Optoreactive tissue stimulation systems

Abstract

Systems, devices, and methods for optically stimulating optoreactive tissue samples are provided. The optical stimulation systems include coherent light sources arrayed in a housing, each coherent light source being configured to precisely focus and align a photon beam on an optoreactive tissue sample in a multi-well cartridge, delivering optical parameters that stimulate physiological responses. The systems, devices, and methods enable parallel stimulation of numerous tissue samples without crosstalk and while maintaining thermal stability, advantageously reducing data acquisition time and operator burden.

Description

OPTOREACTIVE TISSUE STIMULATION SYSTEMSGOVERNMENT LICENSE RIGHTS

[0001] This invention was made with government support under project numbers 1U44TR004795-01 and 4U44TR004795-02 awarded by the National Institutes of Health. The government has certain rights in the invention.CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 637,225, filed April 22, 2024, the entirety of which is hereby incorporated by reference.TECHNICAL FIELD

[0003] The present disclosure relates to the field of tissue research.BACKGROUND

[0004] Measurements of movement of biological samples, including Engineered Tissues (ETs) cultured in vitro, can be useful indicators of the health and functionality of said tissues. Such tissues may move spontaneously or may be moved by stimuli.

[0005] Electrical stimulus of tissues cannot selectively stimulate subpopulation of cells in multicellular cultures; rather, electrical current indiscriminately stimulates an entire tissue sample. This complicates analysis of different cell populations within a tissue sample. For example, with electrical stimulation, it is not possible to determine the existence of a healthy neuromuscular junction via selective stimulation of motor neurons.

[0006] On the other hand, stimulating optoreactive tissue samples (e g., optogenetic tissues) with light enables the selective analysis of different cell populations. However, light sensitive tissues respond best to stable and precise delivery of photons, at a sufficiently high photon concentration, in order to obtain a measurable stimulation response. Incoherent light sources such as light emitting diodes (LED)s cannot provide sufficient, localized photon intensity and also cannot accurately target specific regions of the cell culture sample.

[0007] Accordingly, there is a need for more effective tissue testing platforms that optically stimulate optoreactive tissue samples.BRIEF SUMMARY

[0008] According to a first aspect, the present disclosure provides systems and devices for parallel optical stimulation of optoreactive tissue samples. The system includes an optical stimulation device with coherent light sources (e.g., coherent light modules) arranged in a housing, where each module contains a laser and lens that focuses a photon beam onto tissue samples in a multi-well cartridge. A controller with a processor and memory executes logical instructions to activate the photon beams and control input waveform parameters like frequency, amplitude, and pulse width independently for each coherent light source. In some embodiments, the system delivers an incident optical power on each tissue sample of at least 20 mW / mm2, pulse widths of 1-8 milliseconds (ms), and / or frequencies of at least about 5 Hz. The housing comprises a lattice with apertures that position the modules to emit photon beams into corresponding wells containing tissue fixtures. When the cartridge is properly positioned relative to the device, each photon beam aligns with the tissue fixtures and focuses on a plane extending through the tissue samples. In some embodiments, the system also includes a tissue contractility measurement instrument for measuring physiological responses of the tissue samples to the photon beams, which responses may be provided as feedback for controlling the photon beams. In some embodiments, the system also includes the multi-well cartridge. In some embodiments, the system includes the device and the controller.

[0009] According to a second aspect, the present disclosure provides methods for parallel optical stimulation of a plurality of optoreactive tissue samples. The methods may be practiced with the systems and devices described herein or independently thereof. The methods involve providing optoreactive tissue samples in a multi-well cartridge and positioning the tissue samples relative to an optical stimulation device with coherent light sources mapped to the tissue samples. The methods includes contemporaneously (e.g., simultaneously) emitting elliptical photon beams from the coherent light sources, with each beam focused on and aligned with the longitudinal dimension of a different tissue sample. In some embodiments, each beam delivers an incident optical power of at least 20 mW / mm2on the respective tissue sample, pulse width of 1-8 milliseconds (ms), and / or frequency of at least about 5 Hz. In some embodiments, each beam's polarity aligns with the tissue sample's longitudinal dimension. In some embodiments, different beams use different parameter combinations, e.g., to facilitate efficient exploration of parameter space. The methods include measuring physiological responses to therespective photon beams and optionally controlling one or more parameters of the photon beams based on those responses.

[0010] According to a third aspect, the present disclosure provides coherent light sources such as laser diode modules. The laser diode modules include a hollow body spacer with first and second internal seats that hold a laser diode and lens at a fixed back-focal distance, along with a collar around the laser diode base. The spacer conducts heat from the laser diode through contact between the first internal seat and the laser diode's radial skirt. The collar's skirt maintains a circumferential clearance from the spacer when the laser diode is fully seated. The module communicatively connects to a printed circuit board through the laser diode terminals, with multiple modules positioned in a spaced array through the circuit board connection.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] 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:

[0012] FIG. 1A illustrates a system for stimulating optoreactive tissue samples according to the present disclosure.

[0013] FIG. IB is a section view of an optical stimulation device of the system of FIG. 1A.

[0014] FIG. 1C is a section view of a coherent light module of the optical stimulation device of FIG. IB.

[0015] FIG. 2 schematically illustrates a coherent light photon beam illuminating a tissue sample.

[0016] FIG. 3 plots contraction forces of a tissue sample spontaneously and under stimulation by the system of FIG. 1A.

[0017] FIG. 4 plots contraction forces of optoreactive tissue samples, one of which is optically stimulated by an optical stimulation device of the present disclosure.

