion or a 25x/0.95NA water immersion objective lens, equipped with a spinning disk scanhead (Yokogawa) and a CMOS camera (Hamamatsu). Sequential images were acquired using a microscope automation software (^Manager 2.0).

[00166] Segmentation and strain analysis. Seedwater Segmenter was used to segment Xenopus epithelial cells. A custom FIJI macro was used to acquire cell ROIs and shape information from the segmented cells. PDMS deformation and tissue level strains were calculated using beta-spline based image registration (Image J plugin bUnWarpJ) and a custom ImageJ macro (StrainMapper); custom MATLAB m-code calculated cell level engineering strain rates. See Arganda-Carreras, I. et al. Consistent and elastic registration of histological sections using vector-spline regularization. CVAMIA: Computer Vision Approaches to Medical Image Analysis; 4241, 85-95 (2006) and Sonavane, P. R. et al. Mechanical and signaling roles for keratin intermediate filaments in the assembly and morphogenesis of Xenopus mesendoderm tissue at gastrulation. Development 144, 4363-4376, doi:10.1242/dev.155200 (2017), the disclosures of which are incorporated herein by reference.

[00167] 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

WHAT IS CLAIMED IS:

1. A system for applying strain to a sample, comprising: a cassette comprising a first end member spaced from a second end member, a sample substrate, which is stretchable, connected between the first end member and the second end member so that a portion of the sample substrate extends across a gap between the first end member and the second end member, the sample substrate being adapted to have the sample deposited on the portion thereof which extends across the gap, the first end member including a first cooperating abutment member; an assembly comprising a body having a sample chamber to removably receive the cassette therein, and a cassette interface, the cassette interface comprising a first abutment member which extends to contact the first cooperating abutment member when the cassette is in the sample chamber; and a first actuator in connection with the body, the first actuator comprising a first movable actuator arm configured to be placed in connection with the first abutment member to impart motion to the first abutment member and thereby impart motion to the first cooperating abutment member when the cassette is in the sample chamber to control a width of the gap, and thereby to control a width of the portion of the sample substrate extending across the gap.

2. The system of claim 1 wherein the second end member includes a second cooperating abutment member, and the cassette interface comprises a second abutment member which extends to contact the second cooperating abutment member when the cassette is in the sample chamber, and the system further comprises a second actuator in connection with the body, the second actuator comprising a second movable actuator arm configured to be placed in connection with the second abutment member to impart motion to the second abutment member and thereby impart motion to the second cooperating abutment member, when the cassette is in the sample chamber, to control the width of the gap and thereby the width of the portion of the sample substrate extending across the gap.

3. The system of claim 1 wherein the cassette interface is removably connectible to the body.

4. The system of claim 3 wherein the sample chamber is adapted to contain a volume of a liquid therein.

5. The system of claim 4 wherein the sample chamber comprises a transparent bottom section, and the body is adapted to be placed in operative connection with a stage of a microscope so that an objective of the microscope is aligned with the sample deposited upon the sample substrate via the transparent bottom section.

6. The system of claim 5 wherein the first end member of the cassette and the second end member comprise at least one extendible member connected therebetween which is configured to provide resistance to torsional motion of the first end member relative to the second end member.

7. The system of claim 5 wherein the first end member of the cassette comprises a first upper member attached to a first lower member, and the second end member comprises a second upper member attached to a second lower member, wherein the first lower member of the first end member and the second lower member of the second end member are connected via two spaced extendible members extending therebetween, the two spaced extendible members providing resistance to torsional motion of the first end member relative to the second end member.

8. The system of claim 7 wherein a first end of the sample substrate is positioned between the first upper member and the first lower member and a second end of the sample substrate is positioned between the second upper member and the second lower member, the sample substrate extending across the gap between the two spaced extendible members, wherein the first end of the sample substrate is optionally attached to at least one of the first upper member and the first lower member via an adhesive, the second end of the sample substrate is optionally attached to at least one of the second upper member and the second lower member via an adhesive, the first upper member and the first lower member are optionally attached via an adhesive, and the second upper member and the second lower member are optionally attached via an adhesive.

9. The system of claim 8 wherein the first lower member, the second lower member, and the spaced extendible members are formed monolithically.

