WO2025106923A1

WO2025106923A1 - Systems and methods for high-throughput, multi-well spatiotemporal illumination microscopy

Info

Publication number: WO2025106923A1
Application number: PCT/US2024/056283
Authority: WO
Inventors: Hamid CHORSI; Daniel AHARONI
Original assignee: University of California Berkeley; University of California San Diego UCSD
Current assignee: University of California Berkeley; University of California San Diego UCSD
Priority date: 2023-11-15
Filing date: 2024-11-15
Publication date: 2025-05-22
Anticipated expiration: 2026-05-15

Abstract

A spatiotemporal illumination microscope system is presented. The system includes a multi-well plate configured to receive live cells and a DMD configured to deliver targeted illumination to selectively illuminate a plurality of the wells. The system further includes a tandem-lens system having an objective lens and an imaging lens positioned facing each other to capture fluorescent light emitted from the cells. The system also includes an image sensor that receives fluorescent light captured by the tandem-lens system, generating imaging data. The system further includes a controller configured to use the imaging data to control closed-loop modulation of the live cells.

Description

SYSTEMS AND METHODS FOR HIGH-THROUGHPUT, MULTI-WELL SPATIOTEMPORAL ILLUMINATION MICROSCOPY

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is based on, claims priority to, and incorporates herein by reference for all purposes, U.S. Provisional Patent Application No. 63/599,199 filed on November 15, 2023.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under NS126050, and MH132651 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

[0003] The surging demand for advanced high-throughput and high-resolution neural imaging, as well as medical screening, results from a convergence of factors that encompass scientific exploration, medical breakthroughs, technological advancements, and the pursuit of more precise diagnostic and therapeutic solutions. To address this demand effectively, a pivotal instrument in use is the multi-well plate. These versatile well-plates enable the streamlined assessment of numerous compounds or biological samples concurrently, significantly expediting the pace of scientific research. Researchers can efficiently screen extensive libraries of potential drug candidates and conduct comprehensive biological assays with unparalleled precision and consistency. Beyond these advantages, multi-well plates play a pivotal role in scaling down experiments, resource conservation, cost reduction, and enabling automation. Consequently, they have become an indispensable component of modern drug discovery processes and high-throughput biological research.

SUMMARY OF THE DISCLOSURE

[0004] The present disclosure addresses the aforementioned drawbacks by providing a system and method for modulating and detecting light from a sample.

[0005] In some aspects, a microscopy system configured to modulate and detect light from a sample is presented. The system includes an excitation module that directs structured excitation light along a first axis towards an excitation lens. The excitation lens magnifies the excitation light and directs the excitation light along the first axis. The system further includes a dichroic mirror that is arranged along the first axis and configured to reflect excitation light within a first range of wavelengths and transmit emission light within a second range of wavelengths. The dichroic mirror is angled with respect to the first axis to reflect the excitation light along a second axis. The system further includes an objective lens arranged to receive excitation light reflected along the second axis and form an intermediate image plane. The system also includes an imaging lens positioned along the second axis to receive the emission light from the intermediate image plane, through the objective lens and transmitted through the dichroic mirror. The imaging lens forms a focal plane along the second axis. The system further includes a wellblock array that is positioned between the intermediate image plane and the sample to increase a field of view of the sample. The well-block array includes a plurality of elements, and each element comprises an achromatic objective lens and a tube lens. The system also includes an imaging sensor that is configured to detect and digitize the emission light focused by the imaging lens at the focal plane.

[0006] In other aspects, a method for modulating and monitoring a sample is presented. The method includes directing modulation light from a light source along a first axis. The first axis includes an excitation lens that magnifies the modulation light and a dichroic mirror that is angled with respect to the first axis and configured to reflect the modulation light along a second axis toward an objective lens. The method also includes using the objective lens and a well-block array having a plurality of achromatic lenses to focus the modulation light onto a first plurality of wells containing a sample such that the modulation light modulates the sample to produce emission light directed back along the second axis towards the dichroic mirror. The method further includes using the wellblock array to form an intermediate image of the emission light from the first plurality of wells. The method also includes using the objective lens to direct the intermediate image of the emission light back along the second axis towards the dichroic mirror, which is configured to transmit the emission light. The method further includes focusing the emission light at a focal plane using an imaging lens positioned along the second axis and directing the emission light using an image sensor positioned at the focal plane.

[0007] In still other aspects, a spatiotemporal illumination microscope system is presented. The system includes a multi-well plate that is configured to receive live cells. The system also includes a digital micromirror device (DMD) that is configured to deliver targeted illumination to selectively illuminate a plurality of wells of the multi-well plate. The system further includes a tandem-lens system having an objective lens and an imaging lens positioned facing each other to capture fluorescent light emitted from the live cells in the multi-well plate. The system also includes an image sensor that receives fluorescent light captured by the tandem-lens system and generates imaging data. The system further includes a controller that is configured to use the imaging data to control closed-loop modulation of the live cells in the plurality of wells.

[0008] These are but a few, non-limiting examples of aspects of the present disclosures. Other features, aspects and implementation details will be described hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Various objects, features, and advantages of the disclosed subject matter can be more fully appreciated with reference to the following detailed description of the disclosed subject matter when considered in connection with the following drawings, in which like reference numerals identify like elements.

[0010] FIG. 1 is a block diagram of an example microscopy platform for multi-well spatiotemporal illumination microscopy.

[0011] FIG. 2 A provides an optical path diagram corresponding to an example well block array.

[0012] FIG. 2B provides a spot diagram corresponding to the optical system of FIG. 2A.

[0013] FIG. 2C shows an example 2x2 well block array.

[0014] FIG. 2D shows an example 4x2 well block array.

[0015] FIG. 3 A is a block diagram illustrating an example control module.

[0016] FIG. 3B is a block diagram illustrating another example control module.

[0017] FIG. 4 provides a flowchart of an example process that can be used for modulating and imaging a sample in accordance with the present disclosure.

[0018] FIG. 5 demonstrates the real-time control provided by the described systems and methods.

[0019] FIG. 6A shows an image of a resolution target slide produced using systems and methods described by the present disclosure.

[0020] FIG. 6B shows an image of an example projection pattern produced using systems and methods described by the present disclosure.

[0021] FIG. 6C shows an image of a brain slice sample produced using systems and methods described by the present disclosure. [0022] FIG. 6D shows FIG 6C enlarged to highlight the single-cell resolution provided by the described systems and methods.

[0023] FIG. 7 provides a flowchart of an example process for spatially calibrating systems described in the present disclosure.

[0024] FIG. 8 is a block diagram of an example microscopy system that can implement the methods of the present disclosure.

[0025] FIG. 9 is a block diagram of example components that can implement the system of FIG. 8.

DETAILED DESCRIPTION

[0026] Before any aspects of the present disclosure are explained in detail, it is to be understood that the invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the following drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of "including,” "comprising,” or "having” and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. Unless specified or limited otherwise, the terms "mounted,” "connected,” "supported,” and "coupled” and variations thereof are used broadly and encompass both direct and indirect mountings, connections, supports, and couplings. Further, "connected” and "coupled” are not restricted to physical or mechanical connections or couplings.

[0027] The following discussion is presented to enable a person skilled in the art to make and use embodiments of the invention. Various modifications to the illustrated embodiments will be readily apparent to those skilled in the art, and the generic principles herein can be applied to other embodiments and applications without departing from embodiments of the invention. Thus, embodiments of the invention are not intended to be limited to embodiments shown but are to be accorded the widest scope consistent with the principles and features disclosed herein. The following detailed description is to be read with reference to the figures, in which like elements in different figures have like reference numerals. The figures, which are not necessarily to scale, depict selected embodiments and are not intended to limit the scope of embodiments of the invention. Skilled artisans will recognize the examples provided herein have many useful alternatives and fall within the scope of embodiments of the invention.