[0018] FIG. 5 illustrates methods for parallel optical stimulation of optoreactive tissue samples according to the present disclosure.DETAILED DESCRIPTION

[0019] The present disclosure provides systems, devices, and methods for stimulating optoreactive tissue samples in a highly repeatable, massively parallel format. FIG. 1A - FIG. IB show such a system 100, which will now be introduced at a high level before specifically detailing features thereof.

[0020] Referring briefly to FIG. 1A, the system 100 includes an optical stimulation device 102 designed to be positioned relative to (e.g., rest atop) a multi -well cartridge 104 that includes a multi-well plate 106. The optical stimulation device 102 is operably coupled to a controller 108 comprising a processor 110, a drive 112, and a memory 114 containing logical instructions that, when executed by the processor 110, cause the drive 112 to control input waveform parameters of numerous coherent light sources (e.g., coherent light modules) therein, causing the device 102 to stimulate physiological responses in the tissue samples as described below.

[0021] Advantageously, the highly polarized coherent light sources emit precisely focused photon beams (e.g., having an elliptical shape) that provide high photon density to stimulate the tissue samples without creating crosstalk between wells (see FIG. 4). Accordingly, the system 100 allows contemporaneous stimulation of all tissues, extending parallelization beyond the typical well-by-well measurement approaches and reducing data-acquisition timeframes by a factor of n wells, greatly facilitating handling hundreds of tissues in a scalable fashion.

[0022] In some embodiments, the memory 114 contains a logical power control module 116 that controls the input current and / or output power of the coherent light sources, and a logical protocol control module 118 that controls the pattern of optical stimulation. The tissue responses are measured by a tissue contractility measurement instrument 120, e.g., MANTARRAY™ sold by Curi Bio, Inc., of Seattle, Washington, USA.

[0023] In some embodiments, the system 100 includes the optical stimulation device 102 and controller 108. In some embodiments, the system 100 also includes one or more of the multiwell cartridge 104, the tissue samples, and / or the tissue contractility measurement instrument 120.

[0024] In some embodiments, any combination of the inputs to the optical stimulation device 102 (e.g., input waveform parameters) and / or measurements of the tissue contractility measurement instrument 120 are displayed on a user interface 122 of remote computing resources 124 (such as a laptop).

[0025] As used herein, “optoreactive tissue sample” means a three-dimensional (nonmonolayer) construct formed at least partially of biological tissue that exhibits a physiological response (e.g., contraction) to defined wavelengths of light due to the presence of lightsensitive molecular components. These components may be introduced via genetic modification (as in optogenetic tissue samples) or through sensitization using optically-reactive compounds. The tissue samples may comprise monocultures of a single cell type or co-cultures of multiple interacting cell types, e.g., motor neurons co-cultured with skeletal muscle tissues as described in Fleming, Jacob W., et al. "An automated platform for simultaneous, longitudinal analysis of engineered neuromuscular tissues for applications in neurotoxin potency testing." Current Research in Toxicology 8 (2025): 100218. In some embodiments, the optoreactive tissue samples are cast using the systems and according to the methods, for example, described in PCT Patent Publication No. WO 2024 / 259120 A2, which is herein incorporated by reference in its entirety. Optoreactive tissue samples include optogenetic tissue samples, in which cells are genetically modified (e.g., by transduction or transfection techniques) to express light-sensitive proteins (opsins) such as channelrhodopsins (for activation) or halorhodopsins (for inhibition), enabling temporally precise and cell type-specific control of cellular functions through selective activation or inhibition using defined wavelengths of light. Optoreactive tissue samples also include chemically sensitized tissue samples, in which light-responsive behavior is conferred by the application of exogenous compounds such as optically-reactive graphene oxide (which transduces light into localized thermal or electrical signals capable of modulating cellular activity) or optically-reactive glutamate derivatives (which undergo conformational or photochemical changes upon illumination to activate or inhibit receptor-mediated pathways). These constructs are designed to exhibit excitatory and / or inhibitory functional responses to light exposure, such as muscle contraction or relaxation. The light-responsiveness can be transient, reversible, and tunable based on parameters such as wavelength, intensity, and duration of the stimulus.

[0026] As used herein, a “coherent light source” means a light source that exhibits both high temporal coherence and high spatial coherence. Temporal coherence refers to the monochromaticity of the light and is characterized by the coherence length (Le), i.e., the distance over which the wavefronts remain phase-correlated. For the purposes of this disclosure, a coherence length Lc > 90 pm (e.g., > 95 pm) is considered to indicate high temporal coherence. Spatial coherence refers to the ability of light emitted from different pointsacross the wavefront to interfere, and is quantified by fringe visibility (p); a value of p > 0.5 (e.g., p > 0.6, 0.7, 0.8, or 0.9) is considered to indicate high spatial coherence. Examples of coherent light sources include, but are not limited to, diode-pumped solid-state (DPSS) lasers and laser diodes, both of which typically exhibit long coherence lengths and high spatial coherence. For the purposes of this disclosure, light-emitting diodes (LEDs) are not considered coherent light sources.

