Optical measurement of labeled and label-free tissue contractility

Abstract

Systems, instruments, and methods are provided that measure contractility of tissue samples in a parallel and repeatable format based on measurements of optical signals emitted by the tissue samples, e.g., autofluorescence. The tissue samples are imaged during respective contraction periods, capturing the optical signals. In some embodiments, from the images, signal amplitude changes of the optical signals in a region of interest during the contraction period are determined, from which contraction force changes are determined. In some embodiments, from the images, signal amplitude profiles across the region of interest are determined, from which edge translations of fiducial markers and contraction forces are determined.

Description

OPTICAL MEASUREMENT OF LABELED AND LABEL-FREE TISSUE CONTRACTILITYCROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present disclosure claims the benefit of U.S. Provisional Patent Application No. 63 / 635,074, filed April 17, 2024, the entire disclosure of which is hereby incorporated by reference in its entirety.TECHNICAL FIELD

[0002] The present disclosure relates to the field of tissue research.BACKGROUND

[0003] Muscle contraction is a physiological process relevant to normal cardiac function, volitional movement and numerous other physiological processes critical to survival. Accordingly, methods for screening drug compounds for activity on tissue sample physiology are of great interest for discovering drug compounds that can correct pathophysiological behavior or for ensuring compounds do not have unwanted side effects. Contractions may be spontaneous or in response to a stimulus, but the underlying signaling cascade comprising a voltage signal producing calcium influx and muscle contraction is conserved. It is often desirable to conveniently measure voltage, calcium, and contractility easily in the same preparation.

[0004] Numerous systems have been developed or proposed to make contractility measurements. The devices include strain gauges, magnetic sensing, and machine vision approaches. These methods all have weaknesses impairing their widespread adoption, particularly for high throughput screening. Mechanisms using strain gauges in particular require tedious, time consuming measurements which are not well suited for screening large compound libraries. Magnetic sensing approaches have been described which are suitable for measurements of up to 24 tissues in a 24 well plate but are difficult to scale beyond that, e.g., due to crosstalk. Machine vision approaches for optically tracking muscle contraction require significant computational resources. High rates of spurious measurements further limit their applicability. These optical measurements are typically impaired by optical distortions caused by high numerical aperture (NA) optics, variations in lighting, and distortion of light through physical components of the system with different indices of refraction. Conversely, low NA telecentric lenses optimal for low distortion machine vision are not suitable for fluorescenceimaging. Therefore, due to these challenges, the foregoing methods generally cannot be combined to measure contractility, voltage, and / or calcium within one apparatus. This is problematic because there are biological relationships between contractility and other tissue properties which cannot be easily understood without contemporaneous measurement. For example, a muscle contracts because calcium binds to a protein. Contractility and calcium measurements therefore enable a complete understanding of that relationship.

[0005] Accordingly, there is a need for new approaches for measuring contractility of tissue samples, particularly approaches that enable measurement of contractility and other tissue characteristics in a common device based upon a unified working principle that enables better understanding between biological relationships.BRIEF SUMMARY

[0006] The present disclosure provides systems, devices, and methods for measuring contractility of un-labeled and labeled tissue samples based upon optical signals of the tissue samples. This stands in contrast to other methods of measuring contractility, including magnetic and computer vision-based methods. In some embodiments, the methods and devices also enable measurement of additional characteristics of the tissues, for example, calcium concentration, sodium concentration, potassium concentration, mitochondrial function, ReDox balance, voltage potential change, any physiological contraction force and associated profile parameters for both contraction and relaxation, beat frequency, tetanus parameters such as electrical stimulus profile, etc. Advantageously, this enables measurement of contractility and additional properties in a single instrument.

[0007] In an aspect, methods for measuring tissue contractility of un-labeled or labeled tissues align a multi-well cartridge with an imaging array, where each well contains a tissue sample between flexible and rigid tissue fixtures. The imaging array captures images of the tissue samples during contraction periods, and a controller determines signal amplitude changes of optical signals emitted by the tissues in regions of interest and corresponding contraction force changes. Optional features include determining the contraction forces changes based on length changes of the tissues, displaying results on a user interface, measuring multiple optical signals for calcium concentration and / or voltage changes in addition to contractility, and calibrating measurements using reference markers to determine absolute contraction forces from the signal amplitude changes.

[0008] In another aspect, systems for measuring tissue contractility of un-labeled and labeled tissues comprise a multi-well cartridge, an instrument with an enclosure containing an imaging array, and a controller. The system images tissue samples during contraction periods and determines signal amplitude changes and contraction force changes. Optionally, the system includes fiducial markers to facilitate determining absolute contraction forces. Optionally, the system is configured to measure multiple optical signals, e.g., to determine calcium concentration and / or voltage changes in addition to contractility.

[0009] In another aspect, methods for measuring tissue contractility of un-labeled and labeled tissues use fiducial markers on flexible tissue fixtures, where signal amplitude profiles across regions of interest are analyzed to determine edge translations of the fiducial markers and corresponding contraction forces.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] For a more complete understanding of the present disclosure, reference is now made to the following descriptions taken in conjunction with the accompanying figures, in which:

[0011] FIG. 1A is a side section view of a system for measuring contractility of tissue samples, according to an embodiment of the present disclosure.

[0012] FIG. IB is a detail view of FIG. 1A.

[0013] FIG. 2A is an image showing a tissue sample disposed between two tissue fixtures, with a region of interest overlaid over a portion of the tissue sample.

[0014] FIG. 2B shows a measured signal amplitude of an optical signal within the region of interest over time.

[0015] FIG. 3 is an image showing a tissue sample disposed between two tissue fixtures, with a region of interest and reference markers overlaid over a portion of the tissue sample.

[0016] FIG. 4 illustrates methods of measuring contractility of tissue samples according to the present disclosure.

[0017] FIG. 5A schematically shows a tissue sample disposed between two tissue fixtures in a relaxed state.

[0018] FIG. 5B schematically shows the tissue sample the tissue sample of FIG. 5 A in a contracted state.

[0019] FIG. 6A plots a signal amplitude profile from experimental data of a tissue sample having the configuration shown in FIG. 5A in the relaxed state.

[0020] FIG. 6B plots a signal amplitude profile from experimental data of a tissue sample having the configuration shown in FIG. 5B in a contracted state.

[0021] FIG. 7 illustrates additional methods of measuring contractility of tissue samples according to the present disclosure.DETAILED DESCRIPTION

[0022] The present disclosure provides systems, instruments, and methods for measuring contractility of un-labeled and labeled tissue samples based upon optical signals in a massively parallel format with relatively little computational resources.

[0023] FIG. 1A - FIG. IB show aspects of representative system 100 for measuring contractility of tissue samples, according to an embodiment of the present disclosure. The system 100 is configurable for use in connection with any methods for measuring contractility in un-labeled and labeled tissue samples according to the present disclosure.

[0024] Turning to FIG. 1A, the system 100 for measuring contractility of tissue samples includes an instrument 102 operably coupled to a controller 104 that, in some embodiments, forms part of the instrument 102 and in other embodiments, is at least partially external to the instrument 102. The system 100 is configured to execute methods described herein that measure relative and / or absolute contractility of tissue samples in a multi -well cartridge 106. In some embodiments, the system 100 includes the instrument 102 and the controller 104. In some embodiments, the system 100 also includes the multi-well cartridge 106. In use, an un- labeled or labeled tissue sample 108 (see FIG. IB and FIG. 2A) extends between the tissue fixtures 110a, 110b in each well of the multi -well plate 112, e g., suspended in a polymeric matrix.

[0025] Instrument 102 comprises a light-tight enclosure 114 containing a movable stage 116 disposed above an imaging array 118 and an illumination system 120. The stage 116 is configured to receive the multi -well cartridge 106 thereupon. The stage 116 is movable such that the tissue samples are positioned in a common plane within a focal plane of the imaging array 118 for imaging optical signals emitted by the tissue samples.

[0026] As used herein, "tissue sample" means a three-dimensional (non-monolayer) construct formed of contractile biological tissue comprising muscle cells capable of generating forcethrough contraction, including but not limited to skeletal muscle tissue, cardiac muscle tissue, and smooth muscle tissue. The tissue sample may be native tissue excised from an organism or an engineered tissue construct generated through cell culture techniques, for example tissue samples cultured from induced pluripotent stem cells, primary cells, or cell lines, including engineered muscle tissues and engineered heart tissues. Representative tissue samples include skeletal muscle tissues comprising striated muscle fibers, cardiac muscle tissues comprising cardiomyocytes arranged in functional syncytium capable of spontaneous contraction (e g., engineered heart tissues (EHTs)). In some embodiments, the tissue samples are cast using the systems and according to the methods, for example, described in U.S. Patent Application Number US 2023-0109347 Al, which is herein incorporated by reference in its entirety.