[0028] In response to the expanding use of multi-well plates in neural imaging, drug discovery, and other areas of research, there is a growing requirement for tools capable of executing high-throughput, high-resolution screening of multi-well plates. Unfortunately, currently available multi-well imaging systems cannot accommodate high-density well plates, such as 96-well plates. Furthermore, these existing systems lack the ability to provide targeted illumination with single-cell resolution. Moreover, some are extremely complex, costly, and computationally expensive while providing modest imaging functionalities. They lack high-throughput and large field-of-view monitoring, imaging, and manipulation of neural activity in live cell and tissue imaging with singlecell resolution. Additionally, they lack closed-loop control, which is advantageous for providing optical stimuli to targeted cells using obtained imaging feedback.

[0029] The present disclosure describes improved systems and methods for microscopy that provides high-throughput imaging with a large field of view (FOV), including parallel imaging and pattern stimulation of spatially distributed subfields with high-resolution at multiple wavelengths. The optical platform and methodology incorporate advanced optical design to capture comprehensive, finely detailed spatially distributed images across large fields-of-view, all while enabling precise pattern stimulation across a range of wavelengths.

[0030] In some aspects of the present disclosure, a multi-well spatiotemporal illumination microscope, which may be referred to as the STIMscope, is described. In some configurations, the STIMscope can be used for live-cell imaging and stimulation for high-throughput, high-resolution studies with multi-well plates (e.g., 96-well plate). As one non-limiting example, simultaneous imaging and targeted illumination can be achieved in several wells (e.g., up to 20 or more). This design enables large FOV continuous studies of organoids and high-speed voltage imaging. The STIMscope can provide multiplexed imaging of a multi-well with single -photon, single-cell resolution. The STIMscope can be used with long-term, closed-loop monitoring and targeted optogenetic manipulation. As one non-limiting example, the closed-loop modulation can be used to monitor activity-dependent changes in fluorescent signals and to modulate (e.g., excite or inhibit) targeted neurons or subsets of neurons. The STIMscope can be used for a wide variety of biomedical research and bioimaging, having particular significance in high-throughput drug development and therapeutic interventions. For example, the STIMscope can be used for the close study of the behavior of individual cells or living brain networks and the investigation of functional interactions and changes over time.

[0031] In some implementations, the STIMscope system uses a modular design, allowing for swapping of components to flexibly achieve the desired imaging and modulation parameters, such as magnification, power output, illumination, light collection efficiency, and field-of-view. This flexible and interchangeable setup and components can allow for operation in various configurations, such as upright and inverted orientations. One example setup, which will be described in further detail below, provides concurrent monitoring of up to 20 wells of a 96-well plate. However, the system can be expanded to image and modulate other combinations or numbers of wells. In some implementations, an automated XY translational stage can be included to facilitate observation of the whole multi-well plate (e.g., 96 wells) with four-dimensional resolution.

[0032] Modulation may be achieved using single-photon sources to reduce tissue damage and photobleaching when compared to multiphoton excitation techniques. Various excitation/emission filters and dichroic mirrors can be used to simultaneously image numerous indicators.

[0033] The described systems provide several advantages compared to existing systems, including high light efficiency, large field of view, high numerical aperture (NA), high spatial resolution, and high throughput.

[0034] To demonstrate the effectiveness of the proposed approach, one can compare light collection capabilities of the STIMscope against conventional platforms. Many of the advantages ofthe STIMscope over conventional microscopes arise from its enhanced light efficiency, which is calculated as the product of light collection efficiency and light focusing efficiency.

[0035] Light collection efficiency is determined by the squares of the numerical apertures of the respective objectives, while light focusing efficiency is assessed by multiplying the squares of the magnification with the squares ofthe reciprocal ofthe pixel size for each image sensor. The comparative total efficiency can be defined as:

T] = (light collection efficiency ratio) X (light focusing efficiency per pixel) [Eq. 1]

where NA_Conv is the numerical aperture (NA) of a conventional one-photon microscope, NA_STiMscope is the numerical aperture of the described STIMscope, Px_STIMscope and P^xconv ^are the pixel sizes for the image sensor of the conventional one-photon microscope and STIMscope, respectively, and M_STIMscope and M_Conv are the magnification of the conventional one-photon microscope and STIMscope, respectively.

[0036] In one non-limiting example, the STIMscope has an NA of 0.3, pixel size of 4 pm², and magnification of 1 compared to a conventional microscope with an NA of 0.8, pixel size of 8.41 pm², and magnification of 8. Thus, the comparative total efficiency is:

0.3² (4 pm² 64

’ ⁼ o^N~^x5s^| ^{= 4}'²⁸

[0037] In this non-limiting example, the STIMscope platform requires 4.76 times less light than a conventional one-photon microscope with a high NA (e.g., 0.8). This theoretical comparison represents a lower boundary of the STIMscope platform's performance. The NA in Equation 2 refers to the acceptance NA of the objective, but the overall NA depends on the entire optical system. Since the STIMscope employs large aperture photographic lenses, it enhances light collection efficiency throughout the optical path compared to systems with low-aperture optics. This increased light collection significantly improves the performance of STIMscope even more. Additionally, the STIMscope can achieve even higher NA values using lenses with lower f-n umbers (e.g., up to 0.625). Moreover, while smaller pixel sizes may reduce overall efficiency, they substantially improve the system's resolution. In this way, the STIMscope can achieve high spatial resolution while maintaining high light efficiency.

[0038] The numerical aperture can be approximated as follows:

1

NA = - - - - —

2 x f-number where the f-number is a well-defined parameter in the context of photographic lenses. By employing low f-number aperture lenses (e.g., ranging from 0.8 to 1.4), NAs between 0.36 and 0.625 can be achieved. These values are considered high compared to the NA of a conventional lOx objective lens, which typically ranges from 0.25 to 0.30 and moderate when compared to a 50x air objective lens, which typically has an NA range of 0.5 to 0.9. [0039] Moreover, the total NA, which represents the NA throughout the entire optical system, is higher for the STIMscope. This improvement can be attributed to the use of large aperture photographic lenses, which effectively minimize the loss of off-axis rays that would otherwise fail to reach the image sensor.

[0040] The FOV provided by the STIMscope is advantageously significantly larger than that of conventional microscopes. As non-limiting examples, the field-of- view may be chosen with a diameter between 1-5 mm, for example 3 mm. In one nonlimiting example provided in FIGS. 2 A and 2B, the FOV is set to 2 mm in diameter. As another non-limiting example, the STIMscope setup can achieve FOVs of up to 3 mm² per well in a 96-well plate, simultaneously exciting and imaging 20 wells. This results in a total FOV of 60 mm² in this example. In contrast, a typical high-magnification objective, such as a 50x objective, usually achieves a FOV of only 0.5 mm² per well, which decreases further with higher magnification objectives.

[0041] The STIMscope platform is also characterized by high spatial resolution, achieving single-cell resolution. In a non-limiting example, the experimental imaging resolution reaches 6 pm, and the excitation resolution reaches 8 pm. The STIMscope platform leverages a small-pixel, backside-illuminated image sensor, enabling it to effectively resolve individual cells within a large F 0V.

[0042] High throughput encompasses speed, automation, and scalability. In terms of speed, high throughput refers to the capability to image more than a single well, as seen in commonly used multi-well plate imaging platforms, such as those utilizing 96-well plates. Regarding automation, the STIMscope enables unattended or automated imaging of multiple samples. The system is also compatible with robotic arms for efficient well plate transfer. As for scalability, the system can be expanded to handle large numbers of samples simultaneously. In this way, the STIMscope platform can be integrated with state-of-the-art drug discovery laboratories. In some implementations, multiple STIMscopes can be positioned side by side to further enhance throughput.

