Microscopy with spatial imaging and beam homogenizer

Abstract

The present invention provides a microscope for imaging samples in wells of a multi-well plate. The microscope of the present disclosure includes a beam homogenizer system that shapes a beam from a light source into a shape specific to the bottom of the well of the multi-well plate. In particular, the microscope of the present disclosure can illuminate the well for imaging by passing light through a prism that is below the sample. The light enters the prism from the side and is refracted into the well at a steep angle such that the light only illuminates about the bottom 10 microns of the well. The beam homogenizer shapes the light from the light source so that instead of hitting the prism as a spot with an irregular shape, the light enters the prism in a roughly rectangular pattern with a uniform level of refractive power across the pattern.

Description

[Technical Field]

[0001] This disclosure relates to a microscope. [Background technology]

[0002] Numerous diseases involve electrically active cells such as nerve cells and cardiomyocytes. Consequently, there is a significant motivation to study the properties and interactions of these cells.

[0003] Fluorescence microscopy, a technique in which a phosphor is bound to a sample to detect phenomena such as cell surface binding, neurotransmitter release, or specific DNA sequences, is often used to study cell behavior. However, phosphors and other compounds in the surrounding culture medium, and even in the optical components of the microscope, can exhibit autofluorescence and overwhelm the fluorescence from the sample. Attempts have been made to reduce background fluorescence by using total internal reflection fluorescence (TIRF) microscopy. TIRF microscopes illuminate only thin areas of the sample so that phosphors in the surrounding culture medium do not receive the excitation energy required for fluorescence. However, existing TIRF microscopes require a prism to be pressed against the sample in a configuration that severely limits the conditions acceptable for the sample. For example, a TIRF prism obstructs any culture medium that might be required for living cells and prevents any physical access to the sample. Therefore, fluorescence microscopy has not proven satisfactory for studying the fine details of viable electroactive cells. Other light microscopy techniques have similarly failed to produce limited results. Therefore, there is a need in this field for improved techniques for the optical measurement of cell activity. [Overview of the project] [Means for solving the problem]

[0004] The present invention provides a microscope for imaging samples in the wells of a multiwell plate. The microscope of this disclosure includes a beam homogenizer system that shapes a beam from a light source into a shape with excellent uniformity to the bottom of the wells of the multiwell plate. In particular, the microscope of this disclosure illuminates the wells for imaging by passing light through a prism beneath the sample. The light enters the prism from the side and is refracted into the well at an angle such that the light illuminates only about 10 microns of the bottom of the well. The beam homogenizer shapes the light from the light source so that, instead of impacting the prism as a spot with an irregular shape, the light enters the prism in a substantially rectangular pattern with a homogeneous refractive power level across the pattern. Thus, cells at the bottom of the well are uniformly illuminated with good refractive power for imaging. The microscope may also include an adjustable optical system that allows the homogenizer component to be positioned and the aperture selected so that the homogenized illumination beam can be matched to a particular well of a multiwell plate or a particular imaging lens and its associated field of view. This allows different types of multi-well plates to be loaded onto the microscope for imaging. Each well can contain a viable sample culture medium with multiple living cells in each well to be imaged by the microscope. The cells can contain fluorescent reporter proteins that emit light in response to cellular electrical activity. A beam homogenizer ensures that the entire sample in each well receives strong and uniform illumination while avoiding off-target light that causes autofluorescence. Using a beam homogenizer, the microscope can successfully image living cells in multiple wells of a multi-well plate and record a video of electrical activity useful, for example, to show action potentials propagating within living nerve cells.

[0005] For example, the microscope may operate with two pre-selected image tube lenses and three types of multi-well plates, such as 96-, 384-, or 1,536-well plates. The beam homogenizer may use a pair of microlens arrays that can be repositioned to one of six pairs of pre-set stops, and the optical system may include a screen or block with six predefined apertures. For each of the six possible combinations of image tube lenses and well plates, appropriate homogenizer microlens array spacing and apertures may be set so that the homogenized illumination beam enters the side of the prism as a homogenized rectangular illumination that is refracted to about 10 microns at the bottom of the well, traversing the relevant field of view. Excessive autofluorescence is avoided because only the relevant portion of the sample (e.g., cells growing on the bottom of the well) is illuminated. The light is restricted to the lower portion of the well by directing the beam into the prism at an angle that promotes nearly total internal reflection (TIR) ​​within the glass bottom of the well. Light does not undergo TIR (Transient Infrared), but instead passes through only the bottom 10 microns or so of the well. The microscope therefore maximizes image clarity and signal-to-noise ratio.

[0006] Using approximate TIR illumination restricts the light to the bottom 10 microns of the sample in a multiwell plate. When the sample contains cells growing on the bottom surface of the plate, the light will illuminate those cells but not the rest of the well plate. When the cells contain fluorescent labels such as electroactive optical reporters, the light is adjusted to the excitation wavelength of the fluorescent reporter. Therefore, approximate TIR imaging is very useful for exciting fluorescent reporters in cells growing at the bottom of the wells in a multiwell plate. The optical system of the microscope can illuminate wells simultaneously using multiple excitation wavelengths.

[0007] In addition, the microscope of the present invention can apply spatially patterned illumination to a sample. The microscope can irradiate light onto a well using a pattern such that cells in the well are illuminated, and only those cells are illuminated. Spatial patterning can illuminate one or more specific cells. Using a digital optical processor or digital micromirror device (DMD), the light can be irradiated using an arbitrarily complex pattern such that one or more living cells can be illuminated without any illumination light irradiating into the portion of the well not occupied by one or more cells. The microscope can also initially image the well, and an associated computer system can be used to identify one or more cells in the image and create a “spatial mask” or digital file that identifies, pixel by pixel, the locations in the image that are occupied and not occupied by one or more cells. The computer system can use the spatial mask to activate the corresponding reflective pixels on the DMD. The microscope irradiates light onto the DMD, which is reflected onto the sample using a pattern that matches the locations of one or more cells growing at the bottom of the well. The mask creation step can be performed by fluorescently labeling the cells of interest, using human user selection, or by implementing an object discovery algorithm in the image. Alternatively, the microscope can iterate across each of multiple wells and create a spatial mask for each. The microscope can then operate an xy translation stage to position each of those wells across the imaging optics system, and for each well, the corresponding spatial mask can be used to spatially pattern the illumination onto one or more cells of interest growing at the bottom of that well.

[0008] Spatially patterned illumination is particularly useful when cells are electroactive cells, such as nerve cells, that express electroactive optical actuators. For example, certain channelrhodopsins can be used as photo-aperture ion channels, which then function as electroactive optical actuators. When these proteins are expressed by cells and then illuminated with light of a certain wavelength, the light causes these proteins to pump ions across the cell membrane, resulting in membrane polarization and electroactivity, such as the transmission of action potentials along the cell's length. When multiple cells in a sample express optical actuators, spatially patterned illumination is useful for inducing electroactivity (such as action potentials) only in selected cells among them. Microscopy is very suitable for optogenetic analysis of electroactive cells in multi-well plates because it can image each well in a plate, create a spatial mask for selected cells in each well, and spatially pattern the illumination onto each well sequentially while imaging those wells using a translation stage and DMD.

[0009] Spatially patterned illumination may be provided at wavelengths specific to optogenetic actuators such as channelrhodopsin, which functions as a photo-aperture ion channel. The microscope may further include a prism and dichroic assembly that receives multiplexed light of different wavelengths, reflecting another wavelength from the DMD while refracting at least one wavelength into the well at approximately a TIR angle, thereby simultaneously providing spatially patterned illumination on the sample. The approximately TIR illumination may be at the excitation wavelength of an intracellular fluorescent reporter in the sample, such as a membrane electroactive optogenetic reporter. Also, using the optical system described herein, the microscope can also simultaneously illuminate the sample with approximately TIR accompanied by light of a second wavelength, such as the excitation wavelength of a second optogenetic protein, such as an ion concentration fluorescent reporter. In addition, the microscope may include an imaging system, such as an imaging lens, optional filters, and an image sensor, which can record light at the emission wavelength of the optogenetic protein.

[0010] Therefore, the microscope can simultaneously handle at least five different wavelengths of light (activation light for optogenetic actuators, excitation light for at least two fluorescence reporters, and emission light for both reporters) through a single prism assembly. The microscope records a video of the electroactivity of cells in the wells of a plate and performs imaging assays sequentially for each of the multiple wells of the plate using a translation stage to displace the plate in the x and y directions across the stage. Using a beam homogenizer, the sample is fully illuminated across the applicable field of view with a light beam formed on the wells of the plate currently being used on the stage, with excellent illumination uniformity, which is crucial for capturing optogenetic reporter activity with a good signal-to-noise ratio. Thus, the microscope provides the ability to perform assays to record a video of neuronal activity from multiple wells of a multi-well plate.

[0011] In one aspect, the present invention provides a microscope comprising a stage configured to hold a multiwell plate, a light source mounted within the microscope for emitting a beam of light, and an optical system for directing the beam from below toward the stage, the optical system comprising a homogenizer for spatially homogenizing the beam. The stage may be an electrically operated xy translation stage. The microscope may include a control system comprising memory connected to a processor operable to move the translation stage and position the individual wells of the multiwell plate within the beam path. The optical system may include a prism beneath the stage, thereby causing the beam to be incident on the side of the prism and pass through the wells of the plate at approximately a TIR angle.

[0012] In some embodiments, when the wells of a plate containing an aqueous sample are positioned above the prism, the prism directs the beam into the sample at an angle that avoids all internal reflections within the bottom of the plate. When the wells of a plate containing an aqueous sample are positioned above the prism, the prism may direct the beam into the aqueous sample at an angle of refraction that restricts the light to approximately a few microns at the bottom of the well in order to reduce background autofluorescence and increase excitation intensity.

[0013] In one embodiment, the microscope includes a plurality (e.g., at least three) light sources for emitting three beams at three distinctly different wavelengths. The optical system includes one or more dichroic mirrors to merge the three beams in space and pass the three beams through a homogenizer. One of the prisms may include a dichroic surface that reflects one wavelength for spatial patterning and refracts the others for substantially TIR illumination. Preferably, the homogenizer forms the beams into illumination of a substantially uniform and rectangular area. The homogenizer may include at least two microlens arrays.

[0014] The optical system may include an opaque screen with multiple apertures, the screen being positioned so that the beam passes through one of the apertures. Optionally, the homogenizer may comprise two microlens arrays, and the optical system may comprise multiple microlens array position stops at predetermined intervals, thereby fixing the distance between the two microlens arrays, and thereby shaping the beam to match each of the apertures.

[0015] The microscope may include a stimulating light source that emits a stimulating beam (for example, at a wavelength that activates a photoaperture ion channel). The optical system may also include a digital micromirror device (DMD) such that the stimulating beam is reflected from the DMD and illuminates the bottom of the plate wells using a pattern defined by the DMD. For example, the beam may be at the excitation wavelength of a phosphor, while the stimulating beam is at a second wavelength for electroactive optogenetic operation, etc.