[0027] As used herein, "contraction period" means a time interval during which a tissue sample undergoes one or more contractile event, including at least the contraction phase between a relaxation state and a maximum contracted state where the tissue sample shortens from a relaxed length to a contracted length, optionally including the subsequent relaxation phase where the tissue sample returns to its relaxed length. The contraction period may include numerous contraction-relaxation cycles and is not limited to the period of time of a single contraction cycle. In some embodiments, the contraction period is the period of time between a relaxation state and a successive maximum contracted state of a tissue (a half contraction cycle), during which time the tissue shortens in length. In some embodiments, the contraction period is the period of time between two consecutive maximum contracted states (a full contraction cycle), during which time the tissue lengthens and then shortens. In some embodiments, the contraction period includes numerous full contraction cycles. In some embodiments, the contraction period results from spontaneous contraction or in response to an external stimulus such as electrical or optical stimulation. For cardiac tissues, the contraction period includes at least half of a single cardiac cycle or beat. For skeletal muscle tissues, the contraction period includes at least a single twitch contraction or a sustained tetanic contraction in response to electrical stimulus.

[0028] The systems 100 measure contractility of the tissue samples based upon the working principle that tissue samples largely comprise incompressible liquid; accordingly, the tissue samples are modeled as solid bodies having a fixed volume. For example, the total volume of a tissue sample remains relatively constant whether the tissue is in a relaxed state or a contracted state (e.g., due to spontaneous contraction or electrical stimulus). In a relaxed state, the tissue'svolume is distributed along a relaxed length Lo. In a maximum contracted state, the same volume is confined to a reduced length Lmaxc < Lo. Accordingly, in tissues with endogenous fluorophores and optionally exogenous fluorophores (including both un-labeled and labeled tissues), the location of those fluorophores moves as the tissue contracts. As relevant to this disclosure, when a tissue is in a contracted state, more of the fluorophores exist within a static three dimensional region of interest (ROI) having a length LROI < Lo, LmaXc overlaid over the tissue (e.g., an ROI encompassing a central portion of the tissue), as compared to the relaxed state. Restated, if a static ROI that partially encompasses the tissue sample is observed in both the relaxed and contracted states, then a greater number of fluorophores exist within the ROI in the contracted state as compared to the relaxed state. This is verifiable by imaging optical signals emitted by the fluorophores. Specifically, given the greater number of fluorophores within the ROI when the tissue is in the contracted state as compared to the relaxed state, the tissue will emit a higher-amplitude optical signal within the ROI in the contracted state, all else equal. Accordingly, there is a measurable relationship between the contractility (displacement) of the tissue and the optical signal. And because the contractile tissue samples are suspended between two beam-like or post-like tissue fixtures, there is a relationship between the displacement of the tissue during contraction and the contraction force exerted by the tissue on the beam(s), according to beam theory.

[0029] As used herein, a tissue is 'un-labeled' if it has not been treated with any extrinsic optical markers, fluorescent dyes, stains, proteins, or other compounds that emit an optical signal. Un-labeled tissues contain endogenous fluorophores that emit optical signals.. In embodiments utilizing un-labeled tissue samples, the optical signal used to measure contractility arises from autofluorescence due to various endogenous fluorophores native to the tissue including but not limited to Nicotinamide Adenine Dinucleotide (NADH), extracellular matrix, myoglobin, hemoglobin amino acids, proteins, or a combination thereof.

[0030] As used herein, a tissue is 'labeled' if it has been treated with one or more extrinsic optical markers, such as fluorescent dyes, calcium detection fluorophores, voltage sensitive fluorescent dyes, or other compounds that emit optical signals or engineered to express proteins which produce an optical signal, including but not limited to Green Fluorescent Protein (GFP) and luciferase. For example, a tissue is considered labeled when loaded with calcium dyes that become brighter upon binding to calcium, treated with voltage-sensitive fluorescent dyes that respond to changes in membrane potential, treated with a lipophilic dye to label plasmamembrane, or engineered to express GFP. The optical signals from these labels may be measured at specific wavelengths corresponding to the emission spectrum maximum for the particular label used, such as green fluorescence for tissues labeled with Fluo-4, DiOCis(3), various other green fluorophores or engineered to express GFP, or red fluorescence for tissues labeled with Tetramethylrhodamine Methyl ester (TMRM), Tetramethylrhodamine Ethyl Ester Perchlorate (TMRE), various other red fluorophores, or tissues engineered to express mCherry.

[0031] In embodiments utilizing un-labeled and labeled tissue samples, the optical signal used to measure contractility arises from fluorescence, autofluorescence, transmitted light, reflected light, scattered light, chemiluminescence, bioluminescence, phosphorescence, interference, 2ndorder or higher harmonic generation, or any other suitable method for generating an optical signal from a tissue sample and / or its surrounding medium, where the amplitude of the signal is perturbed by a change in tissue geometry. To clarify, in some such embodiments, different labels can be utilized as different channels to measure different types of information. For example, in an embodiment, a tissue's autofluorescence emit a first optical signal having a first wavelength, whereas calcium detection fluorophores emit a second optical signal having a different second wavelength, and a voltage sensitive fluorescent dye emits a third optical signal having a different third wavelength.

[0032] Having described the working principle of the system 100, its elements and methods will now be detailed.

[0033] Multi-well cartridge 106 is an assembly comprising a multi-well plate 112 coupled with an array of tissue fixtures (e.g., 110a, 110b) such that a plurality of the tissue fixtures 110a, 110b extend into each well. The multi-well plate 112 is constructed from a transparent or translucent polymer having a plurality of wells 122 arrayed in a Society For Biomolecular Screening (SBS) - compatible layout formed of polystyrene, polycarbonate, PET, or similar material and having wells 122 (e.g., 24, 96, 384, 1536, or other number of wells 122), each being sized and otherwise configured to receive a plurality of tissue fixtures 110a, 110b therein. To facilitate imaging, the wells 122 each have a closed and transparent bottom which may be the same as the material comprising the multi-well plate 112 or may be comprised of glass, polyolefin, or other materials suitable for optical imaging. Representative multi-well plates 112 include, but are not limited to, NanoSurface plates sold by Curi Bio, Inc. of Seattle, Washington, USA and Corning® and Falcon® cultureware plates sold by Coming Incorporated of Corning, New York, USA.

[0034] The tissue fixtures 110a, 110b generally include cantilever beams or posts, e.g., flexible posts and rigid posts. In some embodiments, at least one flexible post 110a and at least one rigid post 110b extends into each well 122 of the multi-well plate 112 such that the respective distal ends of the flexible and rigid posts are adjacent and spaced apart. In use, a tissue sample is suspended in an extracellular matrix within each well 122 such and attaches between the distal ends of the flexible post 110a and rigid post 110b. Although the representative embodiments described herein include two tissue fixtures 110a, 110b having different properties in a single well 122 (e.g., a rigid post and a flexible post having different stiffness properties in each well 122), in some embodiments, at least two flexible tissue fixtures extend into each well 122.

[0035] The term “rigid post” / rigid tissue fixture and “flexible post” / flexible tissue fixture may be defined absolutely and / or relatively. For example, in some embodiments, each rigid tissue fixture (e.g., tissue fixture 110b) has a greater force-to-displacement relationship, e.g., at one point along the length thereof (e.g., a greater stiffness at the distal end) and / or a different Young's modulus than the flexible tissue fixture 110a (e.g., flexible post 110a) in the same well 122. In some embodiments, the foregoing relationship (e.g., between the stiffness of the relatively rigid tissue fixture 110b and the relatively flexible tissue fixture 110a) have a ratio of lx - l,000x, e.g., about lx to about 500x, about 5x to about 500x, or about lOx to about 300x. In absolute terms, by way of example, not limitation, in some embodiments, each relatively rigid tissue fixture 110b has a stiffness of about 1,000 N / m to about 10,000 N / m, for example about lON / m to about 30N / m (e.g., about 12N / m or 24N / m). In some embodiments, each relative flexible tissue fixture 110a has a stiffness of about 0. IN / m to about 5N / m, e.g., about 2N / m. Additional characteristics of the tissue fixtures are described in U.S. Patent Application Number US 2023-0109347 Al, which is again incorporated by reference in its entirety.

[0036] Instrument 102 is designed to illuminate and image optical signals emitted from tissue samples 108 in order to provide optical signal data and to determine contractility of the tissue samples 108 from that signal data.

[0037] Enclosure 114 is an opaque metallic or polymeric housing creating a local environment in which the multi -well cartridge 106 can be repeatedly imaged by the imaging array 118 and illuminated by the illumination system 120 without interference from outside light. The stage 116, imaging array 118, and illumination system 120 are arranged within the enclosure 114 in a configuration which provides unhindered access, via a lid 124, to the top of the multi-wellcartridge 106. This enables fitting of additional instrumentation, e.g., electrical stimulators, optogenetic stimulators, liquid handling robots, etc. A rigid frame 126 supports the stage 116 above the imaging array 118. A carriage 128 enables z-axis movement of a camera 130 of the imaging array 118 relative to the stage 116.

[0038] Stage 116 comprises a flat and stable platform 132 upon which the multi-well cartridge 106 is positioned in use. A sample aperture extending through the platform 132 is sized such that when the multi -well cartridge 106 is positioned upon the platform 132 over the sample aperture, the wells of the multi-well cartridge 106 are unobstructed by the platform 132 and can be imaged by the imaging array 118. Stage 116 is an XY stage configured with one or more drives (e.g., a stepper motor) operable to translate the platform 132 in x-, y- , and (optionally) z- dimensions. Representative and non-limiting stages 116 include the SCAN IM™ line of microscope stages manufactured by Marzhauser Wetzlar GmbH & Co. KG of Germany.