[0043] Referring now to FIG. 1, a non-limiting example arrangement of a spatiotemporal illumination microscope system 100 is provided. For the purposes of the present disclosure, the system can be described by defining two axes, the imaging axis 196 and the modulation axis 198. Along the imaging axis 196, forward refers to a direction towards the imaging sensor, and backward refers to a direction toward the sample. Along the modulation axis 198, forward refers to a direction toward the emission/excitation source, and backward refers to a direction toward the imaging axis 196. [0044] The system 100 includes a tandem-lens configuration to increase the numerical aperture, enhance light collection efficiency per pixel, and drastically improve the FOV compared to other systems. In this arrangement, two large-aperture photographic lenses are positioned facing each other. The first lens is an imaging lens 102, which may have an NA in the range of 0.3-0.625 and a focal length in the range of 10- 50 in some implementations. The second lens is an objective lens 104, which may have an NA in the range of 0.17-0.28 and a focal length in the range of 100-400 as non-limiting examples. The objective lens 104 can be arranged backward of the imaging lens 102 along an imaging axis 196. In some implementations, the imaging lens 102 and objective lens 104 can offer flexibility to achieve the appropriate demagnification based on the image sensor format requirements.

[0045] As a non-limiting example, to accommodate the imaging and excitation of a 5x4 well array with intermediate image plane of 45 mm x 36 mm, the STIMscope can employ a medium format (e.g., 48 mm x 36 mm sensor size) objective lens with a large aperture and an extended focal length of 135 mm. As another non-limiting example, to collect the emitted signals from the intermediate image plane, the STIMscope may utilize a 15 mm imaging lens 102 along with a 135 mm objective lens 104, resulting in a 9x demagnification. This example configuration properly maps the intermediate image onto a 5.2 mm x 3.9 mm CMOS image sensor. In other configurations, the focal lengths of the imaging lens and objective lens can be chosen to achieve a sufficient demagnification (e.g., at least 8X, at least 9X, at least 10X, and so forth) to map the intermediate image onto the image sensor based on the sizes of the intermediate image and the image sensor in use.

[0046] The use of photographic lenses is not common in microscopy. Instead, standard microscopy systems utilize lenses that achieve high magnification in order to achieve high resolution. The present disclosure recognizes that a large aperture size can be used to compensate for a lower NA, increasing light collection efficiency. In some implementations, the disclosed system achieves a larger FOV with little to no magnification, or even minification.

[0047] A stage arrangement 106, which contains the sample during use, can also be arranged along the imaging axis 196 backward of the objective lens 104. Thus, the light emitted by the sample, which may be referred to as emission light 190, is captured by the objective lens 104 and directed forward through an infinity-corrected zone 110 to reach the imaging lens 102. The imaging lens 102 subsequently concentrates the gathered light beams at its focal plane, where the image sensor 112 resides.

[0048] To direct the fluorescent light effectively, a dichroic mirror 114, is positioned between the objective lens 104 and imaging lens 102 along the imaging axis. The dichroic mirror 114 can be angled (e.g., 45°) with respect to the modulation axis 198. The dichroic mirror 114 is utilized to reflect light at the desired excitation/emission wavelengths or range of wavelengths. For example, the dichroic mirror 114 can reflect excitation light 188 while allowing fluorescent emission or emission light 190 to pass through. In this way, light within a reflection range of wavelengths is reflected by the dichroic mirror 114, and light within a transmission range of wavelengths is transmitted through the dichroic mirror 114. The light collected by the imaging lens is then transmitted through an emission filter 116 configured to block unwanted light (e.g., excitation light, environmental light) and precisely focused onto the image sensor 112 for digital image capture.

[0049] The system 100 can be designed to accommodate a variety of image sensors 112 so that the image sensor 112 can be flexibly chosen based on the desired application. For example, options range from low-cost, small format complementary metal-oxide semiconductor (CMOS) image sensors to high-end scientific CMOS (sCMOS) sensors, as well as custom-designed image sensors for specialized applications. In some implementations, CMOS sensors with small form factors and small pixel sizes can be used as the system provides high light efficiency per pixel, thus achieving sufficient spatial resolution. In contrast, many standard systems require the use of specialized and highly expensive CMOS sensors that have large pixel size to achieve magnification.

[0050] The stage arrangement 106 includes a stage 120 that can receive a well plate 122 containing a sample. As non-limiting examples, the well plate 122 may be a 6-, 12-, 24-, 48-, 96-, 384-, or 1536-well plate. The stage arrangement 106 also includes several optical components, including a well-block array 124 and an optional electrowetting tunable lens array 126 that create an intermediate image plane 128. The well-block array (WBA) 124 includes several elements (e.g., 2x2, 2x4, 4x5, and so forth) to simultaneously produce a field-of-view (FOV) within several wells (e.g., 4, 8, 20, and so forth) to simultaneously modulate and detect light from multiple wells.

[0051] The Well-Block Array (WBA) 124 can also be configured to generate an intermediate image. Each element can include miniaturized optics to generate the intermediate image, enabling a large FOV within each well. As non-limiting examples, the FOV in each well may be set between 0-3 mm in diameter. This intermediate image enables a large field-of-view within each well and provides the necessary magnification to cover a medium-format sensor.

[0052] In some configurations, the size of each WBA element and its corresponding intermediate image does not exceed the pitch between wells in the well plate. For example, the size of each WBA element and intermediate image can be restricted to 9 mm, as dictated by the 9 mm pitch between wells in a 96-well plate.

[0053] Each individual element within the array can encompass both illumination and collection optics, including achromatic objective lenses, and tube lenses tailored to provide the appropriate magnification. Each element can also include one or more electrowetting tunable lenses. These optics can be meticulously designed to meet the requirements for field-of-view, working distance, and the constraints of available space. Moreover, an electrowetting tunable lens array can be integrated into the optical pathway for each well block, as needed. This addition serves to monitor individual wells with reduced optical aberrations and ensures optimal or improved focus. Notably, the electrowetting tunable lens enables per-well optical sectioning and z-stacking.

[0054] The WBA can be designed using cutting-edge optical design and simulation software facilitated with a computer processor. This design process can be used to ensure that the WBA fulfills the optical prerequisites and achieves the desired FOV and resolution for single-cell imaging and targeted stimulation or modulation. For example, in some implementations, the WBA can be configured to provide at least 4X magnification.

[0055] FIG. 2A provides an illustration of the optical path 200 and components of an example WBA 124 arrangement. In this non-limiting example arrangement, the optical pathway includes four achromatic lenses 202, which collectively yield a 4.5X magnification. This magnification is designed to accommodate a 2 mm-diameter object space field-of-view, effectively filling a 9 mm-diameter space on the image plane. FIG. 2B provides a spot diagram corresponding to the optical system shown in FIG. 2 A, which yields a calculated 4.43 IX magnification.

[0056] The integration of the WBA with the tandem lens configuration of large- aperture photographic lenses allows for the effective partitioning of the field of view into multiple spatially distributed subfields, whether organized spatially or angularly. The WBA consists of a grid-like arrangement of lenses, each optically connected to specific subfields within the field of view. Each lens or ‘well’ in the array can be independently controlled for both imaging and stimulation, enabling the parallel processing and analysis of multiple samples or regions. This approach significantly accelerates data acquisition by facilitating simultaneous imaging and stimulation. The WBA supports patterned illumination for each subfield, allowing for the projection of the desired light patterns onto the sample. This capability enhances both throughput and efficiency by enabling excitation via complex light patterns and simultaneous processing of multiple subfields. Moreover, the STIMscope platform provides dynamic adjustment of illumination patterns based on real-time feedback, which can ensure precise and adaptive stimulation. Such closed-loop control can be facilitated by the synchronized real-time control module, which will be described further below.