[0016] The microscope may include an imaging lens beneath the stage to direct light from the sample in the wells of the plate onto an image sensor mounted within the microscope. Preferably, the optical system includes a prism directly beneath the stage, thereby directing a beam (e.g., as an excitation beam) onto the side of the prism, which directs the beam into the aqueous sample in the wells of the plate at an angle of refraction that restricts the light to the bottom of the wells at approximately 10 microns. The microscope further includes a stimulating light source that emits a stimulating beam (e.g., specific to an optogenetic actuator), and the optical system includes a digital micromirror device (DMD) from which the stimulating beam is reflected, illuminating the bottom of the wells of the plate using a pattern defined by the DMD. The stimulating and excitation beams may be multiplexed on the same optical path and separated by dichroic mirrors that form part of the prism assembly. Alternatively, the stimulating and excitation beams may pass towards the prism assembly along distinctly different, spatially separated paths.

[0017] Aspects of this disclosure provide a method for imaging a sample. The method includes the steps of: positioning a multiwell plate on a microscope stage, the plate having at least one viable cell on the bottom surface of each well; acquiring an image of the cell; and processing the image to create a spatial mask that identifies the area of ​​the bottom surface occupied by the cell and the area not occupied by the cell. The microscope control system then uses the spatial mask to selectively activate the micromirrors of a digital micromirror device (DMD) relative to the cell, causing the microscope to illuminate the DMD with light, thereby specifically reflecting the light onto the area of ​​the bottom surface occupied by the cell, while not reflecting any light onto the area not occupied by the cell. The method may also include the steps of: creating a spatial mask relating to the cells in each of the multiple wells of the multiwell plate; storing the spatial mask in memory; and using the spatial mask and the DMD to selectively illuminate the cells in the multiple wells in a continuous manner. Preferably, the DMD is computer-controlled, comprising a process of being coupled to a non-transient memory system, the memory system having the spatial mask stored therein. Preferably, the light is activation light at a wavelength that activates photo-aperture ion channels within the cell.

[0018] This method is useful when the photo-aperture ion channels are located within presynaptic neurons connected to non-selective cells via synapses. Non-selective cells may include optical reporters of synaptic activity. In a preferred embodiment, the stage comprises an electrically powered xy-translation stage, and a computer instructs the stage to sequentially position each of a plurality of wells across the imaging lens of the microscope and activate the micromirrors of the DMD according to a spatial mask uniquely created with respect to the cells in that well and stored in a memory system. The cells may include neurons such that the step of selectively illuminating the cells in the plurality of wells initiates electrical activity in the illuminated cells. The computer may record a video for each well using an imaging sensor positioned below the imaging lens and store the resulting videos in a memory system. Preferably, the microscope includes a light source and an optical system that directs an excitation beam of light into each well from below at an angle that restricts the light to approximately 10 microns at the bottom of the well. The wells may contain nerve cells expressing electroactive optical reporters, which fluoresce when they fire action potentials. Videos may record the fluorescence activity indicating nerve cell firing. Optionally, cells express fluorescent proteins (e.g., GFP), and a computer system analyzes the signals from the fluorescent proteins and automatically creates spatial masks. In a preferred embodiment, the computer system can automatically create spatial masks for the labeled cells in each of the multiple wells and store the spatial masks in memory, while the microscope uses the DMD and individual spatial masks to sequentially illuminate each well, creating spatially patterned illumination specific to the cells in that well.

[0019] In one aspect, the microscopes of the present invention and methods of using them include an autofocus system. An exemplary method of using an autofocus system includes positioning a multi-well plate on a microscope stage, the plate having at least one cell living on the bottom surface of a well, and the microscope including an autofocus system. Images of the multi-well plate from a plurality of fields of view (FOVs) are acquired. The position of each FOV is determined using the autofocus system. The system then generates a plate focus map of the curvature of the wells of the multi-well plate from a subset of the FOVs. Measurements from cells living on the bottom of the wells are acquired by interpolating the position of the FOVs between a subset of the FOVs.

[0020] In one aspect, the autofocus system is a laser-based autofocus system. An exemplary method of using a laser-based autofocus system may further include moving the microscope stage to a first position, irradiating a laser beam onto the well using the laser of the laser-based autofocus system, and determining the position of the FOV based on the displacement of the laser beam reflected from the sample using the autofocus system. The well with the sample may be moved and the laser beam may be detected for each FOV acquired by the autofocus system.

[0021] In one aspect, the autofocus system is an image-based autofocus system. An exemplary method of using an image-based autofocus system may include recording the X, Y coordinates of n wells across a microplate from a subset of the FOVs, measuring an image quality metric in the n wells across a series of Z steps, and finding the Z step that provides the highest image quality metric across the n wells. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] [Figure 1] FIG. 1 shows components of the microscope of the present disclosure. [Figure 2]Figure 2 shows a prism. [Figure 3] Figure 3 shows an optical patterning system. [Figure 4] Figure 4 shows the output level and shape of a light beam without a homogenizer. [Figure 5] Figure 5 diagrammatically shows the components of an optical system with a homogenizer. [Figure 6] Figure 6 shows the result of spatially homogenizing a beam. [Figure 7A] Figure 7A shows a side view of the hardware related to the optical patterning system. [Figure 7B] Figure 7B shows a top view of the hardware. [Figure 8] Figure 8 shows the housing of the prism system. [Figure 9] Figure 9 illustrates the geometry of the optical path. [Figure 10] Figure 10 shows an aperture fragment. [Figure 11] Figure 11 shows a focusing system. [Figure 12] Figure 12 illustrates how the focusing system functions. [Figure 13] Figure 13 diagrammatically shows a method of imaging a sample. [Figure 14A] Figure 14A is a perspective view of the light source. [Figure 14B] Figure 14B is a top view of the light source. [Figure 14C] Figure 14C is a side view of the light source. [Figure 15] Figure 15 shows a glass-bottom microplate that receives spatially patterned light. [Figure 16] Figure 16 diagrammatically shows a system for the control and use of a microscope. [Figure 17] Figure 17 is a part of the computer-aided design (CAD) drawing of the stage of a 96-well microscope. [Figure 18] Figure 18 shows the optical path for coupling red laser light to a cell sample. [Figure 19]Figure 19 shows hiPSC. [Figure 20] Figure 20 shows an illustrative voltage record. [Figure 21] Figure 21 is a raster plot. [Figure 22] Figure 22 shows the averaged spike rate across cells. [Figure 23] Figure 23 shows the spike shape. [Figure 24] Figure 24 shows the spike timing properties. [Figure 25] Figure 25 shows the averaged fit across cells. [Figure 26] Figure 26 shows the excitability extracted from the data. [Figure 27] Figure 27 shows radar plots of disease phenotype and compound effects. [Figure 28] Figure 28 shows the compound effect projected onto an axis orthogonal to the phenotypic vector. [Figure 29] Figure 29 shows the results from the two compounds. [Figure 30] Figure 30 is a radar plot showing drug-induced changes in neuronal spiking behavior. [Figure 31] Figure 31 shows the concentration response, with two independent titrations plotted. [Figure 32] Figure 32 shows high SNR fluorescence voltage recordings of nerve cells on a 96-well microscope. [Figure 33] Figure 33 is a raster plot showing spikes from the rows of wells. [Figure 34] Figure 34 shows the average ignition rate between lamps in each well. [Figure 35] Figure 35 is a heatmap showing the number of spikes between ramps for each well. [Figure 36] Figure 36 shows the average number of spikes per cell for the ramp portion of the well-by-well protocol in Figure 35. [Figure 37] Figure 37 shows sensory neurons in the dorsal root ganglion (DRG) of a rat. [Figure 38] Figure 38 shows that WT cells, which contain CRISPR / Cas9, were used to knock out the gene, and multiple isogenic clones were proliferated and converted into nerve cells. [Figure 39] Figure 39 shows that a consistent change in spike shape was observed across multiple rounds and KO cell lines. Treatment with clinically effective compounds shifted the behavior back toward the WT state. [Figure 40] Figure 40 shows similar but less severe phenotypes observed in heterozygous patient cell lines and healthy familial controls. [Figure 41] Figure 41 is a multidimensional radar plot revealing changes in neuronal morphology, action potential shape, and spike train behavior. [Figure 42] Figure 42 shows that dimensionality reduction results in a robust phenotype. [Figure 43] Figure 43 shows that CRISPR / Cas9 is used to introduce gain-of-function mutations in ion channels. [Figure 44] Figure 44 shows that CheRiff and QuasAr are expressed in certain neurons. [Figure 45] Figure 45 shows fluorescence images on Firefly, illustrating QuasAr and CheRiff. [Figure 46] Figure 46 shows a single-cell fluorescence trace indicating postsynaptic potentials (PSPs). [Figure 47] Figure 47 shows the modulation of single-cell PSP by control agonists and blockers. [Figure 48] Figure 48 shows the average PSP trace for control pharmacology. [Figure 49] Figure 49 shows a dot density plot. [Figure 50] Figure 50 shows a method for high-performance screening. [Figure 51] Figure 51 shows an exemplary workflow using a laser-based autofocus system. [Figure 52] Figure 52 shows nine fields of view (FOV). [Figure 53] Figure 53 shows a plot of image quality metrics for the wells, captured across a series of Z (axial) steps. [Figure 54] Figure 54 shows an exemplary workflow using an autofocus system. [Figure 55] Figure 55 shows an exemplary plate focus map acquired using an autofocus system. [Modes for carrying out the invention]

[0023] Detailed explanation The present invention provides a multi-well plate microscope for illuminating a sample using substantially TIR light in a configuration that allows living cells to be observed and imaged within the wells of a plate. The present invention is particularly useful for observing fluorescence in biological parts with rapid dynamics and in phosphors with low quantum efficiency. The microscope illuminates the sample from the side rather than through the objective lens, which allows for stronger illumination and correspondingly lower numerical aperture and a larger field of view. By using illumination light at a wavelength distinctly different from the fluorescence wavelength, the TIR microscope allows the illumination wavelength to be almost completely removed from the image using an optical filter, resulting in an image with a dark background and a bright area of ​​interest. The microscope can observe fluorescence and provide a means of indicating underlying chemical or biochemical processes, such as gene expression, the presence of antibodies, or the location of specific proteins within cells.

[0024] Fluorescent reporters, such as modified archrhodopsin proteins known as QuasAr2 and QuasAr3, require strong excitation light to emit fluorescence. Low quantum efficiency and rapid dynamics require strong light to measure potential. The illumination subsystem is therefore configured to emit light at high wattage or high intensity. Phosphor properties such as quantum efficiency and peak excitation wavelength change in response to their environment. Strong illumination allows it to be detected. Autofluorescence caused by strong light is minimized in several ways by the microscope. The use of approximate TIR illumination exposes only the bottom portion of each well to the illumination light, thereby reducing the excitation of the culture medium or other components of the device. In addition, the microscope is configured to provide illumination light that is distinctly different from the imaging light. Optical filters in the imaging subsystem filter out the illumination light, removing unwanted fluorescence from the image. Cyclic olefin copolymer (COC) dishes for culturing cells allow for reduced background autofluorescence compared to glass. Prisms are coupled to multi-well plates through refractive index-matched low autofluorescence oil. The prism is also made of low-autofluorescence fused silica.