10. The system of claim 8 wherein the second end member includes a second cooperating abutment member, the first cooperating abutment member extends upward from an upper surface of the first upper member, and the second cooperating abutment member extends upward from an upper surface of the second upper member.

11. The system of claim 8 wherein the first upper member and the first lower member of the first end member, and second upper member and the second lower member of the second end member have a thickness in the range of 38 — 127 μm.

12. The system of claim 2 wherein the cassette interface comprises a first end, a second end, a first flexible crossbeam extending between the first end and the second end, and a second flexible crossbeam extending between the first end and the second end and spaced from the first flexible crossbeam, the first abutment member extending downward from the first flexible crossbeam, the second abutment member extending downward from the second flexible crossbeam, the first moveable actuator arm being configured to contact the first crossbeam and the second moveable actuator arm being configured to contact the second crossbeam.

13. The system of claim 2 wherein the first actuator comprises a first piezo linear actuator and the second actuator comprises a second piezo linear actuator.

14. The system of claim 8 wherein the cassette interface comprises a first end, a second end, a first flexible crossbeam extending between the first end and the second end, and a second flexible crossbeam extending between the first end and the second end and spaced from the first flexible crossbeam, the first abutment member extending downward from the first flexible crossbeam, the second abutment member extending downward from the second flexible crossbeam, the first moveable actuator arm being configured to contact the first crossbeam and the second moveable actuator arm being configured to contact the second crossbeam.

15. The system of claim 5 wherein the microscope is an inverted brightfield or confocal microscope.

16. The system any one of claims 1 through 15 wherein the sample is a biological sample.

17. The system claim 16 wherein the sample comprises live tissue or live cells.

18. The system of any one of claims 1 through 15 wherein strain applied by the system comprises at least one of extension strain, compressive strain, and shearing strain.

19. The system of any one of claim 1 through 15 further comprising a control system in communicative connection with the first actuator and in communicative connection with the second actuator, when present, the control system comprising a processor system and a memory system in operative connection with the processor system, the memory system having one or more algorithms stored therein which are executable by the processor system.

20. The system of any one of claim 5 through 11, 14 or 15 further comprising a control system in communicative connection with the stage of the microscope, the control system comprising a processor system and a memory system in operative connection with the processor system, the memory system having one or more algorithms stored therein which are executable by the processor system to control a position of the stage of the microscope to align the objective with a determined portion of the sample during application of strain.

21. A method of stretching a sample, comprising: providing a system comprising: a cassette comprising a first end member spaced from a second end member, a sample substrate, which is stretchable, connected between the first end member and the second end member so that a portion of the sample substrates extends across a gap between the first end member and the second end member, the sample substrate being adapted to have the sample deposited on the portion thereof which extends across the gap, the first end member including a first cooperating abutment member, an assembly comprising a body having a sample chamber to removably receive the cassette therein, and a cassette interface, the cassette interface comprising a first abutment member which extends to contact the first cooperating abutment member when the cassette is in the sample chamber, and a first actuator in connection with the body, the first actuator comprising a first movable actuator arm configured to be placed in connection with the first abutment member to impart motion to the first abutment member and thereby impart motion to the first cooperating abutment member when the cassette is in the sample chamber to control a width of the gap, and thereby to control a width of the portion of the sample substrate extending across the gap; inserting the cassette including the sample in the sample chamber; placing the first abutment member in operative connection with the first cooperating abutment member after the cassette is inserted in the chamber, and imparting motion to the first abutment member via the first actuator.

22. The method of claim 21 wherein the second end member includes a second cooperating abutment member, and the cassette interface comprises a second abutment member which extends to contact the second cooperating abutment member when the cassette is in the sample chamber, and the system further comprises a second actuator in connection with the body, the second actuator comprising a second movable actuator arm configured to be placed in connection with the second abutment member to impart motion to the second abutment member and thereby impart motion to the second cooperating abutment member, when the cassette is in the sample chamber, to control the width of the gap and thereby the width of the portion of the sample substrate extending across the gap.