[0057] Overall, the WBA notion and design significantly improves high- throughput imaging and patterned stimulation through parallel processing of multiple subfields, making it particularly valuable for large-scale experiments involving a variety of samples or conditions. The combination of the WBA with the tandem lens configuration offers flexibility in adjusting imaging parameters. By interchanging lenses, users can finetune magnification, resolution, field of view, and working distance to meet the specific requirements of their experiments.

[0058] FIGS. 2C and 2D illustrate example WBA designs that can be used with the STIMscope platform. A 2x2 design and 4x2 design are shown in FIG. 2C and FIG. 2D, respectively. These example WBA designs can be used to achieve simultaneous excitation and imaging of 4 and 20 wells, respectively. However, other arrangements can be used. FIGS. 2C and 2D illustrate the example WBAs, featuring their realization with fluorescence and highlighting how the STIMscope setup can achieve simultaneous imaging and stimulation across multiple light fields.

[0059] Referring again to FIG. 1, the STIMscope platform also provides structured or targeted illumination. The illumination optics in the disclosed setup may consist of several components, including light-emitting diodes [LEDs], excitation filters, collimating/condenser lenses, light homogenizers, total internal reflection prisms, and a digital micromirror device (DMD) or other spatial light modulator.

[0060] The modulation components can include an excitation module 130 aligned along the modulation axis 198 forward of an excitation lens 142. The excitation module 130 produces excitation light 188 that can be directed toward the sample and focused onto the sample plane 150 (via the intermediate image 128 and WBA 124). The excitation light 188 can be used to excite or modulate the sample being imaged. In this way, the described system provides simultaneous and synchronized excitation and imaging of the sample of interest. Such excitation refers to the modulation of the sample and may also include inhibition. Thus, the excitation may also be referred to as modulation, stimulation, or inhibition. Similarly, the excitation components and corresponding excitation light may be referred to as modulation, stimulation, or inhibition components and light, respectively.

[0061] In some implementations, one or more excitation filters 144 may be included along the modulation axis 198. For example an excitation filter 144 can be arranged between the excitation module 130 and excitation lens 142. The excitation filter 144 can be used to tailor the excitation light 188 based on a desired wavelength range. For example, the filter may be configured to allow light with a wavelength corresponding to a desired fluorophore through while blocking other wavelengths.

[0062] The excitation module 130 is responsible for projecting patterned illumination or structured excitation light onto the sample being imaged. The illumination pattern can be configured for the particular application, and the illumination can be targeted anywhere within the field ofview. The excitation module 130 may include a light or illumination source 132, such as LEDs. The illumination source 132 enables multi-color imaging and optogenetic stimulation. The excitation module 130 may also include various excitation filters, collimating/condenser lenses, light homogenizers, and total internal reflection prisms. The excitation module 330 features a modular design that allows for further customization. For example, the excitation module 130 can provide integration of a various optical components, such as SLMs 134, LEDs or other light source 132, light pipes, custom-designed optics (e.g., for telecentric illumination), total internal reflection (TIR) prisms, and other optical components. This flexibility ensures that the STIMscope can be adapted to a wide variety of experimental setups and specifications.

[0063] The excitation module 130 may also include a spatial light modulator (SLM) 134, such as a digital micromirror device (DMD). As a non-limiting example, a DLPTM4710 DMD may be used as part of a DLP4710EVM-LC development board, a Digital Light Processing (DLP) system that can be easily reconfigured to meet the specific needs of the application. The DLP4710EVM-LC supports multiple frequency modes, including a default 60 Hz mode, a high-speed 180 Hz mode, and a monochrome 1440 Hz mode with 24 patterns per frame. This flexibility allows the STIMscope to achieve high temporal resolution in imaging and stimulation, making it suitable for a wide range of applications.

[0064] In some implementations, the DMD’s area may be smaller than that of the intermediate image plane 128. Thus, the excitation can be magnified using the excitation lens 142 in conjunction with an objective lens to cover the entire intermediate image plane 128. As a non-limiting example, the full-size DLPTM4710 has an area of 10.4 mm x 5.8 mm. The excitation lens 142 may include a 20 mm lens used in conjunction with a 135 mm objective lens, resulting in a 6.75x magnification and coverage of an area measuring 70.2 mm x 39.15 mm. In this way, the system can provide magnification of the excitation light 188. The focal lengths ofthe objective and excitation lenses can be chosen to provide desired magnification, such as at least 5X, at least 6X, and so forth. The excitation light 188 that can be directed from the excitation module 130, backward along the modulation axis 198 to the dichroic mirror 114. The angled dichroic mirror 114 can redirect the excitation light 188 backward along the imaging axis 196 to the sample.

[0065] The use of paired imaging with structured light excitation can benefit from precise temporal synchronization and spatial calibration. The disclosed system includes a synchronized real-time control module, which ensures precise coordination among the software and hardware components. The system can also be used with a calibration method to precisely calibrate excitation and imaging in space.

[0066] Referring now to FIG. 3A, an example control module or control system is presented along with the optical components as described in FIG. 1. Such control module can be used to precisely synchronize and coordinate the software and hardware components of the system, providing synchronization of the excitation and emission measurement. Moreover, the control module can provide real-time control ofthe system. For example, a user may be able to adjust imaging and excitation parameters in real-time as images are being acquired.

[0067] The control module or control system integrates various hardware components, including a microcontroller unit (MCU) 352, a host machine 354 (e.g., PC or Nvidia Jetson), an excitation module 330 (e.g., DMD module), and an image sensor 312. The various connections and communication pathways between the system components are illustrated in FIG. 3A as a non-limiting example. However, it is contemplated that other configurations of control modules may be implemented to synchronize the excitation and emission provided by the STIMscope platform.

[0068] The MCU 352 can act as a central hub of the STIMscope’s control and synchronization module. It serves as the master controller, coordinating the timing and actions of the other components to ensure that imaging and stimulation are perfectly synchronized.

[0069] As one non-limiting example, the MCU 352 may be a 32 -bit ARM Cortex microcontroller. However, the system is flexibly configured to allow for the use of other MCUs that have a sufficient number of general-purpose input/output (GPIO) pins to be used. The MCU 352 may be configured with several communication interfaces. For example, the communication interfaces may include universal asynchronous receivertransmitter (UART), inter-integrated circuit (12 C), pulse width modulation (PWM) ports, external interrupt request (IRQ) lines, and so forth. In a non-limiting example configuration, the UART interface can be used to enable serial communication with the host machine (e.g., PC, Nvidia Jetson, etc.). Such connection may be made using USB connection or directly via GPIO pins if available.

[0070] The MCU 352 can be controlled using a custom-programmed firmware that is run on the MCU 352. Such control can be used to manage the GPIO pins and coordinate communication with the other components. In this way, the host machine 354 sends commands to the MCU 352. In some configurations, communication between the host machine 354 and MCU 352 can be facilitated using a Python-based or other interface, allowing for dynamic configuration and control of the system during operation.

[0071] The host machine 354 serves as an interface between a user and the hardware system components, executing commands and relaying data to and from the MCU 352. In this way, the control module can be used to control parameters of the excitation light, such as wavelength, frequency, pulse duration, intensity, illumination or excitation pattern, and start time. The control module can also be used to control parameters of the detection of the image sensor, including gain, exposure time, frame rate, etc. Such parameters may be defined by a user via a user interface of the host machine 354. In some example configurations, the host machine 354 can be a standard PC, Nvidia Jetson, and so forth.

[0072] A custom software (e.g., Python-based script) can be used to facilitate communication between the host machine 354 and the MCU 352 (e.g., via the UART interface). Such software can allow the user to send various commands to the MCU 352. For example, the host machine 354 can allow the user to configure the excitation module 330 and image sensor 312 (e.g., exposure time, frame rate, illumination source controls, gain, or other parameters). The host machine 354 also handles the transmission of raw video frames to the excitation module, which may be communicated via an HDMI connection, for example. This connection ensures that the projected patterns are precisely synchronized with the image acquisition process.