[0025] Microscopes are configured to optically characterize the dynamic properties of cells. Microscopes achieve the full potential of all-optical characterization by simultaneously achieving (1) a large field of view (FOV) to enable the measurement of interactions between cells in a network or to measure many cells in parallel for high processing capacity, (2) high spatial resolution to detect the morphology of individual cells in a well and to facilitate selectivity in signal processing, (3) high temporal resolution to distinguish individual action potentials, and (4) a high signal-to-noise ratio to facilitate accurate data analysis. Microscopes can provide a field of view sufficient to capture tens or hundreds of cells. Microscopes and associated computer systems provide an image acquisition rate of at least about 1 kilohertz, which corresponds to a very short exposure time of about 1 millisecond, thereby enabling the recording of rapid changes occurring in electroactive cells such as nerve cells. Microscopes can therefore acquire fluorescence images using optics enumerated over substantially shorter periods than conventional microscopes.

[0026] Microscopes meet all of these stringent requirements to facilitate the optical characterization of the dynamic properties of cells. Using low numerical aperture (NA) objective lenses, microscopes provide a large field of view with sufficient resolution and light-gathering capability. Microscopes can image at magnifications ranging from 2x to 6x using high-speed detectors such as sCMOS cameras. To achieve high-speed imaging, microscopes typically use, for example, 50 W / cm² at a wavelength of approximately 635 nm. 2 It exceeds that, with a maximum output of approximately 2,000 W / cm². 2 Use extremely strong lighting accompanied by fluence.

[0027] Despite high output levels, the microscope avoids the excitation of nonspecific background fluorescence in the sample, cell growth medium, refractive index-matched fluid, and sample container. Approximate TIR illumination limits autofluorescence to unwanted areas of the sample and sample medium. Optical filters in the imaging subsystem prevent unwanted light from reaching the image sensor. In addition, the microscope prevents unwanted autofluorescence of glass elements in the objective lens by illuminating the sample from the side rather than passing illumination light through the objective lens unit. The microscope's objective lens is physically large, having a front aperture of at least 50 mm and a length of at least 100 mm, and may contain a large number of glass elements.

[0028] Microscopes can be used to observe fluorescent indicators that are sensitive to the specific physical properties of their environment, such as calcium ion concentration or membrane potential. The time-varying signals produced by these indicators are repeatedly measured to illustrate the progression of the chemical or electronic state of living cells. One example of an environmentally sensitive fluorescent indicator for use in conjunction with the present invention is the archrhodopsin-based protein QuasAr2, an optogenetic reporter that is excited by red light and produces a signal that varies in intensity as a function of the cell membrane potential. The optogenetic reporter can be introduced into cells using genetic engineering techniques such as gene transfer or electroporation to facilitate optical measurements of the membrane potential.

[0029] In addition to fluorescent indicators, microscopes can be used to optically activate photosensitive compounds to chemically or electrically perturb cells. The present invention can be used in conjunction with voltage-indicating proteins, such as those disclosed in U.S. Patent No. 10,613,079 and U.S. Publication No. 2014 / 0295413 (their respective contents incorporated by reference). Using photoregulatory activators, stimuli can be applied to the entire sample, a selected region, or individual cells by varying the illumination pattern. One example of a photoregulatory activator is the channelrhodopsin protein CheRiff, which produces a current whose magnitude increases approximately proportionally to the intensity of the blue light shone upon it. In one study, CheRiff produced approximately 22 mW / cm². 2 When illuminated with blue light, all cells expressing the protein generated a current of approximately 1 nA.

[0030] Optically modulated activators, when combined with fluorescent indicators, can enable the complete optical characterization of specific cellular characteristics such as excitability. For example, channelrhodopsins such as CheRiff can be combined with fluorescent indicators such as QuasAr2. Microscopes provide light of different wavelengths to illuminate and activate reporter and activator proteins, respectively, allowing membrane potential to be measured simultaneously with the initiation of action potentials by light.

[0031] Useful samples using a near-TIR microscope include cells expressing electroactive optical activators and electroactive optical reporters. In the wells of a multi-well plate, the first cell expresses the activator, and the second cell expresses the reporter. The microscope can activate the photosensitive activator protein (e.g., optogenetic actuator) using an activation beam, causing a conformational change in the protein, thereby initiating a change in the membrane potential in the cell. The result is that the cell "fires," i.e., an action potential propagates within the electroactive cell. The microscope can simultaneously illuminate the fluorescent optical reporter protein using an activation beam and a spectrally distinct illumination beam, causing the reporter to fluoresce. The microscope's imaging subsystem can measure the fluorescence emitted by the reporter and the corresponding change in membrane potential.

[0032] The microscope employs at least several optical subsystems, including an illumination light subsystem for exciting reporter proteins and an imaging light subsystem for imaging the light emitted by the reporters. The microscope may also include an activation light subsystem for activating activator proteins, such as photo-aperture ion channel proteins. In some embodiments, the illumination subsystem refers to a subsystem that emits illumination light to activate reporter proteins so that they can fluoresce in response to changes in their action potential. The imaging subsystem refers to a subsystem that receives light emitted by reporters when they fluoresce, and the light they emit is referred to as imaging light. The activation subsystem refers to a subsystem that emits light to activate photo-aperture ion channel proteins to initiate changes in membrane potential.

[0033] Figure 1 shows the components of the microscope 101 of this disclosure. The microscope includes a stage 105 configured to hold a multiwell plate 109, an excitation light source 115 mounted within the microscope for emitting a beam of light, and an optical system 601 for directing the beam from below toward the stage. The optical system includes a homogenizer 125 for spatially homogenizing the beam. The microscope 101 includes, or is communicably coupled to, a computer 171 or computing system hardware for performing or controlling various functions. The microscope 101 may also include an optical patterning system 301, which will be discussed in more detail below. The stage 105 is preferably an electrically powered xy translation stage.

[0034] The microscope 101 includes an image sensor 135. The image sensor may be supplied as a digital camera unit, such as the ORCA-Fusion BT digital CMOS camera sold by Hamamatsu Photonics KK (Shizuoka, JP) as part number C15440-20UP, or the ORCA-Lightning digital CMOS camera sold by Hamamatsu Photonics KK as part number C14120-20P. Another suitable camera for use with sensor 135 is a back-illuminated sCMOS camera sold by Teledyne Photometries (Tucson, AZ) under the trademark KINETIX.

[0035] The microscope may also include an imaging lens 137, such as a suitable tube lens. The lens 137 may be an 85mm tube lens, such as a ZEISS Milvus 85mm lens. Using such imaging hardware, the microscope can image an area with a diameter of 5.5mm in a 96-well plate and an area with the entire well width of 3.45mm in a 384-well plate.

[0036] The microscope 101 preferably includes a control system with memory connected to a processor capable of moving a translation stage and positioning individual wells of a multiwell plate within the beam path. Optionally, the microscope 101 includes an excitation light source 115 mounted within the microscope to emit a beam of light 121. The optical system 601 directs the beam 121 from below toward the stage.

[0037] The microscope 101 may optionally include a secondary light source 153. The secondary light source 153 may have its own optical system, sharing some similarities with the optical system 601. However, including the optical system 601 and the secondary light source 153 with its own optical system allows the systems to operate independently, whether simultaneously or not. In some embodiments, the secondary light system operates at a different (e.g., much higher) output than the optical system 601. The secondary light source 153 and its system may be used for calibration or to address optogenetic proteins that operate best at a different output than the set of optogenetic proteins addressed by the optical system 601.

[0038] Figure 2 shows a prism 201 that guides a beam 121 toward a sample 205. The optical system 601 includes the prism 201 directly below the stage, thereby causing the beam to be incident on the side of the prism and pass through the well 211 of the plate. As shown, the aqueous sample 138 contains living cells 113 on the bottom surface 112 of the well 211. Optionally, refractive index matching lens oil 119 optically couples the prism 201 to the bottom 112 of the well. Preferably, when the well 211 of the plate containing the aqueous sample 138 is positioned above the prism 201, the prism directs the beam 121 into the sample at an angle theta that avoids all internal reflections within the bottom 112 of the well of the plate. As shown, when the well of the plate containing the aqueous sample is positioned above the prism, the prism directs a beam into the aqueous sample at an angle of refraction that restricts the light to the bottom of the well by approximately 10 (optionally, 20) microns.

[0039] The present invention provides a microscope in which all optical components are positioned beneath the wells of a multi-well plate, and illumination occurs from the side rather than through the objective lens. Side illumination allows the microscope to have stronger illumination and a larger field of view. Optionally, the area above the stage is not obstructed by optical elements such as prisms. Its configuration allows for physical access to the sample and control over its environment. Thus, the sample may be, for example, living cells in a nutrient medium. Its configuration solves many of the problems associated with conventional TIRF microscopes. In particular, thin areas of sample cells can be illuminated using a substantially TIR beam without the need to physically interfere with the cells by loading them into a fluid chamber. Alternatively, living cells in aqueous media such as maintenance culture medium can be observed. The sample can be further analyzed from above using electrodes or other instruments, as desired. The microscope can be used to image cells expressing a fluorescence voltage indicator. Because the components do not interfere with the sample, living cells can be studied using the microscope of the present invention. If a sample contains electroactive cells expressing a fluorescent voltage indicator, a microscope can be used to visualize the voltage changes, and therefore the electroactivity, of those cells. Since the electroactivity of cells such as nerve cells and cardiomyocytes can be studied using the devices and methods of the present invention, the present invention will help researchers understand the diseases affecting those cells and will enable researchers to discover new preventive and therapeutic methods for diseases such as Alzheimer's disease and heart disease.

[0040] The devices and methods of the present invention can be used in conjunction with optogenetics, in which light is used to control and observe certain events in living cells. For example, photoresponsive genes, such as fluorescent voltage indicators, can be introduced into cardiomyocytes. The reporter may be a rhodopsin-type transmembrane protein that generates an optical signal in response to a change in membrane potential and thereby functions as an optical reporter. For example, a modified version of the microbial rhodopsin protein archrhodopsin 3 (Arch) from Halorubum sodomense may be used as an optical reporter. Examples of optical reporters are the microbial rhodopsins QuasAr2 and QuasAr3. When excited with illumination light at one wavelength, the reporter is activated and emits light of different wavelengths indicating a change in membrane potential. The microscope therefore includes an illumination system for illuminating a sample with light of one wavelength and an imaging system for recording images of the light emitted by the reporter at different wavelengths. The illumination system includes a light source and a prism. Illumination light from a light source is transmitted through a prism, which provides approximate TIR illumination on the sample. The imaging system includes an objective lens and an image sensor for recording the light emitted by the reporter.

[0041] Approximately TIR illumination can provide illumination of thin areas of cells, approximately 10 microns thick. This means that it can illuminate all cells in contact with the bottom of the wells in a multi-well plate. The illumination is emitted from the side rather than through the objective lens, and therefore the microscope can illuminate all cells with strong illumination without illuminating the culture medium or other optical components.

[0042] Optionally, the cells in the sample may also contain optogenetic activators such as photo-aperture ion channels. The optical actuator may be a genetically encoded or modified rhodopsin, such as a microbial channelrhodopsin. For example, sdChR, a channelrhodopsin from Scherffelia dubia, may be used, or an improved version of sdChR called CheRiff may be used as the optical actuator. "CheRiff" refers to a version of sdChR that uses mouse codon optimization, trafficking sequences, and the E154A mutation as described herein. The activators respond to light of a specific wavelength and initiate action potentials in electroactive cells. To activate these optogenetic proteins, the microscope may include an activation system. The activation system directs light onto the sample at wavelengths capable of activating the channelrhodopsin. The activation light may be patterned light corresponding to a certain area of ​​the sample, such as specific cells in the sample. Patterns can be imparted to a light beam using a spatial light modulator (SLM), such as a digital micromirror device (DMD). Light is shone onto the SLM, which reflects a defined pattern of light that is imaged onto the sample. The light pattern can correspond to specific cells that express activators.