[0039] An optional heater plate 134 (see FIG. 2B) is disposed upon the stage 116 and operably coupled to the controller 104 in some embodiments. The multi-well plate 112 sits atop the heater plate 134, which is comprised of an electrically conducting film deposited on a glass substrate so as not to interfere with imaging of the multi -well cartridge 106 by the imaging array 118. In some embodiments, the heater plate 134 comprises a glass substrate having a lOOnm thick indium tin oxide film deposited thereon.

[0040] Illumination system 120 comprises an array of light sources selected, positioned, and oriented to uniformly illuminate the wells 122 of the multi-well cartridge 106 with low temporal noise (e.g., RMS optical noise < 0.05% DC-lMHz) in order to facilitate imaging of the optical signals emitted from the tissue samples 108. In the embodiment shown, the illumination system 120 comprises four LED modules 136, each comprising an LED light source 138 (e.g., models Mic-LED-470CG, UHP-T-WSS-DI, and CombiLED manufactured by Prizmatix, Limited of Israel) that projects light through one or more bandpass excitation filters 140 (e.g., model ET470 / 40X manufactured by CHROMA® of Bellows Falls, Vermont, USA) and a high NA aspheric lens 142. In some embodiments, the aspheric lens 142 has a 50mm diameter, F=40mm, and NA=0.60. In some embodiments, one or more of the LEDs includes a plurality of light emitting diodes. Accordingly, “LED" does not limit the present disclosure to single diode light sources. In some embodiments, the LEDs are configured to emit white light at wavelengths ranging from about 400nm to about 700nm. In some embodiments, the illumination system 120 includes one or more LEDs configured to emit light at a firstwavelength or combination of wavelengths (e.g., white light) and one or more second LEDs configured to emit light at a second wavelength (e.g., blue light). In some embodiments, one or more of the LEDs is configured to emit light at two or more wavelengths (e.g., white and blue light).

[0041] In the illustrated embodiment, the illumination system 120 comprises four LED modules 136, i.e., one positioned proximal to each corner of the rectangular stage 116, providing an illumination power of at least 100 mW or at least 120 mW measured at the tissue sample. In some embodiments, the illumination system 120 includes a greater or fewer number of light sources 138, and a higher or lower illumination power (depending on the application).

[0042] To improve thermal stability, each LED module 136 comprises a heat sink 144 with a plurality of radial cooling fins disposed below the base. Fans 146 circulate air through the enclosure 114, thus removing thermal energy from the illumination system 120. Consequently, illumination system 120 exhibits a high degree of thermal stability conducive to reproducible imaging.

[0043] Imaging array 118 collects optical signals emitted from the tissue samples 108. The imaging array 118 is operably coupled to the controller 104 and includes an optical train 148 comprising a high numerical aperture (NA) photographic collector lens 150 designed for computer vision photography and at least one computer vision camera 130 in a configuration to image all wells 122 of the multi-well cartridge 106 through the sample aperture of the stage 116 (and through the heater plate 134 in equipped embodiments). Optionally, the optical train 148 includes one or more bandpass emission filters 152, dichroic mirrors 154, and accessory lense(s). Representative collector lenses 150 include machine vision lenses sold under the NAVITAR® brand by Ametek, Incorporated of Berwyn, Pennsylvania, USA, for example SKU DO-2595. Camera 130 is a low-noise, high speed, scientific imaging camera comprising a plurality of image sensors, for example scientific CMOS cameras. Representative cameras 130 include KINETIX™ models sold under the Teledyne Photometries brand by Teledyne Technologies Incorporated of Thousand Oaks, California, USA. Representative emission filters 152 include, e.g., models ET525 / 50M, ET520 / 40M , AT620 / 60M manufactured by CHROMA® of Bellows Falls, Vermont, USA). Representative dichroic mirrors 154 include, e.g., models ZT488 / 561rpc, ZT532 / 660rpc, and ZT355 / 650rpc manufactured by CHROMA®.

[0044] In some embodiments, imaging array 118 and illumination system 120 have a fixed excitation and emission wavelength. Accordingly, the light source 138 and bandpass excitationfilter 140 are selected to excite the tissue samples at the selected wavelength. Likewise, the optical train 148 includes an emission filter 152 selected to pass the selected wavelength to the camera 130.

[0045] In some embodiments, imaging array 118 and illumination system 120 are configured for selectable excitation and emission wavelengths for sequential multicolor imaging of different wavelengths (e.g., to image contractility, voltage, and / or calcium concentration via different optical channels). In such embodiments, the bandpass excitation filter 140 and the emission filter 152 each comprises, e.g., a variable bandpass excitation filter or a filter wheel. In some such embodiments, the filters 152, 140 are mounted in a motorized united operably coupled to the controller 104, allowing the user to select from among several filters, e.g., via a user interface.

[0046] In some embodiments, imaging array 118 and illumination system 120 are configured for simultaneous multicolor imaging with one or more cameras. In such embodiments, the light source 138 comprises a multi-wavelength excitation source that emits light through variable bandpass excitation filter 140 and optional diaphragm or pinholes and the aspheric lens 142. In some embodiments, an accessory lens is placed after collector lens 150 forming an infinite conjugate lens system. In such configurations, the collected light is subsequently split by a dichroic mirror 154 in short pass (SP) and longpass (LP) wavelength bands. The SP and LP bands are subsequently passed through appropriate bandpass emission filter 152, and imaged by an auxiliary lens onto one or more cameras 130. By way of illustration, not limitation, a tube lens may be placed into a 4F optical configuration with the collector lens 150, split into LP and SP bands by dichroic mirror 154, with SP band filtered through a bandpass filter and imaged by another tube lens onto a first camera of camera(s) 130. The LP band may be filtered through a bandpass filter and imaged via another tube lens onto a second camera of camera(s) 130. In another embodiment, via appropriately aligned dichroic mirror 154, the SP and LP bands are imaged in tandem onto a camera 130.

[0047] Controller 104 includes circuitry, computing elements, and logical instructions that control the imaging array 118, illumination system 120, and other aspects of the system 100, including imaging array 118, illumination system 120, stage 116, etc. In some embodiments, aspects of the controller 104 are disposed locally in the instrument 102 (e.g., as a printed circuit board, internal control unit, electronic control unit, on-board computer, etc.) disposed externally to the instrument 102 in remote computing resources 156 (e.g., a server, laptop,cloud service). Aspects of the controller 104 disposed locally on the instrument 102 include circuitry elements for control of the illumination system 120 and imaging array 118, which in some embodiments are disposed on one or more printed circuit boards. Such elements include, for example: LED drivers and power control circuits for controlling illumination intensity of the LED modules 136; analog-to-digital converters for processing optical signals from the imaging array 118; digital signal processors for real-time image processing; multiplexers and demultiplexers for handling multiple optical signal channels; temperature control circuits for the heater plate 134; motor control circuits for the stage 116 movement; voltage regulators and circuit protection components; timing and synchronization circuits for coordinating illumination and image capture; and data acquisition interface circuits for communicating with external elements of the controller 104 and remote computing resources 156. Controller 104 is configured to display information on a user interface 162 according to the methods described herein.

[0048] The illustrated implementation of controller 104 includes a processor 158 that includes one or more central processing units (CPU) and / or graphics processing units (GPU), such as a processor from the CORE® family of processors available from Intel Corporation, a processor from the ATHLON® family of processors available from Advanced Micro Devices, Inc., a processor from the POWERPC® family of processors available from the AIM Alliance, etc. However, the present disclosure is not restricted by the architecture of the controller 104 as long as the same supports the operations as described herein. For example, in some embodiments, controller 104 comprises one or more special purpose processors, such as an application specific integrated circuit (ASIC), a GPU such as from the BLACKWELL® and HOPPER® families of GPUs available from NVIDIA®, a field programmable gate array (FPGA), etc.

[0049] Memory 160 includes a non-transitory computer-readable memory, including read-only memory (ROM) devices, random access memory (RAM) devices, one or more hard disk drives (HDDs), flash memory devices, solid state drives (SSDs), and / or other devices configured to store data in a persistent or non-persistent state, or a combination of different memory devices.

[0050] Memory 160 stores logic comprising instructions that, when executed by the processor 158, cause the system 100 to perform operations that determine the relative and / or absolute contractility of the tissue samples 108, optionally in connection with other parameters such as calcium concentration and voltage. In particular, the logic comprises instructions that, whenexecuted by the processor 158, cause the system 100 to perform any of the operations shown and / or described with respect to FIG. 2A - FIG. 7.

[0051] For example, when the multi-well plate 112 is aligned on the stage 116 such that the tissue samples 108 are within the field of view and image plane of the imaging array 118, the controller 104 is configured to image, with the imaging array 118, each of the tissue samples during respective contraction periods. In some embodiments, the imaging array 118 images all wells 122 at once.