[0073] Communication between the MCU 352 and excitation module 330 can be facilitated using I2C commands sent from the MCU 352. For example, the excitation module 330 may include a SLM 334, such as a DMD, configured with a microcontroller and DLP chip that receive I2C commands from the MCU 352. The excitation module 330 may also include a light source 332, such as LEDs. The excitation module 330 can also include trigger outputs. For example, the excitation module 330 may feature two trigger outputs. One trigger can be used to indicate the start of each imaging frame while the other trigger can be used to indicate the start of each excitation pattern. In this way, the trigger signals can be used to synchronize the image sensor with the projection of modulation patterns, ensuring that data acquisition is precisely timed with stimulation.

[0074] In some configurations, the image sensor 312 can connect to the host machine 354 using an interface such as a mobile industry processor interface camera serial interface (MIPI-CSI) or a USB interface. For additional flexibility, the system can include a MIPI-CSI to USB bridge, which allows for the image sensor 312 to be easily connected to a wide range of host machines with USB ports. Synchronization between the image sensor 312 and the excitation module 330 can be achieved through the trigger outputs sent from the excitation module 330 to the MCU 352 and then relayed to the image sensor 312. This ensures that the timing of image acquisition aligns precisely with the projection of modulation patterns, enabling accurate and reliable data capture.

[0075] The synchronized real-time control module provides the system with the ability to perform complex imaging and stimulation tasks with high precision. By coordinating the actions of the MCU 352, host machine 354, excitation module 330, and image sensor 312, the control module ensures that all components work in unison, eliminating any potential delays or discrepancies that could compromise the quality of the data.

[0076] Another major advantage of the disclosed system is its ability to handle real-time adjustments during experiments. For example, if a change in the stimulation pattern, excitation parameters, or imaging parameters is required, the user can make these adjustments on the fly through the host machine's (e.g., Python-based) user interface. The MCU will then execute the necessary commands to reconfigure the system, all while maintaining synchronization. This level of control may be particularly important in life sciences and neuroscience research, where precise timing and coordination are critical to capturing accurate data. Whether studying fast neural dynamics, optogenetic stimulation, or other complex processes, the STIMscope platform provides the tools needed to achieve high-resolution, real-time imaging and stimulation.

[0077] Moreover, the system can be configured with a modular design that allows for easy customization and scalability, enabling researchers or other users to tailor the system for their specific needs. For example, the excitation module can be configured with a DMD or another desired spatial light modulator (SLM) and can integrate other optical components to modify the illumination pattern as desired. Similarly, several different types of image sensors can be used interchangeably to achieve a desired resolution, frame rate, sensitivity, and so forth. This flexibility extends to the MCU and host machine as well. Users can choose different microcontrollers or processing units based on the complexity of their experiments and the level of control desired. The user interface on the host machine is also designed to be highly customizable, allowing users to develop their own scripts and workflows to automate specific tasks or processes.

[0078] In some configurations, the STIMscope platform's control module can be integrated with a wide range of external hardware, such as motorized stages, temperature controllers, and environmental sensors. This integration can be facilitated through the MCU's GPIO pins and communication interfaces, which can be configured to interact with various external devices. For example, motorized stages (e.g., 120) can be used to precisely position well plates during imaging, while temperature controllers can maintain optimal conditions for live cell experiments. The real-time control module ensures that these external devices are synchronized with the imaging and stimulation processes, providing a comprehensive and coordinated experimental setup. This capability further enhances the versatility of the STIMscope platform, allowing researchers to conduct more complex and multifaceted experiments without the need for additional control systems.

[0079] FIG. 3B provides another example arrangement of a control system configured to control the microscope system. The control system includes a host machine 360 (e.g., NVIDIA Jetson) that communicates with a graphical user interface 362 and external devices 364, such as a stage. The host machine 360 controls an SLM driver 366, which can be electrically coupled to and control an SLM 372. The SLM 372 can modulate the excitation light to produce a desired light pattern. The host machine 360 can also control a light source driver 368 (e.g., dual LED driver), which is electrically coupled to the light source 370 (e.g., LEDs) to drive the light source 370. The host machine 360 can also communicate back and forth with an image sensor 374 (e.g., CMOS sensor).

[0080] Referring now to FIG. 4, a process 400 for modulating and monitoring a sample is presented. The process 400 can be used to simultaneously modulate and detect light from multiple wells of a multi-well plate. Such process can be achieved using a microscopy system as previously described (e.g., in FIG. 1, FIG. 3A, or FIG. 3B). A sample is positioned in a sample plane 150, as in block 402. For example, the sample may include live cells positioned within a well plate, such as a 96-well plate. Modulation, stimulation, or excitation light is directed toward the sample, as in block 404. For example, modulation light can be produced by the excitation module 130 using LEDs or another light source 132. The modulation light may have a prescribed spatial pattern achieved by a DMD or another spatial light modulator. Thus, the modulation light may be referred to as structured modulation light or structured excitation light. The modulation light may be directed through filters (e.g., 144) and lenses (e.g., 142) backward along a first axis, such as the modulation axis 198. The excitation light can be redirected by an angled dichroic mirror (e.g., 114) backward along the second axis, referred to as the imaging axis 196, and through the well-block array 124. In this way, the optical components of the system (e.g., 144, 142, 114, 104, 126, 124) can function together to direct the excitation light toward the sample, as in block 404, and to focus the light at a sample plane 150, as in block 406. The excitation light 188 modulates the sample such that the sample produces emission light 190 forward along the imaging axis 196 and focused onto an intermediate image plane 128, as in block 408. From the intermediate image plane 128, the objective lens 104 can direct the emission light 190 of the intermediate image through the infinity corrected zone 110. The emission light 190 can be allowed to pass through the dichroic mirror 114 to the imaging lens 102. The emission light 190 may also be directed through an emission filter 116 to collect the desired light (e.g., a specified wavelength range). In some implementations, the emission filter 116 can block remaining traces of excitation light 188 or other unwanted light (e.g., from the environment) that may be reflected along the imaging axis 196 so thatthe excitation light 188 does notcorruptthe measured signal. The imaging lens 102 can focus the light onto a focal plane 152 located at the image sensor 112, as in block 410. Thus, the desired emission light 190 can be measured or detected by the image sensor 112, as in block 412.

[0081] Process 400 provides an example process for modulating and monitoring a sample, such as live cells. In general, the STIMscope provides closed-loop control of a manipulation and observation feedback loop, as illustrated in FIG. 5. For example, the STIMscope allows for precise stimulation of a specific cluster of neurons or other cell types at the level of individual cells while also allowing for observations of the circuit's response. The experimental parameters can also be adjusted in real-time based on the images acquired.

[0082] FIGS. 6A-6D show example images measured by an example implementation of the STIMscope system. FIG. 6A shows an image of a resolution target slide measured by simultaneously imaging a 2x2 well-block array. FIG. 6B shows an example image acquired while projecting a custom illumination pattern, configured as "UCLA”, onto a single well. FIG. 6C shows an example image of a brain slice sample in a single well. The image of FIG. 6C is enlarged in FIG. 6D to illustrate the single-cell resolution capability of the STIMscope.

[0083] Process 400 can be used with the STIMscope platform for many different applications. For example, the STIMscope can be used for many different experimental or clinical applications in the fields of life sciences and neuroscience, such as high- throughput phenotyping of biological processes, genetic screening, drug development, cell-specific life-cell imaging, neuromodulation, and so forth.