[0043] The microscope may include additional reporters and associated systems for activating them. Proteins that report changes in intracellular calcium levels, such as genetically encoded calcium indicators (GECIs), may be used. The microscope may include subsystems for providing activation light for GECIs, such as yellow light for RCaMPs. Exemplary GECIs include, for example, GCaMPs or RCaMP variants such as jRCaMP1a, jRGECO1a, or RCaMP2. A key challenge when combining multiple optical modalities (e.g., optical excitation, activation, voltage imaging, calcium imaging) is to avoid optical crosstalk between modalities. The pulse of light used to induce optical activation should not induce fluorescence of reporters, the light used to image reporters should not activate photo-aperture ion channels, and the fluorescence of one reporter should be easily distinguishable from the fluorescence of other reporters. In some aspects of the present invention, this separation of modalities is achieved by selecting activators and reporters with little or no spectral overlap. In one embodiment, the activator is activated by blue light, the Ca2+ reporter is excited by yellow light and emits orange light, and the voltage reporter is excited by red light and emits near-infrared light.

[0044] Furthermore, the microscope includes a system for spatially patterned illumination, which is useful for selectively illuminating only specific cells within a sample.

[0045] Figure 3 shows an optical patterning system 301 for spatially patterning light of multiple wavelengths onto a sample. The optical patterning system 301 includes a first light source 313 for emitting a beam of light 302. The beam of light is reflected from a digital micromirror device (DMD) 305. The DMD 305 shapes the beam 302 into a pattern. The patterned beam is imaged onto the sample. The DMD would allow for a fully synchronized 100 μs pattern refresh for fast single-cell stimulation to measure individual synaptic connections or for slightly delayed pulses on connected neurons to examine spike timing-dependent plasticity. The optical patterning system may optionally include a second light source 314. The first light source preferably delivers light of a first wavelength into the beam 302. This may be done using a filter 323 with respect to the first wavelength.

[0046] A dichroic mirror 343 can selectively reflect light of a second wavelength from a second light source 314 into the beam 302. The light patterning system 301 may include one or any number of lens elements 341, such as a 30 mm achromatic doublet, to guide light onto any dichroic mirror 343 or to collimate the beam 302. The second light source 314 may provide light at the second wavelength using a second filter 324 specific to the second wavelength. The light patterning system 301 may include a third light source 315, a third filter 325, and optionally a fourth light source 316 and a fourth filter 326. In a preferred embodiment, once light from various wavelengths is combined into the beam 302, the beam 302 is passed through an optical pipe 321.

[0047] One optional embodiment uses four light sources with four wavelengths: UV (380 nm), blue (470 nm), yellow / green (560 nm), and red (625 nm). UV (380 nm) may be useful for imaging EBFP2 or mTagBFP2 or intracellular calcium. 50 mW / cm 2The output may be sufficient. Blue (470 nm) can be used to image CheRiff (e.g., at 250 - 500 mW / cm to open >95% of the channel), Chronos (at 500 mW / cm to open most of the channel), FLASH (registered trademark), or other such proteins. Yellow / green (560 nm) light can be used to image jRGECO1a (80 mW / cm at 560 nm for neurons or 25 mW / cm for cardiomyocytes), VARNAM, or other proteins. Red (625 nm) can be useful for measuring target proteins with Alexa647 (e.g., at 50 mW / cm) or cell activity with BeRST (e.g., 1 - 20 W / cm for neurons). 2 In), Chronos (at 500 mW / cm to open most of the channel), 2 In), FLASH (registered trademark), or other such proteins. Yellow / green (560 nm) light can be used to image jRGECO1a (80 mW / cm at 560 nm for neurons or 2 25 mW / cm for cardiomyocytes), 2 ), VARNAM, or other proteins. Red (625 nm) can be used to image target proteins with Alexa647 (e.g., at 50 mW / cm 2 In) or cell activity with BeRST (e.g., 1 - 20 W / cm for neurons). 2 ) may be useful.

[0048] The optical patterning system 301 may include one or any number of circular mirrors 326 to direct the beam 302 from the light source 313 (typically mounted on a solid frame or substrate) to the sample. The optical patterning system 301 includes an adjustable circular mirror 327 that controls the final angle at which light approaches the prism assembly 309. In a preferred embodiment, the optical pattern system 301 includes a prism assembly 309 that includes one or more prisms to direct light onto the DMD 305 and onto the sample. The prism may preferably have a refractive index that matches the refractive index of the material forming the bottom of the multi - well plate. For example, the microscope 101 may be designed for use in combination with plates such as glass - bottom microplates with 24, 96, 384, or 1,536 wells sold under the trademark SENSOPLATE by MilliporeSigma (St. Louis, MO). Such microplates have dimensions including a length of 127.76 mm and a width of 85.48 mm. The microplate includes borosilicate glass (175 μm thick).

[0049] The prism assembly 308 may include a dichroic mirror 308 that reflects light of a selected wavelength from the DMD 305, allowing other selected wavelengths to pass through it at approximately a TIR angle, thereby illuminating the sample only over the bottom 10-20 microns of the well. Here, approximately TIR can be understood to mean an angle that is below, but nevertheless very close to, the critical angle at which light emitted from the side would exhibit total internal reflection in a portion of the multiwell plate hardware (e.g., would not exhibit TIR at the bottom of the borosilicate glass of the plate), for example, preferably within 10 degrees of the critical angle, more preferably within 5 degrees of the critical angle with respect to TIR, and most preferably within 2 degrees of the critical angle.

[0050] As shown, the sample being imaged emits light 338 that passes toward the imaging sensor 335 (for example, not drawn but through the tubular lens). Due to the dichroic mirror, the sample can be illuminated with spatially patterned light and also illuminated from the side by approximate TIR light that passes through only about 10 microns at the bottom of the sample well (both from beam 302), and can also emit emitted light 338 that is captured by the sensor 335 for recording motion.

[0051] Any suitable digital optical processor or spatial patterning mechanism may be used as the DMD305. In some embodiments, the DMD305 is a Vialux V9601-VIS DMD system with a 1,920 × 1,200 pixel array of micromirrors at a 10.8 μm pitch and a 20.7 × 13 mm array size. The optical patterning system may optionally include a tubular lens such as a Zeiss Milvus 135 mm to provide a reduction ratio (e.g., 2.7x) on the sample.

[0052] In the embodiment described, each light source 313 is a 3×3mm Luminus LED, imaged onto a 6×6mm optical pipe 321 that maintains source etendue. A four-lens design from the LED to the optical pipe (24-f imaging system) increases light collection efficiency and minimizes angular components. The described light patterning system 301 includes at least three (e.g., four) light sources 313, 314, 315, 316 to emit at least three beams at three distinctly different wavelengths. Preferably, the light patterning system 301 has one or more dichroic mirrors 343 to combine the three beams in space and pass the three beams through a homogenizer and / or optical pipe 321. The optical pipe 321 homogenizes the sources and ensures good overlap of the four LED colors. Light from the optical pipe 321 passes along it toward the DMD.

[0053] The microscope 101 includes an excitation light source 115 mounted within the microscope to emit a beam of light 121. The optical system 601 directs the beam 121 from below toward the stage at a certain angle. One potential problem is aberration, which can affect the shape of the beam 121.

[0054] Figure 4 shows the output level of the light beam from the excitation light source 115, with the aperture square drawn on the image illustrating the optical aperture used in the microscope 101. The amount of light is not uniform across the aperture square. The result would be non-uniform illumination of the cells 113 in sample 205. Microscope 101 avoids non-uniform illumination of the cells 113 by including a homogenizer 125 within the optical system 601 to spatially homogenize the beam 121.

[0055] Figure 5 illustrates the components of the optical system 601. As shown, a first set of components 604 is substantially in a horizontal plane, fixed, for example, to a printed circuit board or other substrate within the base of the microscope 101. A second set of components 605 may have components oriented so that the beam of light travels upward toward the sample.

[0056] Within the optical system 601, the beam 121 passes through a homogenizer 125. Preferably, the homogenizer 125 forms the beam 121 into a substantially uniform and rectangular illumination area. Different methods of laser beam homogenization may be used to create a uniform beam profile. Homogenization may be performed using a lens array optical system or an optical pipe rod.

[0057] A lens array consists of separate lens elements stacked side-by-side, which are then placed in the laser beam path to form an array. Homogenization is achieved by splitting the initial wavefront into separate beams using each lens element, and then focusing these new beams onto a target area using a focusing lens. The separate beams are superimposed on the illumination plane to which they are focused using the focusing lens. Preferably, the shape of the illumination area is identical to that of the lens element aperture. To avoid blurred edges, a second lens array may be used. One lens array is conjugated to another such that the focal point of one array coincides with the pupil plane of the next, and the focal point of the second array coincides with the pupil plane of the first. This setup improves edge sharpness, but the arrays must be very precisely matched with each other for this to work properly. The homogenizer 125 may contain one or any number of microlens arrays. Passing light through microlens arrays can give the light output profile a "top hat" shape. A beam homogenizer smooths out irregularities in the laser beam profile, creating a more uniform profile. A suitable beam homogenizer may use a multifaceted mirror with square facets. The mirror reflects light at different angles, creating a beam with uniform output across the entire beam profile ("top hat" profile). The homogenizer may include a diffraction beam homogenizer or an MLA (microlens array). See Voelkel 2008, Laser beam homogenizing: limitations and constraints, Proc SPIE 7102, Optical Fabrication, Testing, and Metrology III, 71020J (incorporated by reference).

[0058] A second method for homogenization is the use of a rod lens. In this layout, multiple reflections from the initial beam are mixed inside the rod (or optical pipe). The length of the optical pipe (homogenization rod) should exceed several times the size of the laser beam cross-section, which determines the longitudinal system size. The input beam can be focused into the optical pipe using a focusing lens, which is an option to reduce the cross-sectional and longitudinal sizes. An optical pipe that provides TIR reflection reduces reflection losses, while the TIR angle limits the numerical aperture (NA) of the input beam.

[0059] After passing through the homogenizer 125, the light passes through the aperture 555. Any suitable hardware mechanism may be used in the microscope 101 to create the aperture 555.

[0060] Figure 6 shows the result of spatially homogenizing the beam 121. Homogenization can be provided by any suitable optical hardware for the homogenizer 125, such as a pair of microlens arrays or optical pipes, which are mounted within the hardware of the optical patterning system.

[0061] Figure 7A shows a side view of hardware 701 for the optical patterning system.

[0062] Figure 7B shows a top view of the hardware 701. As shown, the hardware 701 includes a prism assembly 708, a mirror 727, and an optical pipe 721. The optical patterning system may include one or any number of lens elements 341, such as a 30 mm achromatic doublet, to guide light onto any dichroic mirror or to collimate the beam. Multiple light sources and mirrors may be present to guide light onto the sample.

[0063] The light patterning system 301 may include one or any number of mirrors to guide the beam from the light source mounted in the hardware 701 to the prism assembly 708 and onto the sample. The prism assembly 708 includes one or more prisms to guide the light onto the DMD 705. The prism assembly 708 may be housed in the housing 800.