[0027] FIG. IB shows a section view of the optical stimulation device 102 of FIG. 1A (here, an optical stimulation lid or "stim lid") positioned relative to the multi-well cartridge 104. The optical stimulation device 102 comprises a plurality of coherent light sources configured as coherent light modules (here, laser diode modules 126a - 1261) arrayed within a housing 128 that sits atop the multi-well cartridge 104 containing numerous optoreactive tissue samples (e.g., 130). A single tissue sample 130 is shown in FIG. IB; however, in practice, a tissue sample 130 is disposed within each well 134. The coherent light sources are each configured to emit a photon beam tuned to stimulate physiological responses in the tissue samples when the optical stimulation device 102 is positioned relative to the multi -well cartridge 104. In some embodiments, the optical stimulation device 102 is “positioned relative to” the multi -well cartridge 104 when the coherent light sources are aligned with the wells of the multi -well cartridge 104 and / or when a housing of the optical stimulation device 102 abuts the multi -well cartridge 104, e.g., when the multi-well cartridge 104 is received within a skirt of the housing. In some embodiments, each of the coherent light sources emits a photon beam in the visible range of the electromagnetic spectrum (e.g., from about 380nm to about 700nm wavelengths). In some embodiments, each of the coherent light sources additionally or alternatively emits electromagnetic radiation in the ultraviolet range (e.g., from about lOnm to about 400mm wavelength) and / or infrared range (e.g., from about 700nm to about 1mm wavelength).

[0028] Multi -well cartridge 104 is an assembly comprising the multi -well plate 106 coupled with an array of tissue fixtures (e.g., beams, posts, hooks, and / or clamps) such that a plurality of the tissue fixtures 132a, 132b extend into each well 134. The tissue fixtures are positioned relative to the corresponding wells by a supporting lattice or cartridge lid 136. The multi-well plate 106 is constructed from a polymer such as polystyrene, polycarbonate, or polyethylene terephthalate (PET) and has a plurality of wells 134 arrayed in a Society For Biomolecular Screening (SBS) - compatible layout, for example a 24, 48, 96, or 384 well layout. In FIG. IB, the multi-well plate 106 is a 24-well plate. Each of the wells 134 is sized and otherwiseconfigured to receive a plurality of the tissue fixtures 132a, 132b therein. In some embodiments, the plate is transparent or translucent to enable imaging by the tissue contractility measurement instrument 120. Representative multi-well plates 106 include, but are not limited to, NANOSURFACE™ plates sold by Curi Bio, Inc. and CORNING® and FALCON® cultureware plates sold by Corning Inc. of Corning, New York, USA.

[0029] The tissue fixtures 132a, 132b generally include beams or posts, e.g., flexible posts and rigid posts. In some embodiments, at least one flexible post 132a and at least one rigid post 132b extends into each well 134 of the multi -well plate 106 such that the respective distal ends of the flexible and rigid posts are adjacent and spaced apart.

[0030] In use, an optoreactive tissue sample 130 is suspended (e.g., in an extracellular matrix) within each well 134 such that it attaches between the distal ends of the flexible tissue fixture 132a and rigid tissue fixture 132b. Although the representative embodiments described herein include two tissue fixtures 132a, 132b having different properties in a single well 134 (e.g., a rigid post and a flexible post having different stiffness properties in each well 134), in some embodiments, at least two flexible tissue fixtures 132a extend into each well 134.

[0031] 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., rigid post 132b) 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 (e.g., flexible post 132a) in the same well 134. In some embodiments, the foregoing relationship (e.g., between the stiffness of the relatively rigid tissue fixture 132b and the relatively flexible tissue fixture 132a) 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 132b 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 relatively flexible tissue fixture 132a has a stiffness of about O. lN / 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 incorporated by reference in its entirety.

[0032] The housing 128 secures the optical elements and certain electronics of the device 102. In the embodiment shown, the housing 128 is an assembly comprising a lattice 138 and an upper lid 140 enclosing a printed circuit board 142 therebetween. The lattice 138 is amonolithic heat sink formed from a relatively thermally conductive material such as aluminum and having an array of apertures 144 formed therethrough in a spatial arrangement or spaced apart array corresponding to (i.e., mapped to) the arrangement of the wells 134 and the tissue samples 130. As stated above, the multi-well plate 106 has 24 wells in the illustrated embodiment; accordingly, the lattice 138 comprises 24 apertures 144. Each of the apertures 144 is sized to receive one of the coherent light modules (in this embodiment, laser diode modules 126a - 126f), which in turn are fastened to the printed circuit board 142. Optionally, the apertures 144 have a diameter sized to provide a friction fit with the respective laser diode module (which also facilitates conduction of thermal energy from the laser diode modules to the lattice 138. Thus, the positioning of each of the laser diode modules 126a - 126f is secured in three orthogonal axes by the apertures 144 and affixation to the printed circuit board 142. Lattice 138 optionally comprises a cartridge alignment feature such as a skirt 146 forming a recess sized to receive the multi -well cartridge 104 therein and to align its well 134 with the laser diode modules 126a - 126f.

[0033] As stated above, in the representative embodiment shown, the multi -well plate 106 and lattice 138 comprise 24 wells and 24 apertures, respectively; accordingly, the optical stimulation device 102 comprises 24 coherent light modules. In the illustrated embodiments, the coherent light modules are configured as laser diode modules 126a - 126f, each of which emits a coherent elliptical photon beam tuned to stimulate physiological responses in the optoreactive tissue samples 130 in each well 134 under the precise input of the controller 108 via printed circuit board 142. In some embodiments, the optical stimulation device 102 includes different types of coherent light sources, for example DPSS lasers instead of laser diodes. Accordingly, laser diodes are utilized throughout the present disclosure as representative and non-limiting coherent light sources. In any embodiment, the term “laser diode” or “laser diode module” may be replaced with “coherent light source” or “coherent light module,” respectively.

[0034] To achieve tissue stimulation, each of the coherent light modules is configured to emit a photon beam 148 at a frequency of at least about 5 Hz (e.g., 5-15 Hz); an output power of at least about 40 mW to about 80 mW (resulting in an incident power at the tissue sample 130 of at least about 20 mW / mm2), and a pulse width of about 1ms to about 8ms (e.g., 3ms to 4ms).