[0084] One non-limiting example use of the describe system lies in the investigation of neurodevelopmental and neuropsychiatric disorders (NPDs). Recent advances in genomics have revealed genetic risk factors associated with NPDs. Leveraging the progress in induced pluripotent stem cell (iPSC) technology, neuroscientists can now cultivate substantial samples of human iPSC-derived neurons within multi-well plates. The STIMscope facilitates large-scale phenotyping of neural cultures from NPD patients. Furthermore, its capabilities empower neuroscientists to explore novel avenues, such as complex light pattern illumination, targeted illumination and closed loop modulation, enabling users to tackle some of the most intricate questions in NPD neuroscience, including inquiries into long-range network connectivity and the effects of genetic modifications on neural activity.

[0085] As another example, an exciting and motivating application for the STIMscope resides in the emerging field of genetic voltage indicators (GEVIs). Unlike calcium signaling, which serves as a proxy for neural activity, GEVIs directly report membrane potential. The STIMscope' s adaptable illumination and imaging modules can be readily customized to accommodate components tailored for large field-of-view voltage imaging. This capability enables high-speed, long-term imaging of live cells using GEVIs, thereby unveiling rapidly evolving neurological and biological processes.

[0086] In the era of precision medicine, there is a growing interest in personalized treatments for neurological disorders. The STIMscope's high-throughput imaging and stimulation capabilities can be harnessed for profiling individual patients' neural circuitry, paving the way for customized interventions and therapies. This becomes especially relevant in light of the significant interest from pharmaceutical companies in discovering novel therapies for neurological conditions, such as Alzheimer's disease, Parkinson's disease, and epilepsy. High-resolution imaging of neuronal networks can aid in screening potential drug candidates and comprehending their impact on neuronal function. This expedites drug development processes and augments the likelihood of identifying effective treatments.

[0087] Additionally, the STIMscope can be used for a range of other applications, including continuous monitoring of self-assembled nanoparticles for drug delivery, large-scale surveillance of two-dimensional material growth, extensive-scale drug encapsulation (e.g., nanogel loading), and monitoring chemical reactions. These diverse applications underscore the versatility and broad impact of the STIMscope platform.

[0088] In general, the STIMscope system may have a mismatch between the excitation module and image sensor that can be caused by magnification, aberrations of the optical system, rotation or shifting of the image sensor, and so forth. However, such mismatch can be corrected using a spatial calibration. Referring now to FIG. 7, a process 700 for spatially calibrating an excitation module (e.g., 130 or 330) with an image sensor (e.g., 112 or 312) is presented. Calibrating the excitation light source with the image sensor can ensure that experiments performed with the system (e.g., as described in FIG. 1, FIG. 3A, or FIG. 3B) accurately and reliably characterize light emission signal produced by a sample modulated by a modulation light source. For example, a user may want to excite a specific single cell when performing optogenetic experiments. Process 700 can be used to determine what pixels of the image sensor correspond to the cell of interest, providing a pixel-by-pixel mapping between the structured light and the sensor. Process 700 includes projecting a known light pattern, as shown in block 702. The known light pattern may be referred to as a calibration pattern, and the corresponding projected light may be referred to as calibration excitation light or calibration modulation light. For example, the known light pattern can be defined by a user using the host machine 354, which communicated with the MCU 352 to control the excitation module 330 to produce the known light pattern. This excitation or modulation light pattern can be directed to a sample, as previously described. The sample, which may be referred to as a calibration sample, maybe chosen based on desired properties. For example, during calibration (e.g., process 700), the user may choose a sample with known emission properties associated with the parameters of the excitation light. As a non-limiting example, the sample may include fluorescent microscope slides or resolution test targets.

[0089] The modulation light causes the sample to produce an emission signal or emission light that is measured as an emission pattern, as in block 704. In the context of calibration (e.g., process 700), the light emitted by the sample may be referred to as calibration emission light. This emission pattern or calibration emission light can be measured using the imaging sensor 312.

[0090] After the emitted light is measured, a spatial registration can be performed in block 706. This spatial registration can produce a spatial mapping between the known projected light with the measured emitted light, accounting for any spatial aberrations of the system between the excitation source and the image sensor. The registration may be a 2D or 3D rigid registration, including translation, rotation, and scaling. The registration may also be a non-rigid registration, including warping. Several methods for image registration may be used as known in the field of computer vision. The registration may be fully automated or may incorporate input from a user.

[0091] Process 700 can be repeated as needed to recalibrate the system. For example, when various optical components are swapped out or the system is moved or otherwise adjusted, the system can be recalibrated calibration prior to experimental use. [0092] Referring now to FIG. 8, an example of a system 800 is shown, which may be used in accordance with some aspects of the systems and methods described in the present disclosure. As shown in FIG. 8, a computing device 850 can receive one or more types of data (e.g., signal evolution data, k-space data, receiver coil sensitivity data) from data source 802. In some configurations, computing device 850 can execute at least a portion of a microscopy system 804 to produce images or other data based on measured emission light or to spatially or temporally calibrate the system. In some configurations, the microscopy system 804 can implement an automated pipeline to provide fourdimensional modulation images or measurements.

[0093] Additionally or alternatively, in some configurations, the computing device 850 can communicate information about data received from the data source 802 to a server 852 over a communication network 854, which can execute at least a portion of the microscopy system 804. In such configurations, the server 852 can return information to the computing device 850 (and/or any other suitable computing device) indicative of an output of the microscopy system 804.

[0094] In some configurations, computing device 850 and/or server 852 can be any suitable computing device or combination of devices, such as a desktop computer, a laptop computer, a smartphone, a tablet computer, a wearable computer, a server computer, a virtual machine being executed by a physical computing device, and so on. The computing device 850 and/or server 852 can also reconstruct or process images from the data.

[0095] In some configurations, data source 802 can be any suitable source of data (e.g., measurement data, images produced from measurement data, processed image data), such as a STIMscope system, another computing device (e.g., a server storing measurement data, images produced from measurement data, processed image data, excitation pattern in time and space), and so on. In some configurations, data source 802 can be local to computing device 850. For example, data source 802 can be incorporated with computing device 850 (e.g., computing device 850 can be configured as part of a device for measuring, recording, estimating, acquiring, or otherwise collecting or storing data). As another example, data source 802 can be connected to computing device 850 by a cable, a direct wireless link, and so on. Additionally or alternatively, in some configurations, data source 802 can be located locally and/or remotely from computing device 850, and can communicate data to computing device 850 (and/or server 852) via a communication network (e.g., communication network 854).

[0096] In some configurations, communication network 854 can be any suitable communication network or combination of communication networks. For example, communication network 854 can include a Wi-Fi network (which can include one or more wireless routers, one or more switches, etc. , a peer-to-peer network (e.g., a Bluetooth network), a cellular network (e.g., a 3G network, a 4G network, etc., complying with any suitable standard, such as CDMA, GSM, LTE, LTE Advanced, WiMAX, etc.), other types of wireless network, a wired network, and so on. In some configurations, communication network 854 can be a local area network, a wide area network, a public network (e.g., the Internet), a private or semi-private network (e.g., a corporate or university intranet), any other suitable type of network, or any suitable combination of networks. Communications links shown in FIG. 8 can each be any suitable communications link or combination of communications links, such as wired links, fiber optic links, Wi-Fi links, Bluetooth links, cellular links, and so on.

[0097] Referring now to FIG. 9, an example of hardware 900 that can be used to implement data source 802, computing device 850, and server 852 in accordance with some configurations of the systems and methods described in the present disclosure is shown.

[0098] As shown in FIG. 9, in some configurations, computing device 850 can include a processor 902, a display 904, one or more inputs 906, one or more communication systems 908, and/or memory 910. In some configurations, processor 902 can be any suitable hardware processor or combination of processors, such as a central processing unit ("CPU”), a graphics processing unit ("GPU”), and so on. In some configurations, display 904 can include any suitable display devices, such as a liquid crystal display ("LCD”) screen, a light-emitting diode ("LED”) display, an organic LED ("OLED") display, an electrophoretic display (e.g., an "e-ink” display), a computer monitor, a touchscreen, a television, and so on. In some configurations, inputs 906 can include any suitable input devices and/or sensors that can be used to receive user input, such as a keyboard, a mouse, a touchscreen, a microphone, and so on.