[0064] Figure 8 shows a housing 800 adjacent to a wireframe drawing of a prism assembly 708 to illustrate how the mirror and prism are housed within the housing in this embodiment. A DMD 705 is connected to the housing 800 adjacent to the prism. The prism assembly 708 reflects light from the DMD 705. The DMD is connected to a cable 816, shown here as a ribbon cable connected to a substrate 817 having a processor for controlling the operation of the DMD. The housing 800 also has an optical dump 815 to receive and extinguish light that has been reflected from the DMD and directed away from the sample by the DMD.

[0065] Figure 9 illustrates the geometric shape of the optical path from the light source (not shown) at the bottom of the microscope 101 upwards to the prism assembly 800.

[0066] Within the optical system 601, the beam 121 passes through the homogenizer 125. Preferably, the homogenizer 125 forms the beam 121 into illumination of a substantially uniform and rectangular area. After passing through the homogenizer 125, the light passes through the aperture 555. Any suitable hardware mechanism may be used in the microscope 101 to create the aperture 555. It may be preferable that the microscope 101 includes a screen or block through which multiple apertures are present.

[0067] Figure 10 shows an aperture fragment 1001 according to one embodiment. As shown, the fragment includes an aluminum block 1002 with seven apertures through it. The fragment may be repositionable on the frame of the microscope 101 to one of seven corresponding predefined stops. When positioned in this manner, a light beam 121 passes through a selected aperture of the fragment 1001. In such embodiments, the optical system 601 includes an opaque screen with multiple apertures, which can be positioned so that the beam passes through one of the apertures. The apertures may be predefined for a specific imaging workflow. For example, each aperture may define a beam profile suitable for imaging wells of a multiwell plate with a certain well count (e.g., 96 or 384).

[0068] The aperture fragment 1001 may include multiple (e.g., seven) apertures for different imaging configurations. Here, as shown, the microscope 101 includes seven pre-selected apertures for imaging each combination of three plate types and two lens types, plus a “wide-opening” option. As shown, from left to right, the fragment 1001 includes the first aperture 1011, the second aperture 1012, the third aperture 1013, the fourth aperture 1014, the fifth aperture 1015, the sixth aperture 1016, and the seventh aperture 1017. The first aperture 1011 is for imaging a 384-well plate at 1 kHz using a 135 mm tube lens and measures 0.90 × 0.89 mm. The second aperture 1012 is for imaging a 384-well plate at 1 kHz using an 85 mm tube lens and measures 0.90 × 1.35 mm. The third aperture 1013 is for imaging a 384-well plate at 500 Hz using an 85 mm tube lens and measures 0.90 × 2.57 mm. The fourth aperture 1014 is for imaging a 96-well plate at 1 kHz using a 135 mm tube lens and measures 1.27 × 0.89 mm. The fifth aperture 1015 is for imaging a 96-well plate at 1 kHz using an 85 mm tube lens and measures 1.27 × 1.35 mm. The sixth aperture 1016 is for imaging a 96-well plate at 500 Hz using an 85 mm tube lens and measures 1.27 × 2.57 mm. The seventh aperture 1017 is for full-open imaging and measures 4.6 mm × 7.2 mm. Block 1002 may include a water channel 1029 with a chilled water connection 1035. Plug 1003 may be used to facilitate the milling of channel 1029 into monolithic block 1002.

[0069] Here, the homogenizer 125 uses two microlens arrays, and the microscope 101 can be designed so that the microlens arrays are repositionable to create defined intervals corresponding to one of the apertures through the fragment 1001. In some embodiments, the homogenizer 125 comprises two microlens arrays, and the optical system 601 comprises a plurality of microlens array position stops at predetermined intervals, thereby fixing the distance between the two microlens arrays and thereby shaping the beam to match each of the apertures.

[0070] The microscope 101 may include multiple optical systems. For example, the optical patterning system 301 may include a stimulating light source 313 (emitting a beam 302) and a digital micromirror device (DMD) 305. The beam 302 is reflected from the DMD and illuminates the bottom of the plate wells using a pattern defined by the DMD. The beam 302 may be at the excitation wavelength of the phosphor. The microscope may also include a secondary optical system with a secondary light source 253 that emits a stimulating beam at a second wavelength.

[0071] The light patterning system 301 includes a prism directly beneath the stage, thereby directing the beam onto the side of the prism, which directs the beam into the aqueous sample in the wells of the plate at an angle of refraction that restricts the light to the bottom of the wells at approximately 10 microns. The microscope 101 may also include a stimulating light source 153 that emits a stimulating beam. Optionally, the stimulating beam is reflected from the DMD and illuminates the bottom of the wells of the plate using a pattern defined by the DMD.

[0072] The microscope 101 may optionally include a secondary light source 153. The secondary light source 153 may have its own optical system, sharing some similarities with the optical system 601. However, including the optical system 601 and the secondary light source 153 with its own optical system allows the systems to operate independently, whether simultaneously or not. In some embodiments, the secondary light system operates at a different (e.g., much higher) output than the optical system 601. The secondary light source 153 and its system may be used for calibration or to address optogenetic proteins that operate best at a different output than the set of optogenetic proteins addressed by the optical system 601.

[0073] In some respects, the microscope of the present invention includes an autofocus system. The microscope may include a laser-based autofocus system. Alternatively, or in addition, the microscope may include an image-based autofocus system. In some respects, the microscope of the present invention includes and uses both laser-based and image-based autofocus systems.

[0074] Figure 11 shows an exemplary laser-based autofocus system that may be included. A red laser beam 1101 is provided by an optical system 601. A yellow laser beam 1105 is provided by a secondary light source 153 (sometimes referred to as the “left path” light source based on how it is depicted on the page). The microscope 101 further includes a laser diode 1109 (e.g., green) and a position-sensing detector 1115. The components depicted (lenses 1, 2, 3, 4, 5, 6, filters 7 and 9, and shutter 8) provide a 4-f imaging system for imaging a sample on a position sensor, so that the curvature of the well plate foil, which would change the beam angle, would not change the position on the sensor. Preferably, lenses 3 and 4 are separated by f3+f4, so that the collimated beam is incident on the sample. Lenses 1 and 2 reduce the beam diameter and avoid clipping on the prism entrance aperture, and the lenses are separated by f1+f2.

[0075] Filter 9 may provide the ability to adjust the output of the green laser light from laser diode 1109 with respect to a given sample type in order to maximize the accuracy of the sensor toward the theoretical submicron limit. Shutter 8 may blink the laser to enable millisecond reading times.

[0076] Figure 12 illustrates how an exemplary autofocus system functions. Sample height varies per field of view (FOV) based on imperfections in plate manufacturing, such as warping of cyclic olefin copolymer (COC) foil (e.g., imperfections in multiwell plates). A green laser enters at a shallower angle and therefore performs a complete TIR. A red laser beam 1101 exhibits a near-TIR. The dashed line 1201 illustrates that the laser position changes as the TIR surface moves. A position-sensing detector 1115 reports the current position of the sample in the well of the multiwell plate to the computing system 171. The microscope 101 may include a z-motor (not drawing) to focus the optical system, under control functions that raise or lower the stage (relative to the optical system) and issue commands to operate the z-motor.

[0077] Figure 51 provides a flowchart of an exemplary method using the laser-based autofocus system of the present invention. As shown, in one aspect, the optical position sensor 1115 may be calibrated before acquiring a reading. The sample is then moved to a fixed position, and the system irradiates the sample with a laser beam. The beam undergoes total internal reflection from the sample and is detected on the optical position sensor 1115. The sample position is calculated based on the displacement of the laser beam. A focus motor moves the camera lens focus to the sample position, at which point the experiment is performed on the sample. Once the experiment is complete, the sample is moved and / or replaced by another sample, and the cycle may be repeated as needed. As shown in Figure 51, the steps of the system occur fast enough to quickly measure the position of all fields of view as the microscope screens the sample, and can enable excellent focusing across all fields of view.

[0078] Exemplary laser-based autofocus systems may utilize micrometer-accuracy position sensors to directly measure the position of the complete total internal fluorescence and FOV on the COC imaging buffer interface.

[0079] In one aspect, the system uses an image-based autofocus system. An exemplary method using an image-based autofocus system includes the step of acquiring a group of images from different fields of view. In one aspect, the image-based autofocus system can indirectly measure the position of the field of view.

[0080] Figure 52 shows an example of nine images of a sample plate acquired from nine different fields of view. The system records the coordinates (X,Y) of n wells traversing a microwell plate, which have good spacing (n=9 in Figure 52). The system then measures the image quality metric in the n wells using a series of Z steps (axial location).

[0081] Figure 53 provides a plot of image quality metrics measured during a series of Z-steps. The system uses the Z-step (value) that provided the highest image quality metric for each well.

[0082] Figure 54 provides a workflow for an exemplary method using an N-point autofocus system employing a laser-based or image-based autofocus system. The N-point autofocus system takes the measured positions of the sample microplate in an arbitrary subset of the field of view (FOV) and generates a curve model representing the curvature of the microplate bottom from these wells. This allows electrophysiological measurements to be performed in a rapid manner by interpolating the FOV positions among an arbitrary subset of the FOV, without requiring the microscope to individually measure the focus for all wells, which would be more time-consuming and limiting in terms of processing power.

[0083] Figure 55 provides an exemplary plate focus map generated by an N-point autofocus system.

[0084] The computer system 171 performs control functions. In some embodiments, the microscope 101 uses a left-path and a secondary light source 153 to image a sample in a multi-well plate in which certain target objects present in the sample are fluorescently labeled. For example, with respect to a neuronal activity assay, each well of the plate may contain a network of living neurons. The microscope may image each well, and the computer may be used to identify all individual neurons in each well, find all synaptic connections between those neurons, and create a record of the location of any presynaptic neurons in each well. If all those neurons are provided with optogenetic actuators such as CheRiff (an algal channel rhodopsin that functions as a photo-appressive ion channel), the computer 171 can create a spatial mask (a digital file with a binary coding of pixels in the image of a well that is occupied or not occupied by a particular neuron) that identifies the location of the presynaptic neurons in each well. The computer can store this information about all the wells in the multi-well plate. The xy motor then parallelizes the plate across the imaging lens, using the DMD to spatially pattern the light onto the wells according to the spatial mask, selectively and exclusively illuminating presynaptic neurons with light at wavelengths that depolarize photo-aperture ion channels, causing the presynaptic neurons (and only them) to fire action potentials. In response to its optical stimulation from the DMD, any postsynaptic neuron will "fire," i.e., exhibit an action potential, and normal neurotransmission with chemical transmission exceeding a threshold will occur. If the neurons contain an electroactive optical reporter, such as one of the modified versions of archrhodopsin 3 known as Arch 3 D95N, QuasAr1, QuasAr2, or QuasAr3, those postsynaptic neurons will emit light as their membrane potential changes as the action potential propagates.The microscope 101 can use the image sensor 135 to record a video of light, which can be analyzed to detect normal neurotransmission and, furthermore, specific features (in the recorded light in the video) that tend to indicate specific biological neurophenotypes such as disease-related issues associated with neuronal activity.

[0085] Therefore, the microscope 101 may be used in various assays on living cells, particularly those relating to the electroactivity of cells. The microscope can assay the activity of various electroactive cell types, including nerve cells and cardiomyocytes. The microscope 101 uses a multi-well plate compatible stage, a computer 171, and a DMD 305, and can store and use spatial masks to spatially pattern illumination onto selected cells from well to well of a plate without redefining the spatial masks between wells (the spatial masks are stored in memory). Thus, the microscope provides a method for imaging a sample.