[0035] In some embodiments, different coherent light modules within the optical stimulation device 102 emit different wavelengths or ranges, e.g., a first plurality of modules emits light ina first wavelength range (e.g., red light or about 620nm to about 750nm), a second plurality of modules emits light in a second wavelength range (e g., green light or about 495nm to about 570nm), and a third plurality of modules emits light in a third wavelength range (e.g., blue light or about 400nm to about 495nm).

[0036] In the illustrated embodiment, laser diode modules 126a - 126f are positioned by the lattice 138 in a spatial arrangement corresponding to wells 134 of the multi-well plate 106 such that each laser diode module 126a - 126f is positioned to emit an elliptical photon beam (e.g., photon beam 148) into one of the wells 134 when the multi-well cartridge 104. Restated, in the illustrated embodiment, there is a 1 : 1 relationship between the number of laser diode modules and the number of wells 134. Accordingly, in some embodiments, optical stimulation device 102 includes 24, 48, 96, or 384 laser diode modules. In some embodiments, the optical stimulation device 102 includes two or more laser diode modules per well 134 (e.g., 2, 3, or 4). In some such embodiments, each of the laser diode modules is configured to emit a photon beam having a different wavelength or other input waveform parameter from the other modules of the same well 134. Accordingly, the number of laser diode modules is not limited to the number of wells 134 or apertures 144 and in some embodiments is a multiple of the number of wells 134 or apertures 144.

[0037] As detailed with respect to FIG. 1C below, each of the laser diode modules 126a - 126f comprises a laser diode and a lens precisely positioned to focus a waist of the polarized photon beam 148 on a focal plane 150 extending through the tissue samples 130 when the multi-well cartridge 104 is positioned relative to the optical stimulation device 102, and further to align each polarized photon beam 148 with the shape of its respective tissue sample 130 to optimize energy delivery. Restated, the focal plane 150 corresponds to the tissue sample plane, i.e., it extends through the tissue samples disposed between distal ends of the tissue fixtures 132a, 132b. In this way, each laser diode module 126a - 126f precisely focuses the respective photon beam 148 of sufficient photon density on the tissue sample 130 in the corresponding well 134 in order to activate optically sensitive cells, e.g., motor neurons.

[0038] Referring again to FIG. 1 A together with FIG. IB, optical stimulation device 102 is communicatively connected to the controller 108 via the printed circuit board 142. Controller 108 is a processor-based system configured to control optical stimulation device 102 and in particular, to control one or more input waveform parameters of each of the laser diode module 126a - 126f. Controller 108 is implemented at least partially internally to the opticalstimulation device 102 (e.g., as the printed circuit board 142, an internal control unit, electronic control unit, on-board computer, etc.) providing control of the laser diode modules 126a - 126f. In some embodiments, controller 108 is implemented at least partially externally with respect to the optical stimulation device 102 as part of remote computing resources 124 (e.g., as a server, personal computer system, notebook computer system, tablet system, smartphone system, etc.) and / or an external drive 112. Control systems implemented internally or externally with respect to optical stimulation device 102 are collectively referred to herein as the controller 108. Controller 108 is configured to display information on the user interface 122 of the remote computing resources 124 according to the methods described herein.

[0039] The printed circuit board 142 includes circuits operably connected to the laser diode modules, to the processor 110, and to the logical modules stored in the memory 114. Said circuits on the printed circuit board 142 enable the stable parallel operation of the laser diode module and include, for example: circuit elements that supply power to the laser diode modules 126a - 126f (e.g., voltage regulators, direct current to direct current (DC-DC) converters, and current drivers); circuit elements that control logic and switching (e.g., field programmable gate array (FPGA), (Metal Oxide Semiconductor Field Effect Transistors (MOSFETS), Digital- To-Analog Converters (DACs), and Pulse Width Modulation (PWM) controllers); signal processing circuit elements (e.g., amplifiers and photodiodes); and protection circuit elements (e.g., electrostatic discharge (ESD) protection diodes, transient voltage suppression (TVS) diodes, current limiting resistors, and thermistors). The foregoing circuit elements are representative and non-limiting.

[0040] Drive 112 is operably connected to the processor 110 and printed circuit board 142, and controls at least one input waveform parameter of each of the laser diode modules 126a - 126f under the direction of the processor 110, e.g., frequency, amplitude, polarity and pulse width. One representative drive is the MANTARRAY™ drive sold by Curi Bio, Inc.

[0041] As shown in FIG. 1A, the illustrated implementation of controller 108 includes processor 110 and memory 114 (e.g., as part of remote computing resources 124). Processor 110 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 Corp., 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 108 as long as the samesupports the operations as described herein. For example, in some embodiments, controller 108 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 Corp, of Santa Clara, California, USA, a field programmable gate array (FPGA), etc.

[0042] The memory 114 includes a non-transitory computer-readable storage medium, e.g., 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.

[0043] Memory 114 contains logical instructions that, when executed by the processor 110, control operations of the optical stimulation device 102 via the drive 112 and printed circuit board 142, namely controlling aspects of one or more input waveform parameters to the laser diode modules 126a - 126f. Representative logical instructions are described herein as modules related to logically different tasks, e.g., a power control module 116 and a protocol control module 118. However, this organization is intended to facilitate understanding and does not limit the architecture of the logical instructions.