[0099] In some configurations, communications systems 908 can include any suitable hardware, firmware, and/or software for communicating information over communication network 854 and/or any other suitable communication networks. For example, communications systems 908 can include one or more transceivers, one or more communication chips and/or chip sets, and so on. In a more particular example, communications systems 908 can include hardware, firmware, and/or software that can be used to establish a Wi-Fi connection, a Bluetooth connection, a cellular connection, an Ethernet connection, and so on.

[00100] In some configurations, memory 910 can include any suitable storage device or devices that can be used to store instructions, values, data, or the like, that can be used, for example, by processor 902 to present content using display 904, to communicate with server 852 via communications system(s) 908, and so on. Memory 910 can include any suitable volatile memory, non-volatile memory, storage, or any suitable combination thereof. For example, memory 910 can include random-access memory ("RAM”), read-only memory ("ROM”), electrically programmable ROM ("EPROM"), electrically erasable ROM ("EEPROM”), other forms of volatile memory, other forms of non-volatile memory, one or more forms of semi-volatile memory, one or more flash drives, one or more hard disks, one or more solid state drives, one or more optical drives, and so on. In some configurations, memoiy 910 can have encoded thereon, or otherwise stored therein, a computer program for controlling operation of computing device 850. In such configurations, processor 902 can execute at least a portion of the computer program to present content (e.g., images, user interfaces, graphics, tables), receive content from server 852, transmit information to server 852, and so on. For example, the processor 902 and the memory 910 can be configured to perform the methods described herein.

[00101] In some configurations, server 852 can include a processor 912, a display 914, one or more inputs 916, one or more communications systems 918, and/or memory 920. In some configurations, processor 912 can be any suitable hardware processor or combination of processors, such as a CPU, a GPU, and so on. In some configurations, display 914 can include any suitable display devices, such as an LCD screen, LED display, OLED display, electrophoretic display, a computer monitor, a touchscreen, a television, and so on. In some configurations, inputs 916 can include any suitable input devices and/or sensors that can be used to receive user input, such as a keyboard, a mouse, a touchscreen, a microphone, and so on.

[00102] In some configurations, communications systems 918 can include any suitable hardware, firmware, and/or software for communicating information over communication network 854 and/or any other suitable communication networks. For example, communications systems 918 can include one or more transceivers, one or more communication chips and/or chip sets, and so on. In a more particular example, communications systems 918 can include hardware, firmware, and/or software that can be used to establish a Wi-Fi connection, a Bluetooth connection, a cellular connection, an Ethernet connection, and so on.

[00103] In some configurations, memory 920 can include any suitable storage device or devices that can be used to store instructions, values, data, or the like, that can be used, for example, by processor 912 to present content using display 914, to communicate with one or more computing devices 850, and so on. Memory 920 can include any suitable volatile memory, non-volatile memory, storage, or any suitable combination thereof. For example, memory 920 can include RAM, ROM, EPROM, EEPROM, other types of volatile memory, other types of non-volatile memory, one or more types of semi-volatile memory, one or more flash drives, one or more hard disks, one or more solid state drives, one or more optical drives, and so on. In some configurations, memory 920 can have encoded thereon a server program for controlling operation of server 852. In such configurations, processor 912 can execute at least a portion of the server program to transmit information and/or content (e.g., data, images, a user interface) to one or more computing devices 850, receive information and/or content from one or more computing devices 850, receive instructions from one or more devices (e.g., a personal computer, a laptop computer, a tablet computer, a smartphone), and so on.

[00104] In some configurations, the server 852 is configured to perform the methods described in the present disclosure. For example, the processor 912 and memory 920 can be configured to perform the methods described herein.

[00105] In some configurations, data source 802 can include a processor 922, one or more data acquisition systems 924, one or more communications systems 926, and/or memory 928. In some configurations, processor 922 can be any suitable hardware processor or combination of processors, such as a CPU, a GPU, and so on. In some configurations, the one or more data acquisition systems 924 are generally configured to acquire data, images, or both, and can include a microscopy system. Additionally or alternatively, in some configurations, the one or more data acquisition systems 924 can include any suitable hardware, firmware, and/or software for coupling to and/or controlling operations of a microscopy system. In some configurations, one or more portions of the data acquisition system(s) 924 can be removable and/or replaceable.

[00106] Note that, although not shown, data source 802 can include any suitable inputs and/or outputs. For example, data source 802 can include input devices and/or sensors that can be used to receive user input, such as a keyboard, a mouse, a touchscreen, a microphone, a trackpad, a trackball, and so on. As another example, data source 802 can include any suitable display devices, such as an LCD screen, an LED display, an OLED display, an electrophoretic display, a computer monitor, a touchscreen, a television, etc., one or more speakers, and so on.

[00107] In some configurations, communications systems 926 can include any suitable hardware, firmware, and/or software for communicating information to computing device 850 (and, in some configurations, over communication network 854 and/or any other suitable communication networks). For example, communications systems 926 can include one or more transceivers, one or more communication chips and/or chip sets, and so on. In a more particular example, communications systems 926 can include hardware, firmware, and/or software that can be used to establish a wired connection using any suitable port and/or communication standard (e.g., VGA, DVI video, USB, RS-232, etc.), Wi-Fi connection, a Bluetooth connection, a cellular connection, an Ethernet connection, and so on.

[00108] In some configurations, memory 928 can include any suitable storage device or devices that can be used to store instructions, values, data, or the like, that can be used, for example, by processor 922 to control the one or more data acquisition systems 924, and/or receive data from the one or more data acquisition systems 924; to generate images from data; present content (e.g., data, images, a user interface) using a display; communicate with one or more computing devices 850; and so on. Memory 928 can include any suitable volatile memory, non-volatile memory, storage, or any suitable combination thereof. For example, memory 928 can include RAM, ROM, EPROM, EEPROM, other types of volatile memory, other types of non-volatile memory, one or more types of semi-volatile memory, one or more flash drives, one or more hard disks, one or more solid state drives, one or more optical drives, and so on. In some configurations, memory 928 can have encoded thereon, or otherwise stored therein, a program for controlling operation of medical image data source 802. In such configurations, processor 922 can execute at least a portion of the program to generate images, transmit information and/or content (e.g., data, images, a user interface) to one or more computing devices 850, receive information and/or content from one or more computing devices 850, receive instructions from one or more devices (e.g., a personal computer, a laptop computer, a tablet computer, a smartphone, etc.), and so on.

[00109] In some configurations, any suitable computer-readable media can be used for storing instructions for performing the functions and/or processes described herein. For example, in some configurations, computer-readable media can be transitory or non- transitory. For example, non-transitory computer-readable media can include media such as magnetic media (e.g., hard disks, floppy disks), optical media (e.g., compact discs, digital video discs, Blu-ray discs), semiconductor media (e.g., RAM, flash memory, EPROM, EEPROM), any suitable media that is not fleeting or devoid of any semblance of permanence during transmission, and/or any suitable tangible media. As another example, transitory computer-readable media can include signals on networks, in wires, conductors, optical fibers, circuits, or any suitable media that is fleeting and devoid of any semblance of permanence during transmission, and/or any suitable intangible media.

[00110] As used herein in the context of computer implementation, unless otherwise specified or limited, the terms "component," "system," "module," "controller," "framework," and the like are intended to encompass part or all of computer-related systems that include hardware, software, a combination of hardware and software, or software in execution. For example, a component may be, but is not limited to being, a processor device, a process being executed (or executable) by a processor device, an object, an executable, a thread of execution, a computer program, or a computer. By way of illustration, both an application running on a computer and the computer can be a component. One or more components (or system, module, and so on) may reside within a process or thread of execution, may be localized on one computer, may be distributed between two or more computers or other processor devices, or may be included within another component (or system, module, and so on).