[0086] Figure 13 illustrates method 1301 for imaging a sample. Method 1301 includes the step 1303 of positioning a multiwell plate on a microscope stage, the plate having at least one viable cell on the bottom surface of each well. The imaging step 1309 is performed to acquire an image of the cells. The image is processed to create a spatial mask that “masks” the surface on the bottom of the wells (1315), i.e., identifies the area of ​​the bottom surface occupied by cells and the area not occupied by cells. Using the mask, the computer signals the DMD to selectively activate the micromirrors of the DMD relative to the cells using the spatial mask (1323). The microscope then illuminates the sample by shining light onto the DMD using a light source 121 (1327), thereby specifically reflecting light onto the area of ​​the bottom surface occupied by cells, while not reflecting any light onto the area not occupied by cells. Method 1301 may include the steps of creating a spatial mask relating to cells in each of several wells of a multiwell plate, storing the spatial mask in memory, and selectively illuminating cells in the several wells in a continuous manner using the spatial mask and a DMD. Optionally, the DMD is computer-controlled, comprising a process of being coupled to a non-transient memory system, the memory system having the spatial mask stored therein.

[0087] The devices and methods of the present invention may be used to utilize fluorescent indicators that are sensitive to the specific physical properties of their environment, such as calcium ion concentration or membrane potential. The time-varying signals produced by these indicators can be repeatedly measured to illustrate the progression of the chemical or electronic state of living cells. One example of an environmentally sensitive fluorescent indicator is the archrhodopsin protein QuasAr2, which is excited by red light and produces a signal that varies in intensity as a function of the cell membrane potential. QuasAr2 can be introduced into cells using genetic engineering techniques such as gene transfer or electroporation to facilitate optical measurements of the membrane potential.

[0088] The present invention provides a large field of view (FOV) optical microscope 100 that can be used to image tens, hundreds, or even thousands of cells simultaneously. Because so many cells can be imaged at the same time, the optical characterization of cell membrane potentials can be increased by orders of magnitude.

[0089] To realize the full potential of all-optical characterization, microscope 100 may be used to simultaneously achieve a large FOV for measuring interactions between cells in a network or for measuring many cells in parallel for high processing capacity, high spatial resolution for detecting the morphology of individual cells and facilitating selectivity in signal processing, high temporal resolution for distinguishing individual action potentials, and a high signal-to-noise ratio to facilitate accurate data analysis. The FOV is preferably large enough to capture tens or hundreds of cells with a resolution of about 1 or 2 microns. To record rapid changes occurring in electroactive cells such as nerve cells, microscope 101 can provide a very fast image acquisition rate of about 1 kilohertz, which corresponds to a very short exposure time of about 1 millisecond (most fluorescence images are acquired over substantially longer periods). The combination of the above requirements places an extreme demand on the instrument for optically characterizing the dynamic properties of cells. The stage comprises an electrically powered xy translation stage, and a computer instructs the stage to sequentially position each of a plurality of wells across the imaging lens of the microscope and to activate the micromirrors of the DMD according to a spatial mask uniquely created with respect to the cells in that well and stored in a memory system. In a preferred embodiment, the cells include nerve cells, and the step of selectively illuminating the cells in the plurality of wells initiates electroactivation in the illuminated cells. Optionally, the computer records a video for each well using an imaging sensor positioned below the imaging lens and stores the resulting videos in a memory system. Preferably, the microscope includes a light source and an optical system that directs an excitation beam of light into each well from below at an angle that restricts the light to about 10 microns at the bottom of the well. The wells may contain nerve cells that express an optical reporter of electroactivation, and the nerve cells fluoresce when they fire action potentials. The microscope may record a video depicting the fluorescence activity indicating nerve cell firing. Therefore, the cells express fluorescent proteins, and a computer system can, at will, analyze the signals from the fluorescent proteins and automatically create a spatial mask.In one embodiment, a computer system can automatically create spatial masks for labeled cells in each of a group of wells and store these spatial masks in memory, while a microscope uses the DMD and individual spatial masks to sequentially illuminate each well, creating spatially patterned illumination specific to the cells in that well.

[0090] In addition to fluorescent indicators, photosensitive compounds have been developed to chemically or electrically perturb cells. Using photoregulatory activators, stimulation can be applied to the entire sample, a selected region, or individual cells by varying the illumination pattern. One example of a photoregulatory activator is the channelrhodopsin protein CheRiff, which produces a current whose magnitude increases roughly proportionally to the intensity of the blue light shone upon it. In one study, CheRiff produced approximately 22 mW / cm². 2 When illuminated with blue light, all cells expressing the protein generated a current of approximately 1 nA.

[0091] Optically modulated activators, when combined with fluorescent indicators, can enable the total optical characterization of specific cellular characteristics such as excitability. For example, the Optopatch method combines electroactivator proteins such as CheRiff with fluorescent indicators such as QuasAr2. The activator and indicator proteins respond to light of different wavelengths, allowing the membrane potential to be measured simultaneously as the cell is excited over a range of photocurrent magnitudes. Optopatch incorporates the content of U.S. Patents 10,613,079 and 9,594,075 (their contents incorporated by reference for any purpose).

[0092] Measuring the electrical properties of cells is crucial for the study, diagnosis, and treatment of diseases involving electrically active cells, such as cardiac and brain cells (neurons and cardiomyocytes, respectively). Conditions affecting these cells include cardiac disease, atrial fibrillation, amyotrophic lateral sclerosis (ALS), primary ALS, and many others. All-optical measurements offer an attractive alternative to conventional methods such as patch clamps, as they do not require precise micromechanical manipulation or direct contact with cells in the sample. Optical methods are far more suitable for high-processing applications. The dramatic increase in processing capacity offered by all-optical measurements has the potential to revolutionize the study, diagnosis, and treatment of these conditions.

[0093] In various aspects, the present invention generally relates to the characterization of the physical properties of cells using fluorescent indicators and photosensitive activators. Examples of applications of the system include studying the effects of potential drug compounds on cardiomyocytes. For example, a microscope may be used to optically acquire action potential (AP) and calcium transient (CT) waveforms from stem cell-derived cardiomyocytes and to characterize arrhythmias in cardiomyocytes. Cardiomyocytes in a sample may be made to express a rhodopsin-type transmembrane optical reporter. The microscope can activate microbial channel rhodopsin using an activation subsystem. APs propagate through the cardiomyocytes. Cells containing the reporter protein are illuminated via an illumination subsystem, and the APs cause a change in the fluorescence of the reporter. Light from the reporter is detected by an imaging subsystem and analyzed to construct an AP waveform. Arrhythmias in the constructed AP waveform can be detected or characterized, for example, by comparison with a known standard or by other analytical techniques.

[0094] A TIRF microscope can therefore be used to study the effects of compounds on cardiomyocytes. Since the subject area can support cells in culture medium, sample cardiomyocytes can be observed using the microscope while exposed to a compound of interest, such as a candidate drug. The detected AP waveform, i.e., any perturbation resulting from arrhythmias, associated with exposure to the compound, can therefore also be observed by the microscope. Since the optical reporter can include a voltage reporter, an ion reporter (e.g., with respect to [Ca2+]), and others or a combination thereof, the microscope can detect the effects of compounds across multiple ion channels in cardiomyocytes, as revealed through all features of the AP waveform.

[0095] Using the microscope 101, cells 113 or any other suitable sample can be imaged. For example, cells 113 may be nerve cells, cardiomyocytes, or other electroactive cells expressing optogenetic reporters.

[0096] Figure 14A is a perspective view of a useful light source 313 in one embodiment of this disclosure.

[0097] Figure 14B is a top view of the light source 313.

[0098] Figure 14C is a side view of a light source suitable for use in combination with the present invention. The light source may include a diode laser, a diode bar laser, other types of lasers, an LED, or other light sources having suitable characteristics.

[0099] Figure 15 shows a glass-bottom microplate 1501 (e.g., a multiwell plate) receiving spatially patterned light from a mask mounted on a DMD305 onto cells 113 in one well of the plate. The cells emit emitted light 338 toward the imaging sensor. Any suitable well plate may be used. As shown, plate 1501 is a glass-bottom microplate with 96 wells, such as a 96-well plate sold by MilliporeSigma (St. Louis, MO) under the trademark SENSOPLATE. Preferably, plate 1501 has dimensions including a length of 127.76 mm and a width of 85.48 mm. Microplate 1501 may include borosilicate glass (175 μm thick).

[0100] As shown, the sample being imaged emits emitted light 338 that passes toward the imaging sensor 335 (for example, not drawn but through the tubular lens). Due to the dichroic mirror, the sample can be illuminated with spatially patterned light and also illuminated from the side with approximate TIR light that passes through only about 10 microns at the bottom of the sample well (both from beam 302), and can also emit emitted light 338 that is captured by the sensor 335 for recording motion.

[0101] An inverted fluorescence microimaging system optically records a large number (e.g., 50–5,000) expression cells or cell clusters within a single field of view. For example, the system may be used to characterize optically induced firing patterns and AP waveforms in electroactive cells expressing Optopatch components. Each field of view is exposed to blue light in pulses of the entire field of view or spatially localized pulses to induce activity (e.g., repeated every 0.5 seconds, 6 seconds, with 9 intensities of 0–10 mW / cm²). 2 (Increases to a certain point, eliciting neuronal firing). The number of steps and output variables depend on the specific study and the expression levels of the activators. Reporter fluorescence, such as that from QuasAr2, is measured at 640 nm, 100 W / cm². 2This can be monitored simultaneously using full-field excitation. Additional useful discussions of microscopes and imaging systems can be found in U.S. Patent No. 10,288,863 (incorporated by reference).

[0102] The method of the present invention may include a step of exciting the cell to be observed, or a step of activating the cell to initiate an action potential. Activation may be direct or indirect (e.g., optical activation of an optical activator or activation of an upstream cell to gap-junction with or synaptically connect with the cell to be observed). Activation may be optical, electrical, chemical, or by any other preferred method. Activation may involve activation of any pattern, including, for example, regular, periodic pulses, a single pulse, an irregular pattern, or any preferred pattern. The method may include a step of varying the optical activation pattern in space or time to highlight a particular aspect of cellular function. For example, the pulse pattern may have an increasing frequency. In one embodiment, the imaging step includes a step of activating an electroactive cell expressing an optical activator using pulses of light.

[0103] Figure 16 illustrates a system 1601 for the control and use of microscope 101. The instrument 100 includes an optical system 600, which is connected to a computer device 171, either directly or via a network 1643. Optionally, system 1601 may include or access a server computer 1609. System 1601 provides fluorescence microscopy for optogenetics. The system includes microscope 101 and computer 171. Microscope 101 preferably includes a stage and an objective lens, located within an imaging optical path including the subject area. Microscope 101 may also include a digital optical processor (DLP) comprising one or more prisms for total internal reflection microscopy, one or more laser bars for providing light, and a digital micromirror device. Computer device 171 may include a touchscreen configured to display the image captured through the objective lens and to control the pattern of activation light based on user input acquired via the touchscreen. Using system 1601, a user can touch a touchscreen and activate electroactive cells displayed on the touchscreen. Computer 171 and the DLP can modify the configuration of the digital micromirror device in response to the user touching the touchscreen. In addition, or alternatively, the microscope stage can respond to pan or zoom gestures made by the user using the touchscreen. For example, the user can touch the screen to adjust the position of the subject area relative to the objective lens.