[0044] Power control module 116 comprises logical instructions that relate to the input command current and / or output power of each of the laser diode modules 126a - 126f. In some embodiments, the module 116 displays, on the user interface 122, an input command current option that enables a user to select the input command current for one, some, or all of the laser diode modules 126a - 126f. In some embodiments, the module 116 displays, in the user interface 122, an optical output power option that enables the user to directly select the optical output power or pulse amplitude for one, some, or all of the laser diode modules 126a - 126f. In such embodiments, the power control module 116 references a lookup table to convert the selected optical output power or pulse amplitude to the corresponding input command current. Accordingly, based on the inputs via the user interface 122, the power control module 116 controls the relevant circuitry of the printed circuit board 142 to control the relevant parameters of the laser diode modules 126a - 126f. In some embodiments, the power control module 116 is configured to control said parameters of each of the laser diode modules 126a - 126f independently from the other laser diode modules 126a - 126f.

[0045] Protocol control module 118 comprises instructions that relate to additional input waveform parameters, including pulse width, pulse repetition rate, frequency, amplitude, polarity, pulse train length, and other characteristics of the photon beams of the laser diode modules 126a - 126f. In some embodiments, the protocol control module 118 displays, on the user interface 122, options to modulate any one or combination of the foregoing parameters. In some embodiments, the protocol control module 118 is configured to control one or more input waveform parameters of each of the laser diode modules 126a - 126f independently from the other laser diode modules 126a - 126f. For example, to accelerate discovery of optimal input waveform parameter combinations for a given experimental setup, in some embodiments, the protocol control module 118 enables each of the laser diode modules 126a- 126f to operate in contemporaneously (in parallel) with a different combination of input waveform parameters relative to the other modules. Thus, in some embodiments, the protocol control module 118 is configured to independently control the frequency, the amplitude, and the pulse width of each of the laser diode modules 126a - 126f. In some embodiments, the protocol control module 118 displays, on the user interface 122, options that enable a user to select the type of tissue sample (e.g., motor neurons) and the desired tissue modulation protocol (e.g., a twitch response). Accordingly, based on the inputs via the user interface 122, the protocol control module 118 controls the relevant circuitry of the printed circuit board 142 to control the relevant parameters of the laser diode modules 126a - 126f over time in order to achieve the desired tissue response.

[0046] In some embodiments, the power control module 116 and protocol control module 118 are together configured to independently control one or more of the following input waveform parameters of the laser diode modules 126a - 126f: a frequency of at least about 5Hz (e.g., 5 to 15 Hz); an input current in order to deliver an incident power at the tissue sample of at least about 20 mW / mm2, e.g., about 20 mW / mm2to about 50 mW / mm2, a pulse width of about 1ms to about 8ms (e.g., 3ms to 4ms), and / or a pulse train length of about 50ms to about 300 milliseconds (e.g., about 50ms to about 100ms). Restated, one or more of said parameters may be controlled both within each well 134 and between the wells 134.

[0047] In some embodiments, the power control module 116 and protocol control module 118 modulates one or more of the foregoing input waveform parameters in response to a physiological response measured by the optional tissue contractility measurement instrument 120 and provided as feedback to the optical stimulation device 102.

[0048] The laser diode module and some or all of the respective wells 134 and the tissue fixtures collectively form what is referred to herein as a laser diode tissue stimulation module 152. Accordingly, system 100 includes a plurality of such laser diode tissue stimulation module 152, which may include structure in permanent and / or consumable aspects of the system.

[0049] FIG. 1C shows a section view of the laser diode module 126a, which is representative of all such laser diode modules of the optical stimulation device 102 of FIG. IB. Laser diode module 126a is a modular optical assembly or capsule comprising a laser diode 154 precisely positioned relative to a lens 156 by a lens spacer 158 configured to focus and align the photon beam with the tissue sample in the focal plane to optimize selective activation of physiological responses (e.g., a twitch contraction) in certain cells of the optoreactive tissue samples, e.g., iPSC-derived motor neurons expressing ChR2. Accordingly, the selection and packaging of the laser diode 154 and lens 156 enable optimal performance.

[0050] Laser diode 154 is selected to emit a photon beam at a wavelength and with a sufficient incident power to stimulate a physiological response in a given tissue sample. Accordingly, the specific features of the laser diode 154 differ for different applications. In some embodiments, laser diode 154 emits a blue light photon beam (e.g., about 400nm to about 495nm wavelength) and / or provides an incident optical power on the tissue sample in the focal plane (see FIG. IB) of at least 20 mW / mm2, at least 30 mW / mm2, or at least 40 mW / mm2. Representative laser diodes 154 include, e.g., model GH04580A2G manufactured by SHARP®. In some embodiments, the laser diode 154 emits a different wavelength, e.g., red light (about 620nm to about 750nm) or green light (e.g., about 495nm to about 570nm). Laser diode 154 has a fast axis and a slow axis that influence the shape of the photon beam as discussed below with respect to FIG. 2.

[0051] Lens 156 is an aspherical collimation focal lens selected to focus a waist of the photon beam on the tissue samples and to preserve the elliptical shape of the photon beam 148. In some embodiments, the lens 156 is selected with a focal length equal to a distance (within the optical stimulation device 102) between the lens 156 and the distal ends of the tissue fixtures.

[0052] Lens spacer 158 is a hollow body (e.g., cylindrical) that receives the laser diode 154 and lens 156. Notably, lens spacer 158 fixes a back-focal distance 160 between an emitter of the laser diode 154 and the lens 156 in order to focus the waist of the photon beam on the focal plane that extends through the tissue samples and distal ends of the tissue fixtures.Additionally, lens spacer 158 is a part of a thermal conduction path that conducts thermal energy from the laser diode 154 to the lens spacer 158 and to the lattice 138. Accordingly, lens spacer 158 is formed of a relatively thermally conductive material such as aluminum in order to conduct thermal energy away from the laser diode 154 to the lattice 138. To fix the back-focal distance 160, lens spacer 158 comprises a diode seat 164 (first internal seat) and an internal lens seat 162 (second internal seat), each being formed as an internal shoulder or stepped bore having a respective diameter sized to receive the lens 156 and the laser diode 154. In the illustrated embodiment, the diode seat 164 is localized to sit against (form a physical contact against) a radial skirt of the laser diode 154 such that the diode seat 164 forms a thermal conduction path to conduct thermal energy from the laser diode 154 to the lens spacer 158.