[00111] In some implementations, devices or systems disclosed herein can be utilized or installed using methods embodying aspects of the disclosure. Correspondingly, description herein of particular features, capabilities, or intended purposes of a device or system is generally intended to inherently include disclosure of a method of using such features for the intended purposes, a method of implementing such capabilities, and a method of installing disclosed (or otherwise known) components to support these purposes or capabilities. Similarly, unless otherwise indicated or limited, discussion herein of any method of manufacturing or using a particular device or system, including installing the device or system, is intended to inherently include disclosure, as embodiments of the disclosure, of the utilized features and implemented capabilities of such device or system.

[00112] As used herein, the phrase "at least one of A, B, and C" means at least one of A, at least one of B, and/or at least one of C, or any one of A, B, or C or combination of A, B, or C. A, B, and C are elements of a list, and A, B, and C may be anything contained in the Specification.

[00113] The present disclosure has described one or more preferred embodiments, and it should be appreciated that many equivalents, alternatives, variations, and modifications, aside from those expressly stated, are possible and within the scope of the invention.

Claims

1. A microscopy system configured to modulate and detect light from a sample, the system comprising: an excitation module configured to direct structured excitation light along a first axis towards an excitation lens, the excitation lens magnifying the excitation light and directing the excitation light along the first axis; a dichroic mirror arranged along the first axis and configured to reflect excitation light within a first range of wavelengths and transmit emission light within a second range of wavelengths, wherein the dichroic mirror is angled with respect to the first axis to reflect the excitation light along a second axis; an objective lens arranged to receive excitation light reflected along the second axis and form an intermediate image plane; an imaging lens positioned along the second axis to receive the emission light from the intermediate image plane, through the objective lens and transmitted through the dichroic mirror, the imaging lens forming a focal plane along the second axis; a well-block array positioned between the intermediate image plane and the sample to increase a field of view of the sample, wherein the well-block array comprises a plurality of elements and each element comprises an achromatic objective lens and a tube lens; and an imaging sensor configured to detect and digitize the emission light focused by the imaging lens at the focal plane.

2. The microscopy system of claim 1, wherein the excitation module comprises a light source and a spatial light modulator configured to produce the structured excitation light.

3. The microscopy system of claim 2, wherein the light source comprises one or more light-emitting diodes.

4. The microscopy system of claim 2, wherein the spatial light modulator is a digital micromirror device.

5. The microscopy system of claim 1, further comprising a multi-well plate containing the sample and wherein the system is configured to simultaneously modulate and detect light from a plurality of wells of the multi-well plate.

6. The microscopy system of claim 5, wherein the multi-well plate comprises at least 96 wells.

7. The microscopy system of claim 5, wherein the system is configured to simultaneously modulate and detect light from at least 20 wells of the multi-well plate.

8. The microscopy system of claim 2, wherein the excitation module further comprises at least one of excitation filter, collimating lens, condenser lens, light homogenizer, or total internal reflection prism.

9. The microscopy system of claim 1, wherein the excitation module is configured to provide multi-color imaging and optogenetic stimulation of the sample.

10. The microscopy system of claim 1, wherein the system provides single-cell resolution of the sample.

11. The microscopy system of claim 1, further comprising an emission filter positioned between the imaging lens and the imaging sensor.

12. The microscopy system of claim 1, wherein the imaging sensor is a complementary metal-oxide semiconductor.

13. The microscopy system of claim 1, wherein the well-block array is configured to provide at least 4X magnification.

14. The microscopy system of claim 1, wherein the excitation lens has a focal length of 20 mm, the objective lens has a focal length of 135 mm, and the excitation lens and objective lens provide a 6x magnification of the excitation light.

15. The microscopy system of claim 1, wherein the imaging lens has a focal length of 15 mm, the objective lens has a focal length of 135 mm, and the imaging lens and objective lens provide at least a 9x demagnification of the emission light.

16. The microscopy system of claim 1, further comprising a control system configured to synchronize the excitation module and the imaging sensor.

17. The microscopy system of claim 1, further comprising a control system configured to control parameters of at least one of the excitation light or the imaging sensor, the parameters including at least one of a wavelength, a frequency, a pulse duration, an intensity, an illumination pattern, a start time, a gain, an exposure time, or a frame rate.

18. The microscopy system of claim 17, wherein the control system is electrically coupled to a user interface, the excitation module, the image sensor, and a stage that receives the sample.

19. A method for modulating and monitoring a sample, the method comprising the steps of: directing modulation light from a light source along a first axis, the first axis comprising an excitation lens that magnifies the modulation light and a dichroic mirror angled with respect to the first axis and configured to reflect the modulation light along a second axis toward an objective lens; using the objective lens and a well-block array comprising a plurality of achromatic lenses, focusing the modulation light onto a first plurality of wells containing a sample such that the modulation light modulates the sample, producing emission light directed back along the second axis towards the dichroic mirror; using the well-block array, forming an intermediate image of the emission light from the first plurality of wells; using the objective lens, directing the intermediate image of the emission light back along the second axis towards the dichroic mirror, wherein the dichroic mirror is configured to transmit the emission light; focusing the emission light at a focal plane using an imaging lens positioned along the second axis; and detecting the emission light using an image sensor positioned at the focal plane.

20. The method of claim 19, the steps further comprising calibrating the light source and image sensor in space.

21. The method of claim 20, wherein calibrating the light source and image sensor in space comprises the steps of: using a control system to define a known pattern of modulation light; directing the known pattern of modulation light from the light source along the first axis; focusing the known pattern of modulation light onto the plurality of wells containing a calibration sample such that the modulation light modulates the calibration sample, producing calibration emission light; using the well-block array and the imaging lens, focusing the calibration emission light at the focal plane; detecting the calibration emission light using the image sensor; and using a control system to map the known pattern of modulation light from the light source to the calibration emission light detected using the image sensor.

22. The method of claim 20, wherein the sample comprises live cells.

23. The method of claim 20, wherein the sample is configured within a multi-well plate; modulation light is configured to simultaneously modulate the sample within the first plurality of wells of the multi-well plate; and the image sensor is configured to simultaneously detect emission light from the first plurality of wells of the multi-well plate.

24. The method of claim 23, further comprising adjusting a position of the sample to simultaneously modulate the sample within a second plurality of wells of the multi-well plate and simultaneously detect emission light from the second plurality of wells of the multi-well plate.

25. The method of claim 20, further comprising adjusting parameters of the modulation light based on the emission light detected by the image sensor, the parameters including at least one of a wavelength, a frequency, a pulse duration, an illumination pattern, an intensity, a gain, exposure time, or a framerate; and repeating the steps using the adjusted parameters.

26. A spatiotemporal illumination microscope system, comprising: a multi-well plate configured to receive live cells; a digital micromirror device [DMD] configured to deliver targeted illumination to selectively illuminate a plurality of wells of the multi-well plate; a tandem-lens system comprising an objective lens and an imaging lens positioned facing each other to capture fluorescent light emitted from the live cells in the multi-well plate; an image sensor configured to receive fluorescent light captured by the tandemlens system and generate imaging data; and a controller configured to use the imaging data to control closed-loop modulation of the live cells in the plurality of wells.

27. The system of claim 26, wherein the controller is further configured to synchronize the DMD and the image sensor.

28. The system of claim 26, wherein the controller is further configured to spatially calibrate the DMD and the image sensor.

29. The system of claim 26, further comprising a well-block array configured to produce a field of view in the plurality of wells, the well-block array comprising a plurality of elements, each including at least one achromatic objective lens.

30. The system of claim 29, wherein each element further includes at least one tube lens.

31. The system of claim 29, wherein the well-block array is configured to produce an intermediate image plane having a magnification with respect to the field of view in the multi-well plate.