[0052] As described below with respect to FIG. 2A - FIG. 4, based on the images, the controller 104 determines, for each of the tissue samples 108: a signal amplitude change during the contraction period of an optical signal emitted by the tissue sample in a region of interest; and a contraction force change during the contraction period based on the signal amplitude change. In some embodiments, the controller determines an absolute contraction force based on the signal amplitude change and movement of the fiducial marker relative to one or more reference markers. Accordingly, the system 100 determines the relative and / or absolute contraction force change during the contraction period. Additionally, this methodology enables measurement of beat frequency in cardiac tissues.

[0053] In some embodiments, described below with respect to FIG. 5A - FIG. 7, based on the images, the controller 104 determines signal amplitude profiles at different time points based on an optical signal emitted from the tissue sample in a region of interest, wherein each of the signal amplitude profiles plots a signal amplitude of the optical signal across the region of interest; an edge translation of a fiducial marker based the plurality of signal amplitude profiles; and a contraction force of the tissue sample based on the edge translation.Accordingly, the system 100 determines the absolute contraction force during the contraction period.

[0054] As described above, the controller 104 includes logical instructions that, when executed by the processor 158, cause the system 100 to perform operations including sensing, with the imaging array 118, an optical signal emitted from each of the tissue samples during a contraction period. Time series images captured by the imaging array 118 encode characteristics of said optical signals over time, e.g., as illuminated pixels. Based on these images, the controller 104 determines contractility of the tissue samples as described below.

[0055] FIG. 2A shows a representative image from high frame-rate video captured by the imaging array 118 of system 100 of FIG. 1A - FIG. IB. In particular, the image of FIG. 2Acaptures autofluorescence signals emitted by an un-labeled tissue sample 108. As the imaging array 118 images a plurality of the wells 122 of the multi-well cartridge 106, the image of FIG. 2A has been cropped and magnified (e.g., by the controller 104) to show a single well 122.

[0056] In particular, FIG. 2A shows the un-labeled tissue sample 108 disposed between two tissue fixtures 110a, 110b. In this embodiment, the tissue sample 108 is a 3D engineered heart tissue (EHT) having a length Lo of approximately 4mm to about 8mm and suspended in an extracellular fibrin matrix. In the illustrated embodiment, the tissue sample 108 is cast with the tissue fixtures 110a, 110b in a casting plate before transference to the multi -well plate 112 for evaluation by the instrument 102. Casting systems include Mantarray™ system sold by Curi Bio, Incorporated of Seattle, Washington, USA. The non-limiting EHT tissue casting protocol involves preparing a fibrin-based extracellular matrix containing cardiomyocytes and fibroblasts in a specific ratio (e.g., at a ratio of 500,000 cardiomyocytes and 75,000 fibroblasts) cast in 156 pL of hydrogel. The process includes surface modification of the tissue fixtures 110a, 110b using poly(ethyleneimine) and glutaraldehyde for tissue adhesion, followed by combining the cell-hydrogel mixture with thrombin solution in a casting plate and allowing the tissue formation between tissue fixtures 110a, 110b during an incubation period (e.g., 80- minutes at 37°C). After formation, the tissue samples 108 are transferred to the multi-well plates 112 with maintenance media.

[0057] As a contractile tissue, tissue sample 108 exhibits periodic contractions spontaneously and / or in response to an electrical or optical stimulus such as may be adapted to the multi-well cartridge 106 within the instrument 102. According, FIG. 2A captures the tissue sample 108 in a relaxed state or in a state of contraction. In the relaxed state, tissue sample 108 has a relaxed length Lo between the tissue fixtures 110a / 110b and an average radius ro between the tissue fixtures 110a / 110b.

[0058] In FIG. 2A, tissue fixture 110a includes a fiducial marker 164. However, said fiducial marker 164 is not a necessary element to measure the relative or absolute contractility of the tissue sample 108, as will be described below. As used herein, a 'fiducial marker' means a physical feature incorporated into or embedded within a tissue fixture that is optically distinguishable from the distal end of the tissue fixture. The fiducial marker has a well-defined geometric shape and optical properties that create sharp contrast with the distal end of the tissue fixture, such as a dark-colored rectangular marker contrasting with a light-colored distalend of the tissue fixture, enabling clear imaging and precise tracking of the tissue fixture's position.

[0059] In FIG. 2A, the tissue sample 108 is overlaid or superimposed (by controller 104) with a static region of interest 166 encompassing a portion of the tissue sample 108. The region of interest 166 represents a theoretical three-dimensional region occupied by the tissue sample 108 in a relaxed state and during a contraction period. Region of interest 166 has a length LROI < Lo, Lmaxc (i.e., shorter than the length of the tissue sample 108 at maximum contraction) and a radius TROI > ro, rmaXc (i.e., a radius greater than the radius of the tissue sample 108 at maximum contraction). The region of interest 166 has a three dimensional volume, but may be represented in images as two-dimensional area. For example, in FIG. 2A, region of interest 166 is shown as a two-dimensional area in which an optical signal emitted by the tissue sample 108 is measured during a contraction period. In any embodiment, the controller 104 may cause the user interface 162 to display one or more of the images overlaid with the region of interest 166, e.g., a two-dimensional shape representing the region of interest 166.

[0060] In this example, the region of interest 166 is represented in the image as a static rectangle encompassing a portion of the tissue sample 108 disposed between the two tissue fixtures 110a, 110b. In some embodiments, the region of interest 166 has a different shape, e.g., elliptical. The region of interest 166 is sized and positioned such that a greater proportion of the overall volume of the tissue sample 108 is bounded by the region of interest 166 in a contracted state (e.g., partial or complete tetanus state) as compared to the relaxed state. Accordingly, a greater number of fluorophores of the tissue sample 108 are bounded by the region of interest 166 in any contracted state as compared to the relaxed state. In FIG. 2 A, the region of interest 166 is localized on an elongate central portion of the tissue sample 108. In some embodiments, the region of interest 166 has a geometric center localized on a center of the tissue sample 108 in the relaxed state, e.g., a half way point between the two tissue fixtures 110a, 110b. The region of interest 166 does not encompass the entire tissue sample 108. In some embodiments, the region of interest 166 encompasses at least part of a single one of the tissue fixtures 110a, 110b, e.g., the at least part of the relatively rigid tissue fixture 110b.

[0061] The image of FIG. 2A is representative of the type of image encoding optical signals emitted by the tissue sample 108 and forming the basis of analytical methods that will now be further described.

[0062] As shown in FIG. 2A, the tissue sample 108 is an elongate, three-dimensional tissue sample suspended between two tissue fixtures 110a, 110b (at least one of which tissue fixtures 110a / 110b is relatively flexible as compared to the other tissue fixture). According to embodiments of the present disclosure, tissue sample 108 is modeled, in a relaxed state, as a cylindrical volume of incompressible fluid with an initial length Lo and a radius ro in a central region thereof between the tissue fixtures 110a / 110b. In some embodiments, the radius ro is the average radius of the tissue sample 108 between the tissue fixtures 110a / 110b in the relaxed state. To facilitate nomenclature, Lo and ro are represented simply as L and r in the following equations. Accordingly, a volume, V, of the tissue sample 108 is:

[0063] Equation 1) V = nr2L

[0064] Because the tissue sample 108 is modeled as an incompressible fluid, there is no change in volume V during a contraction of the tissue sample 108. Accordingly:

[0065] Equation

[0066] As shown below in FIG. 2B, the optical signal emitted from tissue sample 108 (e.g., autofluorescence) is imaged within the region of interest 166 between the two tissue fixtures 110a / 110b. The region of interest 166 is sized in relation to the corresponding tissue sample 108 and has a length ZROI < L and radius FROI > r. An amplitude of the optical signal in the region of interest 166, F, changes as the tissue sample 108 contracts either spontaneously or in response to an external stimulus (e.g., an electrical stimulus). As described above, this occurs because a greater number of the fluorophores are located within the region of interest 166 during a contraction. Therefore, F is proportional to the volume, V, of the tissue sample 108 located in the region of interest 166. Accordingly:

[0067] Equation 3) F = cnr2LROhwhere c is a proportionality constant determined by the excitation intensity of the LED module 136, density and intrinsic brightness of fluorophores in the tissue sample 108, characteristics of the collection optics (e.g., collector lens 150), and the camera 130 sensitivity and settings.

[0068] Further, a change in the optical signal amplitude, F, in the region of interest 166 produced by a contraction of the tissue sample 108 is given by the derivative of F with respect to L. Substituting the result of Equation 2 yields:

[0069] Equation

[0070] Rearranging Equation 4 and dividing by Equation 3 yields:

[0071] Equation

[0072] While Equation 5 models the relationship between changes in the optical signal amplitude and length in media with a relatively high mean free path, in a highly scattering media such as muscle tissue, light scattering prevents fluorophores from the interior of the tissue sample 108 from contributing to the measured fluorescence. However, in the case where mean free path approaches zero, the optical signal amplitude is proportional to the surface area of the tissue sample 108. Accordingly, the signal amplitude, F, in such media is modeled as follows:

[0073] Equation 6) F = 2cnrLR0I

[0074] A similar derivation to the previous case for highly scattering tissue, e.g., muscle tissue, yields:

[0075] Equation 7) — = —F 2L

[0076] In some embodiments, a tissue sample 108 with an intermediate scattering cross section is modeled as a linear combination of Equations 5 and 7, resulting in an intermediate scaling factor:

[0077] Equation 8) - = c -y

[0078] However, it has been discovered that the scattering cross section for visible light in muscle tissue is sufficiently high that Equation 7 accurately models the relationship between signal amplitude and length.