[0053] A collar 166 disposed around a base of the laser diode 154 and against a terminal face of the laser diode 154 functions, along with a terminal extension 168, to set a calibrated distance between the laser diode 154 and the printed circuit board 142 such that the waist of the photon beam is focused on a focal plane that extend through the distal ends of the tissue fixtures when the multi-well cartridge is received within the optical stimulation device. Additionally, the collar 166 facilitates insertion of the laser diode 154 into the lens spacer 158. To prevent mis-seating the laser diode 154 at a suboptimal back-focal distance from the lens 156, a central body height and a skirt thickness of the collar 166 are sized to create a circumferential clearance 170 between the collar 166 skirt and a terminal end of the lens spacer 158 when the laser diode 154 is fully seated in the diode seat 164. Accordingly, the laser diode 154 fully seats in the diode seat 164 before the skirt of the collar 166 contacts the lens spacer 158. Restated, when the laser diode 154 is seated in the diode seat 164, the collar 166 is spaced apart from the lens spacer 158 by the clearance 170.

[0054] The laser diode module 126a further includes toroidal seal toroidal seals 172a, 172b disposed at either end thereof that apply even compression of the components when the laser diode module 126a is secured (e.g., screwed) to the printed circuit board 142. This compression helps ensure the accurate back-focal distance 160, provides effective heat sinking from the laser diode 154, and isolates the laser diode 154 from shock and ingress.

[0055] Turning to FIG. 2, operation of the representative laser diode module 126a is shown. The laser diode module 126a emits the photon beam 148 (dotted line) having an oblong or elliptical shape with a longitudinal dimension and a short dimension. For example, in some embodiments, the photon beam 148 has elliptical dimensions of about 2.0-3.0mm x 0.5-1.5mm.As shown, in some embodiments, the photon beam 148 is aimed and focused on a localized section of the tissue sample 130. For example, in FIG. 2, the photon beam 148 is aimed and focused on a central section of the photon beam 148 disposed between the tissue fixtures 132a, 132b. In some embodiments, the photon beam 148 is aimed and focused on an end section optionally encompassing one of the tissue fixtures 132a, 132b). The shape and size of the photon beam 148 is characterized by the divergence angles of the laser diode 154 along a long axis or fast axis 174 (dashed line) and a slow axis that are respectively perpendicular and parallel to the diode junction. Restated, the longitudinal dimension of the elliptical photon beam 148 is aligned with (e.g., parallel to) the fast axis 174 of the laser diode 154. In some embodiments, the polarity of the laser diode 154 is aligned with the fast axis 174 and the longitudinal dimension of the elliptical photon beam such that the electrical field of the photon beam oscillates in the same dimension of max divergence (i.e., the fast axis 174). The optical stimulation device 102 leverages this property by clocking or rotationally orienting the laser diode module 126a within its respective aperture 144 of the lattice 138 to align the fast axis 174 and / or the polarity with the tissue fixtures 132a, 132b and a longitudinal dimension of the tissue sample 130 in the corresponding well. In this way, the longitudinal dimension of the tissue sample 130 is aligned with the tissue fixtures 132a, 132b and with the longitudinal dimension of the photon beam 148, i.e., with the fast axis 174. Advantageously, this optimizes incident optical power on the portion of the tissue sample 130 disposed between the tissue fixtures 132a, 132b. Restated, the fast axis 174 aligns with an axis extending through the tissue fixtures 132a, 132b.

[0056] FIG. 3 plots spontaneous contraction forces of optoreactive tissue samples and contraction forces of the same tissue samples when stimulated with the optical stimulation device 102 as described above. In FIG. 3, the optoreactive tissue samples are optogenetic tissue samples containing motor neurons expressing ChR2 co-cultured with skeletal muscle tissue as described in Fleming, Jacob W., et al. "An automated platform for simultaneous, longitudinal analysis of engineered neuromuscular tissues for applications in neurotoxin potency testing." Current Research in Toxicology 8 (2025): 100218. Specifically, to create the tested tissue samples, co-culture motor neuron-skeletal muscle tissues were created through a multi-step process combining engineered muscle tissues (EMTs) with neurospheres. The EMTs were first generated using a 9: 1 ratio of iPSC-derived skeletal muscle myoblasts to primary human dermal fibroblasts, with about 420,000 cells per tissue. These cells were suspended in ahydrogel mixture containing fibrinogen, Dulbecco's Modified Eagle Medium (DMEM), and an extracellular matrix hydrogel derived from Engelbreth-Holm-Swarm (EHS) mouse sarcoma (here, MATRIGEL® manufactured by Corning, Inc ), which was then combined with thrombin solution to create the initial muscle tissue structure. Separately, neurospheres were formed using iPSC-derived motor neurons (250,000 cells per well) that were cultured for 10 days in specialized casting trenches. At day 10, the mature EMTs were combined with the neurospheres using a linking hydrogel composed of 1 mg / mL type-I rat tail collagen with 5% Matrigel. Following hydrogel addition, the EMT tissue lattice was submerged into the neurosphere casting plate, incubated for 1 hour, and maintained in specialized media containing both muscle maintenance components and neuronal supplements. The co-cultures were maintained in situ for 24 hours before transfer to maintenance medium supplemented with 2 pg / mL doxycycline to enable channelrhodopsin-2 expression in the motor neurons.