[0104] In some respects, this disclosure provides a high-resolution, large-field imaging system. The imaging system may include an objective lens, a tubular lens, an optical filter, a mirror, a focusing mechanism, and other optical elements for forming an image on the image plane. The imaging system may also include an image detector present on the image plane of the imaging system for recording a fluorescence image of a sample.

[0105] Built-in by reference References and citations to other documents, such as patents, patent applications, patent publications, journals, books, articles, and web content, are made throughout this disclosure. All such documents are incorporated herein in their entirety by reference to this specification for any purpose.

[0106] Equal parts In addition to those shown and described herein, various modifications of the invention and many further embodiments will be apparent to those skilled in the art from the entirety of this publication, including references to scientific and patent documents cited herein. The subject matter of this specification contains important information, examples, and guidance that can be adapted to the practice of the invention in its various embodiments and equivalents. [Examples]

[0107] (Example 1) Example 1: Overview This disclosure provides an optogenetic microscope compatible with 384-well plates, streamlining automation and implementing a novel, more sensitive voltage-trace excitation algorithm, enabling a fourfold increase in measurement efficiency. The microscope can automate several processes in cell production, reducing labor and reagent usage and improving reproducibility. Together, these improvements make the microscope a robust and practical tool for screening excitability and synaptic transmission modulators in human iPSC-derived neuronal disease models. The microscope may be used to perform screening in human cell disease models of loss-of-function (LOF) mutations in KCNQ2 and provide data showing strong pharmacological modulation.

[0108] Embodiments of this disclosure provide a custom ultra-wide-field fluorescence microscope (sometimes referred to as "Firefly") for all-optical measurements of cellular electrophysiology. The microscope leverages the Optopatch toolkit (optical stimulation + optical voltage reporting, e.g., CheRiff and QuasAr), which uses engineered proteins to enable simultaneous optical stimulation and recording of neuronal action potentials (APs). The channelrhodopsin CheRiff enables AP stimulation using blue light, and the voltage-sensitive fluorescent protein QuasAr enables high-speed electrical recording using red light. The Firefly microscope routinely performs simultaneous voltage recording from >100 individual neurons over a large (0.5 × 4 mm) field of view (FOV) with a time resolution of 1 ms and a high signal-to-noise ratio (SNR). A digital micromirror device (DMD) projects a fully reproducible optical pattern, allowing individual cells to be stimulated sequentially while recording from many postsynaptic partners. A computer system provides a fully automated analysis, identifying each individual neuron and calculating its voltage trace. In all voltage traces, spikes are detected, and important spike shape and timing parameters are calculated. Since each cell fires many APs, a wealth of information can be extracted to distinguish cell type, cellular state, disease phenotype, and pharmacological response. In addition, electrodeless recording minimizes cell perturbation, allowing recording of the same neuron before and after compound addition. This enables the identification of compound effects on different neuronal subtypes and overcomes the biological "noise" of highly heterogeneous neuronal responses. Beyond the autonomous excitability and firing patterns of cells, it is possible to study synaptic transmission, long-term potentiation / inhibition, and network and circuit behavior.

[0109] Figure 17 is a portion of the computer-aided design (CAD) drawing of the stage of the 96-well microscope of this disclosure.

[0110] Figure 18 shows the optical path for coupling red laser light to a cell sample via a prism for low background voltage imaging, and the blue light path for focal stimulation via a digital micromirror device.

[0111] For robust, high-performance operation, the Firefly system may include software tools, such as automation and control software for applying blue light stimulation, recording high-speed video data, moving between wells, and operating a pipetting robot for automated compound addition. The tools may also include analysis software for extracting voltage-versus-time traces from each neuron in each multi-gigabyte video. The reduced data (voltage traces, identified action potentials, and extracted characteristics) and associated metadata such as cell type, compound, and compound concentration may be stored in a relational database.

[0112] Embodiments of this disclosure provide instruments that enable analytical workflows for phenotypic screening and rich multidimensional assay readout for neuronal excitability in rodent or human-induced pluripotent stem cell (hiPSC)-derived neurons. The methods provide the high reproducibility and stability required for pharmacological measurements and screening. Microscopy is useful for measurements in many cell types and for the detection of multiple disease phenotypes. In addition to neuronal excitability measurements, microscopy is useful for assays relating to synaptic transmission. Synaptic measurements are robust and ready for implementation in multi-well plates. They represent another powerful assay modality relating to the proposed 384-well instrument.

[0113] (Example 2) Example 2: Data Analysis Pipeline Figure 19 shows a Firefly image with an overlay (colored region) of hiPSCs. Firefly images with an overlay (colored region) of motor neurons derived from hiPSCs are identified by automated analysis.

[0114] Figure 20 shows exemplary voltage recordings from derived motor neurons identified by automated analysis. Exemplary voltage recordings from selected cells and the blue stimuli used to induce firing, i.e., steps, pulse trains, and ramps, are shown. Time is used to select cells and the blue stimuli used to induce firing, i.e., steps, pulse trains, and ramps.

[0115] Figure 21 is a raster plot where each point is an identified action potential, and each row is a neuron from a single field of view. The images show the state before (green) and after (orange) the addition of ML213 at 1 μM, a potassium channel opener that reduces the resting potential and suppresses firing.

[0116] Figure 22 shows the averaged spike rate across cells.

[0117] Figure 23 shows the spike shape properties.

[0118] Figure 24 shows the spike timing properties.

[0119] Figure 25 shows the averaged fit across cells.

[0120] Spike shape, spike timing properties, and fit can be automatically extracted on a cell-by-cell basis. Excitability is automatically extracted on a cell-by-cell basis.

[0121] Figure 26 shows the excitability extracted from the step waveform in Figure 22, demonstrating suppression of firing at all stimulus intensities.

[0122] Analysis Overview: Figures 19–26 illustrate the automated analysis workflow for measuring intrinsic excitability and pharmacological responses in hiPSC-derived motor neurons. Neurons are measured for response using a stimulation protocol (blue) designed to examine a wide range of spiking behavior. All pixels capturing fluorescence from a single neuron covariate over time, following the cell's unique firing pattern (Figure 20). Time covariance is used to generate a cell-weighted mask (colored area in Figure 19), and the masked pixels are averaged frame by frame in the video to calculate a voltage trace. Each FOV was recorded twice, before and after the addition of potassium channel opener ML213. The illustrative trace in Figure 20 demonstrates the underlying variability in neuronal behavior, and recordings from many neurons must be averaged to capture the compound effect. From fluorescence-time traces, each action potential in the dataset is identified (Figure 21), and its firing rate (Figure 22), spike shape parameters (Figure 23), and relative timing (Figures 24 and 25) are measured as a function of the stimulus.

[0123] Figure 26 shows a clear reduction in neuronal excitability induced by ML213. All parameters are automatically extracted by parallelized analysis in the cloud, stored in a database, and the figure is automatically generated. Stimulus-dependent extracted values, significantly reduced in number and complexity from raw video data, serve as a basis for more detailed analysis to distinguish cell type, cell state, disease phenotype, and pharmacological response. Approximately 300 parameters can be extracted per cell.

[0124] (Example 3) Example 3: Dimensionality In compound screening, multidimensional data can be reduced to several parameters that can be used to rank and select compounds to pursue. In disease modeling, for example, hiPSC-derived neurons from patients and healthy controls are used to screen for compounds that induce disease cells to behave like healthy cells. Dimensionality reduction is illustrated in Figures 27–29.

[0125] Figure 27 shows a radar plot that enables visualization of disease phenotypes and compound effects. This phenotype is more fully explained by vectors in the 300-dimensional parameter space of the recording.

[0126] Figure 28 shows the compound effects projected along and orthogonal to the phenotypic vector (Figure 5B), providing metrics for phenotypic inversion and unwanted "side effects."

[0127] Figure 29 shows the results from two compounds in the context of such phenotypes. These two values ​​provide far more information when ranking compounds than simple ID effect sizes from most target-based screens.

[0128] Figures 27-29 show multidimensional analysis using iPSC-derived excitatory cortical neurons (NGN2) cultured on day 30.

[0129] Figure 27 is a radar plot showing a selected subset of parameters normalized to values ​​for wild-type (WT) neurons. The data show the behavior of WT neurons, disease model neurons with reliable LOF mutations (knockout / KO), and KO neurons in the presence of the disordered potassium channel blocker 4-AP and the disordered sodium channel blocker lamotrigine. The difference between WT and KO traces is functional phenotype. 4-AP substantially reversed the phenotype, while lamotrigine perturbed the behavior but did not reverse the phenotype.

[0130] Figure 28 is a schematic diagram illustrating phenotypic inversion and "side effects." In the 300-dimensional space of recorded parameters, only two are shown: WT wells are clustered, and KO wells are clustered. The vectors between these groups represent the phenotype (red). The drug effect (blue) is decomposed into components along the phenotypic vector (phenotypic inversion) and components orthogonal to it (side effects). An ideal drug would reverse the effect of the mutation and move the wells from the KO group to the WT group.

[0131] Figure 29 is a plot showing many wells projected onto the phenotype / adverse reaction space. The WT and KO wells are separated along the phenotype direction. The application of the two compounds from Figure 27 (eight concentrations from 0.28 to 600 μM) has an increasing effect on KO cell behavior as the concentration increases. 4-AP shifts cell behavior toward and beyond WT behavior, while lamotrigine shifts behavior toward both WT and KO. The connected drug points are in order of increasing concentration, and the two lines represent experimental reproduction for consecutive experiments over two weeks.

[0132] (Example 4) Example 4: Application The apparatus and methods described herein are useful for demonstrating the effects of compounds on various targets (Figures 30 and 31). Note that the voltage-opening potassium channel Kv7.x blocker XE-991 and Kv7.x opener ML-213 drive behavior in opposite directions, as expected. Concentration response curves are readily measured using the Optopatch platform (Figure 31). All compounds show large effect sizes and good reproducibility on two independent plates. Each symbol represents >100 cells in one well. All data shown were measured on a single day.

[0133] This system is also ready for high-capacity drug screening. To test system uniformity and reproducibility, ML-213 was added to alternating rows in a 96-well plate (Figures 32-37). The change in spike rate between ramps was easily visible in each well and was uniform across the plate (Figures 34-35). Using one parameter, the number of spikes between ramps, a Z' of 0.31 indicates a failed decision in one of the 73,000 wells, which is sufficient for screening tens of thousands of compounds for phenotypic screening in neurons. A slight drift in time was present because the wells were read out continuously at room temperature (Figure 36). Recent improvements in the automated control software have reduced the scan time per FOV from 30 seconds to 10 seconds, which will reduce scan time artifacts. Furthermore, the microscope has a 5x increased imaging area and the number of FOVs per well has been reduced to 1, which will further increase the screening speed.

[0134] Figures 30 and 31 show, for example, the pharmacology of E18 rat hippocampal neurons cultured for 14 days.

[0135] Figure 30 is a radar plot showing drug-induced changes in neuronal spiking behavior along multiple dimensions, tabled on the right. For each parameter, the medium is normalized to 1 (green), and the drug effect is shown in orange.

[0136] Figure 31 shows the concentration response, with two independent titrations plotted (square and circle).