[0079] Accordingly, a change in the signal amplitude of an optical signal emitted from a tissue sample 108 during a contraction period is directly related to a change in the length of the tissue sample 108. Restated, F is minimized when the tissue sample is in a relaxed state, and maximized when the tissue sample is in a maximum contracted state. Accordingly, in some embodiments (e.g., cardiac tissue contracting spontaneously), when F is plotted against time, minima and maxima respectively correspond to relaxation states and maximum contracted states of the tissue. Accordingly, the difference or change between successive minimum and maximum F values of a tissue conveys information about the strength and health of the tissue. Likewise, changes over time in maximum F values (amplitude peaks), and changes over time in local minimum F values (amplitude troughs), convey similar information. Further still,changes over time in the difference between successive minimum and maximum F values convey similar information.

[0080] In embodiments where each tissue sample 108 is suspended between a pair of relatively flexible and rigid tissue fixtures 110a, 110b, the change in length of the tissue sample 108 is, in turn, directly related to the displacement in the distal end of the flexible tissue fixture 110a, which is directly related to the contraction force exerted by tissue sample 108 on said tissue fixture 110a according to beam theory. For example, one representative beam theory equation is:F L3

[0081] Equation 9) 8max—c lxat x = L3EI

[0082] In Equation 9, x is a location of perpendicular force on a beam (i.e., the location along the flexible tissue fixture 110a where the contraction force is applied by the tissue sample 108), dmax is the maximum displacement of the distal end of the flexible tissue fixture 110a (i.e., the maximum length change of the tissue sample 108), Fcis the contraction force of the tissue sample 108 acting on the flexible tissue fixture 110a, Lnxis the length of the tissue fixture 110a, E is the elastic modulus of the tissue fixture 110a (e.g., Young's Modulus), and 1 is the second moment of area of the cross section of the tissue fixture 110a.

[0083] Accordingly, a change in the signal amplitude of an optical signal emitted from a tissue sample 108 during a contraction period is directly related to a change in the contraction force exerted by the tissue sample 108 on the relatively flexible tissue fixture 110a. For example, the minimum signal amplitude and the next-in-time maximum signal amplitude correspond, in time and relative value, to the relaxation state and maximum contracted state of the tissue, respectively. In this way, measuring the relative or absolute difference or change in these two signal amplitude values enables direct measurement of contraction force changes of the tissue sample 108, i.e., relative contraction force changes (e.g., expressed as a percentage). Stated differently, a decrease in the signal amplitude (a negative signal amplitude change) indicates a negative change in the volume of the tissue sample 108 located in the region of interest 166, i.e., the tissue sample 108 has become weaker, for example, in the presence of a drug candidate. Alternatively, a decrease in the change between local signal amplitude maxima and minima over time could indicate that the tissue sample 108 has become weaker. While the signal amplitude change and contraction force change are linked by a length change of the tissue sample, in practice, contraction force changes may be directly inferred from the signal amplitude change.

[0084] The systems 100, instruments 102, and methods provide actionable information that is useful, for example, in drug discovery and tissue engineering applications. Firstly, it represents a novel and robust method to measure tissue contractility that does not rely on complex magnetic or optical systems. Secondly, this approach is readily integrated into devices that are also configured to measure other electrophysiological properties, e.g., calcium concentration and voltage. Third, the foregoing methodology is effective on un-labeled tissues, which promotes repeatability and efficiency. Accordingly, the systems 100, instruments 102, and methods described herein enable a direct understanding of biological relationships between optical signals (e.g., fluorescence) and physiological properties of tissues.

[0085] FIG. 2B shows a measured signal amplitude (in arbitrary units) of the autofluorescence signal emitted by the tissue sample 108 in the region of interest 166 of FIG. 2A, plotted against frames captured by the camera 130 at a frame rate of 250 frames per second. The plotted signal amplitude represents the average value of the pixels located within the region of interest 166. In some embodiments, the system 100 displays plots such as FIG. 2B on the user interface 162

[0086] From the signal amplitude plotted in FIG. 2B, signal amplitude changes can be directly determined over time from the underlying data. As described above, changes in the signal amplitude are directly proportional to changes in fluorophores of the tissue sample 108 located within the region of interest 166 over time. And as demonstrated above, e.g., via Equation 7 and Equation 9, changes in signal amplitude are directly related to changes in the length of the tissue sample 108 and changes in the contraction force. Restated, signal minima and maxima shown in FIG. 2B respectively represent local minimum and maximum contraction forces of the tissue sample 108.

[0087] Advantageously, this methodology is effective with un-labeled tissue samples, has few failure modes, is robust to lighting variations, computer vision inaccuracies, and other factors, and does not require significant computing resources. Following from these advantages, in some embodiments, the relative changes in the contraction force are output by the controller 104 to user interface 162 contemporaneously with data acquisition (e.g., in “real time”) to provide the user immediate data, without time-intensive post processing). Moreover, the controller 104 is configured to contemporaneously record signal amplitudes and determine contraction force changes of all of the tissue samples 108 in the multi-well plate 112 in parallel.

[0088] FIG. 3 illustrates an alternative embodiment of the present disclosure that builds upon FIG. 1 A - FIG. 2B and enables efficient measurement of absolute contraction force of a tissue sample 108 based upon signal amplitude changes during the contraction period.

[0089] FIG. 3 is an image showing the tissue sample 108 disposed between two tissue fixtures 110a, 110b, as imaged by the system 100. Tissue fixture 110a is a flexible tissue fixture and tissue fixture 110b is a rigid tissue fixture. Tissue fixture 110a is provided with a fiducial marker 164 at its distal end (here, a dark-colored rectangular marker that contrasts with a lightcolored distal end of the tissue fixture 110a).

[0090] In FIG. 3, the image of the tissue sample 108 is overlaid or superimposed (e.g., by the controller 104 on the user interface 162) with a region of interest 166 encompassing a portion of the tissue sample 108 as described above with respect to FIG. 2A - FIG. 2B. Additionally, the controller 104 superimposes a plurality of reference markers 168a, 168b, 168c over the tissue sample 108 between the tissue fixtures 110a, 110b. The reference markers 168a - c are disposed in a contraction path of the fiducial marker 164, i.e., between the fiducial marker 164 and the region of interest 166. In FIG. 3, the reference markers comprise three spaced-apart parallel marks. In some embodiments, the reference markers are more numerous (e.g., 4, 5, 6, etc.) or less numerous (e.g., 2 markers).

[0091] The reference markers 168a - c are spaced apart from the fiducial marker 164 by known distances when the tissue sample 108 is in the fully relaxed state, e.g., by 200, 400, 600 pm. In some embodiments, these known distances are calibrated periodically, e.g., prior to commencing data recordation of a multi -well plate 112. In use, the imaging array 118 senses the optical signal of the tissue sample 108 during contraction cycles, e.g., as plotted in FIG. 2B. The optical signal amplitude is calibrated with movement of the fiducial marker 164 relative to the reference markers 168a, b, c periodically, e.g., at the beginning of an experiment.Accordingly, this calibration establishes a relationship between the optical signal amplitude and movement of the fiducial marker 164.

[0092] From this information, contraction force changes and peak contraction times are determined by the controller 104. As the tissue sample 108 contracts, the fiducial marker 164 moves toward the reference markers 168a - c. Therefore, in some embodiments, the controller 104 determines the absolute position of the fiducial marker 164 (e.g., at peak contraction) from the time series images (e.g., as shown in FIG. 3), based upon the known distances of the reference markers 168a - c and the resolution of the camera 130. In some embodiments, thecontroller 104 determines the absolute position of the fiducial marker 164 (e.g., at peak contraction) based upon the times at which a leading edge of the fiducial marker 164 (e.g., the right edge) crosses one or more of the reference markers 168a - c and optionally an imputed contraction velocity. Based upon the absolute length change of the tissue sample 108 at peak contraction, the controller 104 determines the absolute contraction force using beam theory, e.g., based upon Equation 9 above. In some embodiments, the controller 104 displays the foregoing information on the user interface 162 of the system 100.

[0093] Advantageously, the foregoing methodology enables accurate measurement of absolute contraction force and provides valuable information regarding the underlying biology. Additionally, this method does not require significant computer vision resources to track the movement of the fiducial marker 164.

[0094] In brief summary, the methodologies described with respect to FIG. 2A - FIG. 3 provide at least three main advantages: first, said methods can be used intermittently to recalibrate the optical signal measured in the static region of interest 166. Second, the calibrated region of interest-based measurement methods described utilize significantly reduced computing power, enabling real-time determination of relevant contraction force information. Third, traditional computer vision techniques applied to fiducial markers under varying conditions (lighting, biological, etc.) need robust, complex algorithms capable of handling failure modes caused by edge case situations. This drawback increases with well density of a given multi -well plate; for example, 96 or 384 well-plates utilizing traditional computer vision techniques require significantly more processing time than a 24 well-plate. In contrast, the careful tracking of the fiducial marker 164 according to the methods above occurs with limited frequency (e.g., once or occasionally during an experiment), and therefore greater computing resources and time can be taken for those occasional calibrations. Subsequently, the optical signal amplitudes in the static regions of interest 166 are easily calculated temporally and not prone to error.