[0057] Blue light stimulation was carried out with the optical stimulation device 102 described above, with a 450 nm wavelength laser focused onto the center of the tissue (as shown in FIG. 2) using monophasic 250 ms pulse trains (90 mA input current producing 41.7 mW / mm2incident optical power on the tissue sample, 8 ms pulse width, and 100 Hz frequency) spaced at 5 second intervals. Peak contraction force for each tissue was derived from an average of 10 consecutive peaks. Calculation of active peak force, half relaxation time, and time from 10% contraction to peak was undertaken using the PULSE™ 3D platform sold by Curi Bio, Inc.

[0058] As shown in FIG. 3, the optoreactive tissue samples (here, motor neuron-skeletal muscle EMTs) show robust and quantifiable response to blue light activation with the system 100 described herein, with SkM-only controls showing no activation, providing confidence that incident blue light acts through the motor neurons expressing ChR2 and not another off-target non-specific activation mechanism.

[0059] FIG. 4 illustrates a distinct advantage of the optical stimulation device 102 described herein, i.e., the absence of crosstalk between wells even when used with a transparent multiwell plate 106. In particular, FIG. 4 plots contraction forces of optoreactive tissues prepared as described with respect to FIG. 3. The optoreactive tissue samples in a subset of six adjacent wells of the 24-well configuration described previously, i.e., wells C3, C4, C5, D3, D4, and D5. The plotted contraction forces were measured with a tissue contractility measurement instrument 120 - here, MANTARRAY™ sold by Curi Bio, Inc.

[0060] In FIG. 4, only the optoreactive tissue sample in well D4 is optically stimulated with the optical stimulation device 102 described above. None of the optoreactive tissue samples in wells C3, C4, C5, D3, or D5 are optically stimulated with the optical stimulation device 102. As shown, the optoreactive tissue sample in well D4 registers robust and quantifiable contraction forces in response to optical stimulation by optical stimulation device 102, whereas all optoreactive tissue samples in the adjacent wells do not register any contractions other than spontaneous contractions.

[0061] This advantage follows from the high temporal and spatial coherence of the laser diode modules 126a - 126f described above and precise focusing on the focal plane in the multi-well plate 106. By comparison, incoherent light sources such as light emitting diodes (LEDs) would cause significant crosstalk between wells, particularly at incident power levels at 20mW / mm2and higher, leading to significant noise and diminished repeatability.

[0062] FIG. 5 illustrates methods 500 for parallel optical stimulation of a plurality of optoreactive 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 optical stimulation device 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.

[0063] Step 502. A plurality of optoreactive tissue samples 130 are provided, e.g., in a multi-well cartridge 104. In some embodiments, each of the tissue samples 130 has an elongate shape and a longitudinal dimension aligned with its longest dimension (e.g., the longitudinal dimension is coaxial with the fast axis 174 shown in FIG. 2).

[0064] Step 504. The multi-well cartridge 104 is positioned relative to an optical stimulation device 102 comprising a plurality of coherent light sources (e.g., 126a - 126f) in a spaced apart array mapped to the spatial arrangement of the tissue samples 130 as described above.

[0065] Step 506. A plurality of photon beams 148 (e.g., elliptical photon beams) are contemporaneously emitted by the plurality of coherent light sources 126a -126f. Restated,each of the coherent light sources 126a - 126f emits a photon beam contemporaneously with the other coherent light sources. In some embodiments, each of the photon beams 148 has a wavelength in the visible light spectrum, infrared spectrum, or ultraviolet spectrum. Each of the photon beams 148 (e.g., a waist thereof) is focused on a different one of the optoreactive tissue samples 130. In some embodiments in which the photon beam is elliptical, each of the elliptical photon beams is focused on, and a longest dimension is aligned with the longitudinal dimension of, a different one of the tissue samples 130 and / or a fast axis 174 of the corresponding coherent light sources 126a - 126f. In some embodiments, each of the photon beams has an incident optical power on the corresponding optoreactive tissue sample 130 of at least 20 mW / mm2, a pulse width of about 1 ms to about 8 ms (e.g., about 3m to about 5m), and a frequency of at least about 5Hz. To facilitate exploration of parameter space, in some embodiments, at least some of the photon beams 148 have different combinations of incident optical power, pulse width, and / or frequency. Restated, in some embodiments, one or more input waveform parameters of each of the coherent light sources 126a - 126f is controlled independently of the other coherent light sources.

[0066] Step 508. One or more physiological responses (e.g., contraction force) of each of the optoreactive tissue samples 130 to the corresponding photon beam 148 is measured, e.g., by a tissue contractility measurement instrument 120. In some embodiments, one or more parameters of one or more of the photon beams 148, and / or more input waveform parameters of one or more of the coherent light sources 126a - 126f, is modulated based upon the measured physiological response(s). Restated, in such embodiments, the measured physiological response(s) is provided as feedback, which is then implemented by varying one or more parameters of the corresponding photon beam 148. In other words, some embodiments further comprise controlling at least one of the incident optical power, the pulse width, or the frequency of at least one of the elliptical photon beams in response to the at least one measured physiological response of the optoreactive tissue sample(s) (e.g., the optoreactive tissue sample(s) on which the relevant photon beams are focused).

[0067] Step 510. In optional step 510, the measured physiological response(s) is displayed on a user interface 122 (e.g., as shown in FIG. 4).

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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 theparticular embodiment (such as when the embodiment is positioned for use) and are not intended to limit the present disclosure to these directions or locations.

[0073] 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, the present 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, i.e., any one of the following conditions satisfy all of the foregoing expressions: A; B; C; AB; AC; BC; ABC.