23. The method of claim 21 wherein the cassette interface is removably connectible to the body.

24. The method of claim 23 wherein the sample chamber is adapted to contain a volume of a liquid therein.

25. The method of claim 24 wherein the sample chamber comprises a transparent bottom section, and the body is adapted to be placed in operative connection with a stage of a microscope so that an objective of the microscope is aligned with the biological sample deposited upon the sample substrate via the transparent bottom section.

26. A microscope system, comprising: a microscope including a stage and an objective; and a strain system for applying strain to a sample, the strain system, comprising: a cassette comprising a first end member spaced from a second end member, a sample substrate, which is stretchable, connected between the first end member and the second end member so that a portion of the sample substrate extends across a gap between the first end member and the second end member, the sample substrate being adapted to have the sample deposited on the portion thereof which extends across the gap, the first end member including a first cooperating abutment member; an assembly comprising a body having a sample chamber to removably receive the cassette therein, and a cassette interface, the cassette interface comprising a first abutment member which extends to contact the first cooperating abutment member when the cassette is in the sample chamber; and a first actuator in connection with the body, the first actuator comprising a first movable actuator arm configured to be placed in connection with the first abutment member to impart motion to the first abutment member and thereby impart motion to the first cooperating abutment member, when the cassette is in the sample chamber, to control a width of the gap and thereby a width of the portion of the sample substrate extending across the gap.

27. The microscope system of claim 26 wherein the second end member includes a second cooperating abutment member, and the cassette interface comprises a second abutment member which extends to contact the second cooperating abutment member when the cassette is in the sample chamber, and the strain system further comprises a second actuator in connection with the body, the second actuator comprising a second movable actuator arm configured to be placed in connection with the second abutment member to impart motion to the second abutment member and thereby impart motion to the second cooperating abutment member, when the cassette is in the sample chamber, to control the width of the gap and thereby the width of the portion of the sample substrate extending across the gap.

28. The microscope system of claim 26 wherein the cassette interface is removably connectible to the body.

29. The microscope system of claim 27 wherein the sample chamber is adapted to contain a volume of a liquid therein.

30. The microscope system of claim 28 wherein the sample chamber comprises a transparent bottom section, and the body is adapted to be placed in operative connection with the stage of the microscope so that the objective of the microscope is aligned with the sample deposited upon the sample substrate via the transparent bottom section.

31. The microscope system of any one of claims 26 through 30 wherein the microscope is an inverted brightfield or confocal microscope.

32. A cassette for use in applying strain to a sample, comprising: a first end member spaced from a second end member, the first end member comprising a first upper member attached to a first lower member, the second end member comprising a second upper member attached to a second lower member, wherein the first lower member of the first end member and the second lower member of the second end member are connected via two spaced extendible members extending therebetween, the two spaced extendible members providing resistance to torsional motion of the first end member relative to the second end member, a sample substrate, which is stretchable, connected to the first end member, between the first upper member and the first lower member, at a first end of the sample substrate, and connected to the second end member, between the second upper member and the second lower member, at a second end of the sample substrate so that a portion of the sample substrate extends across a gap between the first end member and the second end member, the sample substrate being adapted to have the sample deposited on the portion thereof which extends across the gap.

33. The cassette of claim 32 wherein at least one of the first upper member and the second upper member comprises a cooperating abutment member via which force can be applied to the cassette.

34. The cassette of claim 32 wherein the first upper member comprises a first cooperating abutment member extending therefrom, and the second upper member comprises a second cooperating abutment member extending therefrom, via which force can be applied to the cassette.

35. The cassette of claim 32 wherein the at least one of the first upper member, the first lower member, and the first end of the sample substrate therebetween are attached via an adhesive, and wherein at least one of the second upper member, the second lower member, and the second end of the sample therebetween, are attached via an adhesive.

36. The cassette of claim 32 wherein the first lower member, the second lower member, and the spaced extendible members are formed monolithically.

37. The cassette of claim 32 wherein the first upper member and the first lower member of the first end member, and second upper member and the second lower member of the second end member have a thickness in the range of 38 - 127 μm.

38. The cassette of claim 32 wherein the first end of the sample substrate, connected between the first upper member and the first lower member, is laterally wider that the portion of the sample substrate which extends across the gap, and the second end of the sample substrate, connected between the second upper member and the second lower member, is laterally wider that the portion of the sample substrate which extends across the gap.