[0095] FIG. 4 illustrates methods 400 of measuring contractility of tissue samples according to the present disclosure. The following methods may be implemented independently of the system 100 described above. However, any of the methods may be restated as a method for using the system 100 or instrument 102 described above. Therefore, to facilitate understanding, reference numerals corresponding to the system 100 are used in the following methodology. Accordingly, those terms have the same meaning as described above. Moreover,in any method, any feature or combination of features of said elements of system 100 may be expressly included as part of the method. Restated, because the following methods are compatible with the system 100, the present disclosure includes variants of the methods that include any feature or combination of features of the system 100 described above, as well as any step or combination of steps described above (e.g., with respect to FIG. 2A - FIG. 3).

[0096] Block 402. A multi-well cartridge 106 is aligned relative to an imaging array 118, e.g., of an instrument 102 for measuring tissue contractility. The multi -well cartridge 106 comprises wells 122, each comprising an optically transparent bottom end 170 and a tissue sample 108 disposed between a plurality of tissue fixtures, e.g., a flexible tissue fixture 110a and a rigid tissue fixture 110b. In some embodiments, at least some of the tissue samples 108 are un-labeled. In some embodiments, at least some of the tissue samples 108 are labeled. The imaging array 118 is aligned to contemporaneously image the wells 122, e.g., by positioning the multi -well cartridge 106 upon a stage 116 of the instrument 102 such that the imaging array 118 can image the well 122 through a sample aperture of the stage 116.

[0097] Block 404. The imaging array 118 captures a plurality of images of each tissue sample 108 during respective contraction periods of each tissue sample 108. In other words, the imaging array 118 captures numerous images of each tissue sample 108 during a time period when that tissue sample 108 experiences one or more contractions. The images capture optical signals emitted by each of tissue samples 108 during contraction and relaxation, e.g., autofluorescence. The imaging array 118 is configured to image all wells 122 contemporaneously. For example, in some embodiments, each image captured by the imaging array 118 includes all wells 122 and tissue samples 108 in the multi -well cartridge 106. In some such embodiments, the images are post-processed by the controller 104 into smaller images, each image isolating one of the wells 122.

[0098] Block 406. Optionally, the controller 104 causes any one or more of the images captured in block 404 to be displayed on the user interface 162.

[0099] Block 408. Based upon the images captured in block 404, the controller 104 (in particular, the processor 158), determines parameters for each of the tissue samples 108 culminating in the determination of relative and / or absolute contraction forces of each tissue sample 108. In some embodiments, the controller 104 determines the parameters for each of the tissue sample 108 contemporaneously and independently.

[0100] The parameters determined from the images by the controller 104 for each tissue sample 108 include, at least, a signal amplitude change during the contraction period of a (first) optical signal emitted by the tissue sample 108 in a region of interest 166, and a contraction force change during the contraction period based on the signal amplitude change. As described above, the optical signal includes, for example, autofluorescence from (NADH) dehydrogenase in un-labeled tissues or fluorescence from GFP in labeled tissues. Accordingly, each optical signal has a particular wavelength or range of wavelengths that expose pixels of the imaging array 118. Accordingly, the optical signal registers in the images at varying brightness or intensity values depending on the signal amplitude, i.e., depending on the strength of the tissue's contraction.

[0101] Each region of interest 166 encompasses a portion of the respective tissue sample 108, e.g., a central portion of the tissue sample 108 between the respective tissue fixtures 110a, 110b. The region of interest 166 should not encompass the entire tissue sample 108 as the number of fluorophores within the region of interest 166 will be constant. Accordingly, for a given tissue sample 108, the corresponding region of interest has a length that is less than a length of the tissue sample 108 and a radius that is greater than a radius of the tissue sample 108.

[0102] Each image registers the amplitude of the optical signal across a plurality of pixels of the camera 130 at a single point in time. Accordingly, in some embodiments, the controller 104 averages or sums the pixel values within the region of interest 166 at a given time point to create a single value representing the signal amplitude in the region of interest 166, i.e., the number of fluorophores in the region of interest 166 at that time point.

[0103] The controller 104 determines the signal amplitude change (difference in value) during the contraction period based upon the underlying data (e.g., average pixel values in the region of interest 166), e.g., resulting in a plot as shown in FIG. 2B. The signal amplitude change is, in some embodiments, a change in signal amplitude between successive minima and maxima, a change over time in signal maxima, a change over time in signal minima, and / or a change over time in the difference between successive minima and maxima. For example, in some embodiments, the controller 104 determines the signal amplitude change during each contraction cycle (i.e., the difference or relative change in amplitude values between successive minima and maxima) in a contraction period comprising numerous contraction cycles. In some embodiments, determining the signal amplitude change comprises determining a plurality ofdifferences or relative changes in the signal amplitude between successive amplitude minima and maxima. In some embodiments, this signal amplitude change is determined in absolute units (e.g., lumens) and / or relative units (e.g., in arbitrary units or as a percentage).

[0104] Based on the signal amplitude change, the controller 104 determines a contraction force change during the contraction period. In some embodiments, the contraction force change is a relative change such as a percentage difference between in amplitude values between successive minima and maxima. In some embodiments, the controller 104 determines the contraction force change by directly inferring from the signal amplitude change, e.g., based on a known relationship between AF (Equation 7) and Fc(Equation 9). For example, in a contraction period comprising numerous contraction cycles, a 5% decrease between the highest maximum signal amplitude value and the lowest signal amplitude value equates to a 10% decrease in contraction force over the contraction period. In some embodiments, the controller 104 determines the contraction force change based upon a length change. For example, based upon the signal amplitude changes between successive minima and maxima, the controller 104 determines length changes of the tissue sample, e.g., based upon Equation 7 above. Then, based upon the length changes, the controller 104 determines contraction force values based upon Equation 9, and differences over time in those values.

[0105] The foregoing steps are applicable both to un-labeled and labeled tissues. In some embodiments, the foregoing methods are utilized with labeled tissues to determine contractility in addition to information such as calcium concentration and / or voltage. For example, in some examples, the tissues are labeled with a first marker (e.g., calcium detection fluorophores) that generate a second optical signal having a different second wavelength, and with a second marker (e.g., voltage sensitive fluorescent dye) that generates a third optical signal having a different third wavelength. In such embodiments, for each of the tissue samples 108, the controller 104 also determines a signal amplitude change of the second optical signal emitted by the tissue sample 108 in the region of interest 166 during the contraction period, based upon the plurality of images, and based on the signal amplitude change of the second optical signal, determines, with the controller, a calcium concentration change of the tissue sample 108 during the contraction period. Optionally, in such embodiments, the controller 104 determines a signal amplitude change of the third optical signal emitted by the tissue sample 108 in the region of interest 166 during the contraction period, based upon the plurality of images; and determines the voltage change of the tissue sample 108 based on the signal amplitude change ofthe third optical signal. In the foregoing example, calcium concentration and voltage are representative examples; in other embodiments, the markers can indicate different el ectrophy si ol ogi cal characteri sti cs .

[0106] Block 410. Optionally, the controller 104 causes the contraction force change, the optical signal amplitude, signal amplitude change, and / or the plurality of images overlaid with the region of interest 166 to be displayed on the user interface 162.

[0107] In some embodiments, each of the flexible tissue fixtures 110a comprises a fiducial marker 164. In some such embodiments, the method 400 further comprises, for each of the tissue samples: calibrating (e.g., as part of block 404), with controller 104, the signal amplitude change with a movement of the fiducial marker 164 relative to at least one reference marker 168a, b, c overlaid by the controller 104 on the images; and determining (e.g., as part of block 408), with the controller 104, an absolute contraction force based on the signal amplitude change and the movement of the fiducial marker 164 relative to the at least one reference marker 168a, b, c. Such additional steps increase the accuracy of the method 400 and enable computation of absolute contraction force of the tissue samples 108. Optionally, the controller 104 causes any of the foregoing to be displayed on the user interface 162.

[0108] FIG. 5A - FIG. 7 illustrate additional methodologies for using optical signals (e.g., fluorescence signals) to measure absolute contraction forces of tissue samples, utilizing a fiducial marker tracking system. The relative optical signal is calibrated, for example, at experiment start and optionally at set intervals during longer experiments, based on the computer vision tracking of the length of the tissue sample based on a known location of the fiducial marker. Advantageously, this method allows for accurate measurement of absolute contraction forces based on tissue length changes, e.g., according to Equation 9.