Claims

CLAIMSWhat is claimed is:

1. A system for parallel optical stimulation of a plurality of optoreactive tissue samples in a multi-well cartridge, the system comprising: an optical stimulation device comprising a plurality of coherent light modules arrayed in a housing, wherein each coherent light module comprises a laser and a lens positioned to focus a photon beam from each laser on a focal plane extending through the multi-well cartridge when the multi-well cartridge is positioned by the housing relative to the optical stimulation device; and a controller operably coupled to the optical stimulation device, the controller comprising a processor and a non-transitory computer-readable storage medium (a memory) containing logical instructions that, when executed by the processor, cause operations, comprising: activating the photon beams and controlling at least one input waveform parameter of each of the coherent light modules.

2. The system of claim 1, wherein the at least one input waveform parameter comprises at least one of a frequency, an amplitude, or a pulse width.

3. The system of claim 2, wherein the at least one input waveform parameter comprises the frequency, the amplitude, and the pulse width, wherein the controller is configured to independently control the frequency, the amplitude, and the pulse width for each of the coherent light modules.

4. The system of claim 1, wherein controlling the at least one input waveform parameter of each of the coherent light modules comprises controlling an amplitude of the photon beam such that the photon beam has an incident optical power on the corresponding optoreactive tissue sample of at least 20 mW / mm2, controlling a pulse width between 1 ms to 8 ms, and controlling a frequency of at least 5Hz.

5. The system of claim 1, wherein the housing is a lattice comprising a plurality of apertures positioning the coherent light modules in a spaced apart array mapped to a plurality of wells of the multi-well cartridge, wherein the photon beam of each of the lasers is emitted into one ofthe wells when the multi-well cartridge is positioned relative to the optical stimulation device by the housing and when the controller activates the photon beams.

6. The system of claim 1, further comprising the multi -well cartridge, wherein the multi-well cartridge comprises a plurality of wells, each of the wells containing a plurality of tissue fixtures therein, wherein each photon beam has an elliptical shape, wherein when the multiwell cartridge is positioned relative to the optical stimulation device by the housing and when the controller activates the photon beams, a longitudinal dimension of each of the photon beams aligns with the plurality of tissue fixtures of the corresponding well.

7. The system of claim 6, wherein the focal plane extends through distal ends of the tissue fixtures when the multi-well cartridge is positioned relative to the optical stimulation device by the housing.

8. The system of claim 7, wherein each of the wells of the multi-well cartridge comprises an optoreactive tissue sample extending between the plurality of tissue fixtures, wherein the focal plane extends through each of the tissue samples.

9. The system of claim 1, further comprising a tissue contractility measurement instrument configured to measure movement of the optoreactive tissue samples disposed in a plurality of wells of the multi-well cartridge when the multi-well cartridge is positioned relative to the optical stimulation device by the housing and when the controller activates the photon beams.

10. A method for parallel optical stimulation of a plurality of optoreactive tissue samples, comprising: providing a plurality of optoreactive tissue samples in a multi-well cartridge, wherein each of the optoreactive tissue samples has a longitudinal dimension; positioning the multi-well cartridge relative to an optical stimulation device comprising a plurality of coherent light sources in a spaced apart array mapped to an arrangement of the plurality of optoreactive tissue samples; contemporaneously emitting an elliptical photon beam from each of the coherent light sources, wherein each of the elliptical photon beams is focused on, and aligned with the longitudinal dimension of, a different one of the optoreactive tissue samples, wherein each of the elliptical photon beams has an incident optical power on the corresponding optoreactivetissue sample of at least 20 mW / mm2, a pulse width of 1 ms to 8 ms, and a frequency of at least 5 Hz; and measuring at least one physiological response of each of the optoreactive tissue samples to the corresponding elliptical photon beam.

11. The method of claim 10, wherein a polarity of each of the elliptical photon beams is aligned with the longitudinal dimension of the respective optoreactive tissue sample.

12. The method of claim 10, wherein at least some of the elliptical photon beams have different combinations of the incident optical power, the pulse width, and the frequency.

13. The method of claim 10, further comprising controlling at least one of the incident optical power, the pulse width, or the frequency of at least one of the elliptical photon beams in response to the at least one physiological response of at least one of the optoreactive tissue samples.

14. The method of claim 10, wherein each of the elliptical photon beams has a wavelength in at least one of the visible light spectrum, ultraviolet spectrum, or infrared spectrum.

15. The method of claim 10, wherein each of the elliptical photon beams is focused on a localized section of the optoreactive tissue samples on which it is focused.

16. A laser diode module for a system for parallel optical stimulation of a plurality of optoreactive tissue samples, comprising: a hollow body spacer comprising: a first internal seat and a second internal seat; a laser diode seated upon the first internal seat; a lens seated upon the second internal seat at a fixed back-focal distance from the laser diode; and a collar disposed around a base of the laser diode and against a terminal face thereof.

17. The laser diode module of claim 16, wherein the hollow body spacer forms a thermal conduction path configured to conduct thermal energy from the laser diode to a thermally conductive lattice, and wherein the first internal seat forms a contact against a radial skirt of the laser diode to conduct thermal energy from the laser diode to the hollow body spacer.

18. The laser diode module of claim 16, wherein the collar comprises a skirt portion spaced apart from a terminal end of the hollow body spacer by a circumferential clearance when the laser diode is fully seated in the first internal seat.

19. The laser diode module of claim 16, further comprising a printed circuit board communicatively connected to a plurality of terminals of the laser diode.

20. The laser diode module of claim 19, wherein the printed circuit board is communicatively connected to a plurality of laser diode modules position in a spaced apart array.

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