[0109] FIG. 5A schematically illustrates a tissue sample 508 at to in a relaxed state disposed between a relatively flexible tissue fixture 510a and a relatively rigid tissue fixture 510b, as described above. A fiducial marker 564 having relatively low fluorescence is embedded within the relatively flexible tissue fixture 510a (e.g., as shown in FIG. 3). In the illustrated embodiment, fiducial marker 564 is a dark-colored rectangular marker (e.g., a magnet) that contrasts with a light-colored distal end of the tissue fixture 510a. The tissue sample 508 is overlaid or superimposed (by the controller 104) with a region of interest 566 encompassing a portion of the tissue sample 508. As compared to the regions of interest of previous embodiments, the region of interest 566 of FIG. 5 A has a reduced area relative to the tissuesample 508. As shown, region of interest 566 has a length LROI < LQ (i.e., a length shorter than the relaxed length of the tissue sample 508) and a radius TROI < ro (i.e., a radius less than the relaxed radius of a central section the tissue sample 508). In the relaxed state, a leading edge of the fiducial marker 564 extends into the region of interest 566. Accordingly, the region of interest 566 is partially occluded by the fiducial marker 564 at to.

[0110] FIG. 5B shows the same tissue sample 508 at ti in a maximum contracted state. Consequently, tissue sample 508 has a reduced length as compared to FIG. 5A. When the tissue sample 508 contracts, the relatively flexible tissue fixture 510a and embedded fiducial marker 564 deflect toward the relatively rigid tissue fixture 510b. Consequently, the leading edge 572 translates to the right and the fiducial marker 564 occludes a greater proportion of the region of interest 566 as compared to to.[oni] FIG. 6A and FIG. 6B respectively plot signal amplitude profiles 602a, 602b from experimental data of tissue samples having the configuration shown in FIG. 5A and FIG. 5B, i.e., the tissue sample is disposed between a relatively rigid tissue fixture and a relatively flexible tissue fixture with an embedded fiducial marker. The underlying tissue samples from which the data are derived are EHTs prepared as described above with respect to FIG. 2A.

[0112] FIG. 6A plots the signal amplitude profile 602a at to (corresponding to FIG. 5A), wherein the tissue sample is in a relaxed state. FIG. 6B plots the signal amplitude profile 602b at ti, wherein the tissue sample is in a maximum contracted state. Each signal amplitude profile 602a, 602b represents a single timepoint (to and ti) and plots, as diamond-shaped data points, the vertically-averaged amplitude of the optical signal (in arbitrary units) across the region of interest 566. Restated, across the region of interest 566, the plotted signal amplitude reflects the average signal amplitude of all pixels of the imaging array at that x-position in the region of interest 566 (i.e., each pixel column is averaged).

[0113] Curves fitted to the data points of the signal amplitude profiles 602a, 602b enable the determination of an edge translation of the fiducial marker 564 between to and ti, i.e., a maximum translated distance of the leading edge 572. Specifically, the position of the leading edge 572 of fiducial marker 564 is determined at any time point by fitting an appropriate mathematical function (e.g., an error function). In some embodiments, the functions are computationally fit by the controller 104, e.g., using a least squares approach, Levenberg- Marquardt algorithm, or similar approach, and optionally displayed on a user interface.

[0114] An image of a one dimensional semi-infinite fluorescent object with a sharp boundary (e.g., the leading edge 572 of fiducial marker 564), imaged through diffractive optics such as instrument 102, is given by:

[0115] Equation 10) F(x) = a + where a is a constant to correct forbackground signal amplitude (e.g., fluorescence), c is a proportionality constant or scaling factor, d is a fitting parameter, and c / ' / 'is the error function: x-x 2 ,X~X° _ 2

[0116] Equation 11) er (—^) = a + — Joderdt, where, xo is the location of theboundary, and x is the coordinate along the direction of motion. Although the tissues described here are of finite size, L » X, where X is the wavelength of the detected light.

[0117] Given diffraction effects in the system 100, the data points of FIG. 6A and FIG. 6B are well described by Equation 10 above. For example, in FIG. 6A and FIG. 6B, the data points are fitted by the respective functions representing Equation 10. The fitted parameter xo gives an estimate of the position of the leading edge 572 of the fiducial marker 564. In FIG. 6A, xo= 0.2565 mm. By comparison, in FIG. 6B, the fitted parameter xo is 0.66356 mm. Taking the difference of the implied edge positions in FIG. 6A and FIG. 6B yields a maximum edge translation at peak contraction of 407.06 pm. This edge translation value is also the maximum displacement of the relatively flexible tissue fixture 510a; therefore, the contraction force of the tissue sample 508 is determinable from beam theory, e.g., based on Equation 9.

[0118] Advantageously, the error function-based approach described above accurately models how an edge appears when imaged through diffracting optics, matches the expected physical behavior of the system 100, and enables tracking the fiducial marker 564 with sub-pixel resolution. Nevertheless, other function-fitting approaches could be used. For example, in some embodiments, a high-order polynomial function is fit the data points instead of an error function.

[0119] FIG. 7 illustrates methods 700 of measuring contractility of tissue samples 108 I 508 according to the present disclosure. The following methods may be implemented independently of the system 100 described above. However, any of the methods may be restated as a method for using the system 100 or instrument 102 described above, particularly embodiments wherein each of the tissue fixtures 110a / 510a comprises a fiducial marker 164 / 564. Therefore, to facilitate understanding, reference numerals corresponding to the system 100 are used in the following methodology. Accordingly, those terms have the same meaningas described above. Moreover, in any method, any feature or combination of features of said elements of system 100 may be expressly included as part of the method. Restated, because the following methods are compatible with the system 100, the present disclosure includes variants of the methods that include any feature or combination of features of the system 100 described above, as well as any step or combination of steps described above (e.g., with respect to FIG. 5 A - FIG. 6B).

[0120] Block 702. A multi -well cartridge 106 is aligned relative to an imaging array 118, e.g., of an instrument 102 for measuring tissue contractility. The multi -well cartridge 106 comprises wells 122, each comprising an optically transparent bottom end 170 and a tissue sample 108 / 508 disposed between a plurality of tissue fixtures, e.g., a flexible tissue fixture 110a / 510a and a rigid tissue fixture 110b / 510b. The tissue fixture 110a / 510b comprises a fiducial marker 164 I 564 disposed at its distal end. In some embodiments, at least some of the tissue samples 108 / 508 are un-labeled. In some embodiments, at least some of the tissue samples 108 I 508 are labeled. The imaging array 118 is aligned to contemporaneously image the wells 122, e.g., by positioning the multi-well cartridge 106 upon a stage 116 of the instrument 102 such that the imaging array 118 can image the well 122 through a sample aperture of the stage 116.

[0121] Block 704. The imaging array 118 captures a plurality of images of each tissue sample 108 I 508 and each fiducial marker 164 / 564 during respective contraction periods of each tissue sample 108 I 508. In other words, the imaging array 118 captures numerous images of each tissue sample 108 / 508 and the fiducial marker 164 / 564 to which it is connected during a time period when that tissue sample 108 / 508 experiences one or more contractions. The images capture optical signals emitted by each of tissue samples 108 / 508 during contraction and relaxation, e.g., autofluorescence or fluorescence. The imaging array 118 is configured to image all wells 122 contemporaneously. For example, in some embodiments, each image captured by the imaging array 118 includes all wells 122 and tissue samples 108 / 508 in the multi-well cartridge 106. In some such embodiments, the images are post-processed by the controller 104 into smaller images, each image isolating one of the wells 122.

[0122] Block 706. Optionally, the controller 104 causes any one or more of the images captured in block 404 to be displayed on the user interface 162.

[0123] Block 708. Based upon the images captured in block 704, the controller 104 (in particular, the processor 158), determines parameters for each of the tissue samples 108 / 508culminating in the determination of absolute contraction forces of each tissue sample 108 / 508. In some embodiments, the controller 104 determines the parameters for each of the tissue sample 108 / 508 contemporaneously and independently.

[0124] The parameters determined from the images by the controller 104 for each tissue sample 108 I 508 include, at least: a plurality of signal amplitude profiles (at different times) from the plurality of images based on an optical signal emitted from the tissue sample 108 / 508 in a region of interest 166 I 566, wherein each of the signal amplitude profiles plots a signal amplitude of the optical signal in the region of interest 166 / 566 (e.g., every pixel column across the region of interest 166 I 566 is averaged); an edge translation of the fiducial marker 164 I 564 (e.g., a leading edge 572) based the plurality of signal amplitude profiles; and a contraction force of the tissue sample 108 / 508 based on the edge translation. The edge translation is determined based upon fitting a function to each the plurality of signal amplitude profiles (e.g., based upon Equation 10). In some embodiments, a first signal amplitude profile is taken when the tissue sample 108 / 508 is in the relaxed state and a second signal amplitude profile is taken when the tissue sample 108 / 508 is in a maximum contracted state. A function is fitted to each of these signal amplitude profiles, and a location of a features of the fiducial marker 164 I 564 (e.g., the leading edge 572) is determined from the fitted function. The difference in the feature location imputed from the fitted functions determines the edge translation of the fiducial marker 164 I 564. In some embodiments, the region of interest 166 I 566 has a length and / or a radius that is less than a length of the tissue sample 108 / 508 in a maximum contracted state.

[0125] Block 710. Optionally, the controller 104 causes the contraction force, the optical signal amplitude, signal amplitude change, and / or the plurality of images overlaid with the region of interest 166 to be displayed on the user interface 162.

[0126] Various changes can be made to the embodiments of the present disclosure as could be reasonably contemplated in view of the above-described description by any person skilled in the art. The following claims are presented as examples of embodiments of the present disclosure, but these claims should not be construed to limit other claims or other embodiments disclosed herein.

[0127] The detailed description set forth above in connection with the appended drawings, where like numerals reference like elements, are intended as a description of representative embodiments of the present disclosure and are not intended to represent the only embodiments.Each embodiment described m this disclosure is provided as an example or illustration and should not be construed as preferred or advantageous over other embodiments. The illustrative embodiments provided herein are not intended to be exhaustive or to limit the disclosure to the precise forms disclosed. Similarly, any steps described herein may be interchangeable with other steps, or combinations of steps, in order to achieve the same or substantially similar result. Further still, one or more features of any embodiment may be combined with one or more features of one or more embodiments to form additional embodiments, which are within the scope of the present disclosure.

[0128] Generally, the embodiments disclosed herein are non-limiting, and the inventors contemplate that other embodiments within the scope of this disclosure may include structures and functionalities from more than one specific embodiment shown in the FIGURES and described in the specification. It will be appreciated that variations and changes may be made by others, and equivalents employed, without departing from the spirit of the present disclosure. Accordingly, it is expressly intended that all such variations, changes, and equivalents fall within the spirit and scope of the present disclosure as claimed. For example, the present disclosure includes additional embodiments having combinations of any one or more features described above with respect to the representative embodiments.

[0129] In the foregoing description, specific details are set forth to provide a thorough understanding of representative embodiments of the present disclosure. It will be apparent to one skilled in the art, however, that the embodiments disclosed herein may be practiced without embodying all the specific details. In some instances, well-known process steps have not been described in detail in order not to unnecessarily obscure various aspects of the present disclosure.

[0130] The present disclosure includes the word “comprising,” which is an open-ended term that means “includes at least” the element(s) that follow. The present disclosure may include references to directions, such as “first,” "second," "vertical," "horizontal," "front," "rear," "left," "right," "top," and "bottom," “below,” “around,” etc. These references, and other similar references in the present disclosure, are intended to assist in helping describe and understand the particular embodiment (such as when the embodiment is positioned for use) and are not intended to limit the present disclosure to these directions or locations.

[0131] The present disclosure may also reference quantities and numbers. Unless specifically stated, such quantities and numbers are not to be considered restrictive, but exemplary of the possible quantities or numbers associated with the present disclosure. Also in this regard, thepresent disclosure may use the term "plurality" to reference a quantity or number. In this regard, the term "plurality" means any number that is more than one, e.g., two, three, four, five, etc. The term "about," "approximately," etc., means plus or minus 5% of the stated value. The term "based upon" means "based at least partially upon." The term "between" includes the values recited in connection therewith. The expressions “at least one of A, B, or C"; “at least one of A, B, and C"; and “at least one of A, B, and / or C" have the same meaning, z.e., any one of the following conditions satisfy all of the foregoing expressions: A; B; C; AB; AC; BC; ABC.

Claims

CLAIMSWe claim:

1. A method of measuring contractility of tissue samples, comprising: aligning a multi-well cartridge relative to an imaging array, the multi-well cartridge comprising a plurality of wells, each well comprising an optically transparent bottom end and a tissue sample disposed between a flexible tissue fixture and a rigid tissue fixture, wherein the imaging array is configured to contemporaneously image the plurality of wells; imaging, with the imaging array, a plurality of images of each of the tissue samples during a respective contraction period; and determining, with a controller comprising a processor and a memory operably coupled to the imaging array, for each of the tissue samples: a signal amplitude change during the contraction period of an optical signal emitted by the tissue sample in a region of interest, based upon the plurality of images; and a contraction force change of the tissue sample during the contraction period based on the signal amplitude change.

2. The method of claim 1, further comprising determining, with the controller, for each of the tissue samples, a length change during the contraction period based on the signal amplitude change, wherein determining the contraction force change during the contraction period is based on the length change.

3. The method of claim 1, wherein each image of the plurality of images comprises the plurality of wells.

4. The method of claim 1, wherein for each of the tissue samples, the region of interest has a length that is less than a length of the tissue sample.

5. The method of claim 4, wherein for each of the tissue samples, the region of interest has a radius that is greater than a radius of the tissue sample.

6. The method of claim 1, further comprising displaying, on a user interface, for at least one of the tissue samples, at least one of the contraction force change, the optical signal amplitude, optical signal amplitude change, or the plurality of images overlaid with the region of interest.

7. The method of claim 1, further comprising for each of the tissue samples: determining, with the controller, a signal amplitude change of a second optical signal emitted by the tissue sample in the region of interest during the contraction period, based upon the plurality of images; and determining, with the controller, a calcium concentration change or a voltage change of the tissue sample during the contraction period based on the signal amplitude change of the second optical signal.

8. The method of claim 7, further comprising for each of the tissue samples: determining, with the controller, a signal amplitude change of a third optical signal emitted by the tissue sample in the region of interest during the contraction period, based upon the plurality of images; and determining, with the controller, the voltage change of the tissue sample based on the signal amplitude change of the third optical signal, wherein the calcium concentration change is determined based on the second optical signal.

9. The method of claim 1, wherein the optical signal is a fluorescence signal and wherein the tissue sample is un-labeled.

10. The method of claim 1, wherein each of the flexible tissue fixtures comprises a fiducial marker, the method further comprising, for each of the tissue samples: calibrating, with the controller, the signal amplitude change with a movement of the fiducial marker relative to at least one reference marker overlaid by the controller on the plurality of images; and determining, with the controller, an absolute contraction force based on the signal amplitude change and the movement of the fiducial marker relative to the at least one reference marker.

11. A system for measuring contractility of tissue samples, comprising: an instrument comprising an enclosure configured to receive a multi-well cartridge comprising a plurality of wells, each well comprising an optically transparent bottom end and a tissue sample disposed between a flexible tissue fixture and a rigid tissue fixture, the enclosure containing an imaging array arranged to contemporaneously image the plurality of wells of the multi-well cartridge when the multi-well cartridge is aligned relative to the imaging array; anda controller comprising a processor and a non-transitory computer-readable memory (a memory) operably coupled to the imaging array, the memory storing logic comprising instructions that, when executed, cause operations, comprising: imaging, with the imaging array, a plurality of images of each of the tissue samples during a respective contraction period; and determining, with the processor, for each of the tissue samples: a signal amplitude change during the contraction period of a first optical signal emitted by the tissue sample in a region of interest, based upon the plurality of images; and a contraction force change during the contraction period based on the signal amplitude change.

12. The system of claim 11, wherein each of the flexible tissue fixtures comprises a fiducial marker, wherein the memory comprises additional instructions that, when executed by the processor, cause additional operations for each of the tissue samples, comprising: determining, with the processor, an absolute contraction force based on the signal amplitude change and a movement of the fiducial marker relative to at least one reference marker overlaid by the controller on the plurality of images.

13. The system of claim 11, wherein the memory comprises additional instructions that, when executed by the processor, cause additional operations for each of the tissue samples, comprising: determining, with the processor, for each of the tissue samples: a signal amplitude change of a second optical signal emitted by the tissue sample in the region of interest during the contraction period, based upon the plurality of images; and a calcium concentration change or a voltage change of the tissue sample during the contraction period based on the signal amplitude change of the second optical signal.

14. The system of claim 13, the memory storing logic comprising instructions that, when executed, cause additional operations, comprising: determining, with the processor, for each of the tissue samples:a signal amplitude change of a third optical signal emitted by the tissue sample in the region of interest during the contraction period, based upon the plurality of images; and the voltage change of the tissue sample based on the signal amplitude change of the third optical signal, wherein the calcium concentration change is determined based on the second optical signal.

15. A method of measuring contractility of tissue samples, comprising: aligning a multi-well cartridge relative to an imaging array, the multi-well cartridge comprising a plurality of wells, each well comprising an optically transparent bottom end and a tissue sample disposed between a rigid tissue fixture and a flexible tissue fixture comprising a fiducial marker, wherein the imaging array is aligned to contemporaneously image the plurality of wells; imaging, with the imaging array, a plurality of images of each of the tissue samples during a respective contraction period; and determining, with a controller comprising a processor and a memory operably coupled to the imaging array, for each of the tissue samples: a plurality of signal amplitude profiles from the plurality of images based on an optical signal emitted from the tissue sample in a region of interest, wherein each of the signal amplitude profiles plots a signal amplitude of the optical signal across the region of interest; an edge translation of the fiducial marker based the plurality of signal amplitude profiles; and a contraction force of the tissue sample based on the edge translation.

16. The method of claim 15, wherein the edge translation is determined based upon fitting a function to the plurality of signal amplitude profiles.

17. The method of claim 15, wherein plurality of signal amplitude profiles plot the signal amplitude across the region of interest at different times.

18. The method of claim 15, wherein for each of the tissue samples, the region of interest has a length that is less than a length of the tissue sample in a maximum contracted state.

19. The method of claim 18, wherein for each of the tissue samples, the region of interest has a radius that is less than a radius of the tissue sample in the maximum contracted state.

20. The method of claim 15, wherein the optical signal is a fluorescence signal and the tissue sample is un-labeled.

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