[0137] Figures 32-37 show the verification of high processing capacity for screening with E18 rat hippocampal neurons cultured for 14 days.

[0138] Figure 32 shows high SNR fluorescence voltage recordings of neurons on a 96-well Firefly in medium control wells or in wells with 1 μM ML-213, a Kv7.x agonist that hyperpolarizes cells and reduces firing. Blue light stimulation is shown below.

[0139] Figure 33 is a raster plot showing spikes from the rows of wells. The ML213 dramatically reduces the ignition rate in all wells tested.

[0140] Figure 34 shows the average ignition rate between lamps in each well. The medium wells (green) and ML213 wells (red) are easily distinguishable.

[0141] Figure 35 is a heatmap showing the number of spikes between ramps for each well.

[0142] Figure 36 shows the average number of spikes per cell for the ramp portion of the well-by-well protocol in Figure 35. The calculated Z' of 0.31 is more than good enough to perform phenotypic screening.

[0143] Figure 37 shows sensory neurons in the dorsal root ganglion (DRG) of rats. When treated with a cocktail of inflammatory mediators found in the joints of arthritis patients, the neurons fire more action potentials. A large phenotype can aid in robust screening. The inflammatory mediator cocktail may be a composition such as that described in WO 2018 / 165577 (incorporated by reference).

[0144] (Example 5) Example 5: Phenotype Determination Figures 38–45 show disease-associated phenotypes detected using the Optopatch platform, including monogenic epilepsy data with undisclosed mutations.

[0145] Figure 38 shows that WT cells, which contain CRISPR / Cas9, were used to knock out the gene, and multiple isogenic clones were proliferated and converted into nerve cells.

[0146] Figure 39 shows that a consistent change in spike shape was observed across multiple rounds and KO cell lines. Treatment with clinically effective compounds shifted the behavior back toward the WT state.

[0147] Figure 40 shows similar but less severe phenotypes observed in heterozygous patient cell lines and healthy familial controls.

[0148] Figure 41 is a multidimensional radar plot revealing changes in neuronal morphology, action potential shape, and spike train behavior. Treatment with clinical compounds shifts knockout (KO) toward the waste state (WT) for all metrics.

[0149] Figure 42 shows that dimensionality reduction results in a robust phenotype consistent across all three cell lines. Drug therapy restores the phenotype.

[0150] Figure 43 shows that CRISPR / Cas9 is used to introduce gain-of-function mutations in ion channels. The mutations alter the action potential shape and firing behavior between disease model neurons and their isogenic control groups.

[0151] In addition to testing diverse pharmacological mechanisms, the platform can be applied to many neuronal types related to different disease models. Microscopy may be used to record from many types of human iPSC-derived neurons, including rodent CNS neurons (e.g., Figure 32), rodent DRG sensory neurons (Figure 37), and NGN2 cortical excitatory (Figures 38–42), inhibitory, motor, interval, and dopaminergic neurons. Microscopy may also be used with Optopatch to detect functional phenotypes in several human iPSC-based and rodent neuronal disease models.

[0152] The examples shown in Figures 38–43 include different neurological disease models. This disclosure includes disease models in an isogenic background using CRISPR / Cas9 methods and gene knockout or knock-in with patient-derived neurons. Multiple disease and control strains and our highly sensitive electrophysiological means may be used to identify phenotypes associated with disease states for statistical significance.

[0153] In addition to intrinsic excitability measurements, the Optopatch platform and Firefly instruments can generate clear measurements of synaptic function. The methods may also be used to measure excitatory and inhibitory postsynaptic potentials (EPSPs and IPSPs) in individual cells, information that cannot be obtained using calcium imaging or microelectrode arrays. While not as mature as our excitability measurements, novel analytical tools and assays can be robustly implemented in 96- and 384-well plates with processing power comparable to that of our excitability measurements.

[0154] (Example 6) Example 6: Assay Figures 44–49 show recent results using a high-processing-capacity whole-field stimulation assay. High-processing-capacity screening of synaptic function was implemented using distinctly different populations of neurons, with presynaptic neurons expressing the actuator CheRiff and postsynaptic neurons expressing the voltage sensor QuasAr using a construct into which Cre recombinase and loxP were introduced. All cells express CreOFF-CheRiff (Cre excises CheRiff, turning off its expression) and CreON-QuasAr (Cre inverts QuasAr to a forward orientation, turning on its expression). Cre was added at low titers to transduce a subset of neurons, creating dissimilar populations of neurons expressing either QuasAr or CheRiff. Short pulses of blue light induced action potentials in presynaptic cells, and PSPs were detected in postsynaptic cells. By using an appropriate postsynaptic channel blocker, the inventors can separate excitatory depolarization voltage changes from AMPA channels (Figures 47 and 48, upper) and NMDA channels from inhibitory hyperpolarization voltage changes from GABAA channels (Figures 47 and 50, lower).

[0155] Figure 49 highlights numerous individual cells measured, demonstrating the clear effects of both positive and negative channel modulators. Additional insights can be gained when cell types are identified using fluorescent labeling. Excitatory and inhibitory cells can be distinguished by transducing cells with a lentiviral construct containing GFP driven by an inhibitory promoter, and excitatory and inhibitory subtypes can be identified using the mouse Cre strain. The synaptic assay under development elucidates individual synapses by stimulating a single presynaptic cell with DMD.

[0156] Figures 44-49 show, for example, a high-processing-capacity synaptic assay using E18 rat hippocampal neurons cultured for 14 days.

[0157] Figure 44 shows that CheRiff is expressed in a subset of neurons (presynaptic neurons 4601) (typically 10–50%), while QuasAr is expressed in the remaining portion (typically 50–90%) (postsynaptic neurons 4602).

[0158] Figure 45 is a fluorescence image on Firefly showing QuasAr fused with citrin (green), CheRiff fused with EBFP2 (blue), and TagRFP transported into the nucleus (red), which are used for automated image segmentation.

[0159] Figure 46 shows a single-cell fluorescence trace indicating postsynaptic potentials (PSPs). The inventors independently examined synaptic signals by pharmacologically separating AMPA, NMDA, and GABA.

[0160] Figure 47 shows the modulation of single-cell PSPs in response to control agonists and blockers for AMPAR and GABAAR assays. CheRiff stimulation is shown on the lower side.

[0161] Figure 48 shows the mean PSP traces for control pharmacologies, i.e., black: pre-drug, blue-green: competing blocker [AMPAR: 100 μM NBQX / CNQX, 389 cells; GABAAR: 20 μM gabadin, 176 cells], green: negative allosteric modulator (NAM) [100 μM GYKI 53655, 291 cells; GABAAR: 30 μM picrotoxin, 176 cells], purple: medium control group [AMPAR: 167 cells; GABAAR: 236 cells], and blue, red, and yellow: positive allosteric modulator (PAM) [AMPAR: 0.1-1 μM cyclothiazide, 512 cells; GABAAR: 0.1-1 μM diazepam, 244 cells].

[0162] Figure 49 gives a dot density plot (each dot represents one postsynaptic neuron) showing drug-induced changes in PSP area normalized to the mean pre-drug response. Black whiskers represent mean ± SEM.

[0163] A method is provided for establishing a robust Optopatch assay with practical cost and speed, and for fully validating those assays using a large-scale phenotypic screen. The microscope of this disclosure is useful with 384-well plates. Using the optics, automation, and sCMOS camera of this disclosure, the inventors will also quadruple their reading speed, maintain a nearly constant plate scanning time, and quadruple their processing capacity. The larger imaging area will capture all neurons in the wells, allowing for highly efficient utilization of these beneficial reagents. The method of this disclosure provides a phenotypic screen.

[0164] This disclosure provides a 384-well optogenetic microscope. Converting from a 96-well plate to a 384-well plate offers a fourfold reduction in cost. The method may be used to record a 4mm wide × 0.5mm high FOV at a 1kHz frame rate, and increasing the height reduces the frame rate with respect to the Hamamatsu ORCA-Flash scientific CMOS (sCMOS) camera. Transitioning to 3.3mm wide wells on a 384-well plate allows 82% of the FOV to be used, moderately reducing the number of cells recorded per minute. The sCMOS camera by Hamamatsu uses chip architecture and data transfer technology to achieve significantly faster readout speeds. Combined with modifications to the imaging optics and upgrades to more powerful excitation lasers, the instrument of this disclosure may be capable of imaging the entire 3.3 × 3.3 mm area of ​​a 384-well plate at frame rates faster than 500 Hz, which is a fivefold increase in recording area. These methods, using Optopatch, make it possible to image all active neurons in each well of a 384-well plate in a single video (with the potential exception of cells around the periphery of the wells), which may represent an unprecedented processing capability for single-cell electrophysiological recordings.

[0165] (Example 7) Example 7: Apparatus Features The microscope may be switched to a 2.7x to 2x magnification tube lens to maximize the number of neurons that can be recorded simultaneously. 2x magnification is sufficient to capture the full 3.3 mm height of the well at 500 Hz. Despite the reduced magnification, the new microscope will have better resolution. Combined with the reduced pixel size, the resolution will be improved from 4.8 μm to 2.8 μm. The Vialux V-9601 DMD provides a fully synchronized 100 μs pattern refresh for fast single-cell stimulation to measure individual synaptic connections or for slightly delayed pulses on connected neurons to examine spike timing-dependent plasticity. Custom prisms for coupling a red laser to the sample may be designed to fit smaller well sizes. Custom 384-well plates, fabricated entirely from cyclic olefin copolymer (COC), may be used. COC is a plastic designed for UV spectrophotometric applications with ultra-low laser absorption and autofluorescence. Like conventional polystyrene culture substrates, COC can be treated with oxygen plasma for tissue culture (TC) and supports healthy and active nerve cell culture. COC essentially eliminates background fluorescence and minimizes laser heating of the substrate. Data was recorded on a COC 96-well plate. Control software may be included to handle the new hardware (camera and DMD) and minimize dead time when scanning a 384-well plate. The new DMD has a display pattern that can be updated using a digital trigger synchronized with the camera frame. The new software also enables multithreading (loading the tip, loading the drug, and moving it to the plate) for controlling the pipetting robot during the performance of other microscopy tasks, so that the microscope can record high-speed video almost continuously. To enable easy multithreading, the control software may be designed in C# or an analogue.

[0166] (Example 8) Example 8: High-capacity screening Plate production may be automated for assays used to identify disease-associated phenotypes and optimize HTS. Heatmap analysis will be used to characterize intra-plate and inter-plate variability, and variations in cell plating and handling, stimulation protocols, and assay duration will be tested to achieve intra-plate and inter-plate variability <20% while maintaining a Z' value >0.3 as described. DMSO tolerance will be defined using concentration-response experiments to identify DMSO levels that produce a <10% change in assay window size compared to a buffer control value. Following confirmation of assay preparation, a small set of five screening plates will be randomly selected from the library to guide the selection of final screening concentrations. Plates of these compounds will be double-screened at 1, 3, 7, and 10 microM. The inventors select compound concentrations that yield a hit compound rate of approximately 1%, and a hit compound will be defined as a change greater than 3 standard deviations (SD) from the control value. Using these concentrations, the inventors aim to capture a number of true hit compounds with some false positives.

[0167] The FDA-approved drug library and tool compound pilot screen may utilize a library of approximately 2,400 drugs approved worldwide. This library is a screening and selection of available tool compounds at chosen screening concentrations. This step serves as the final test for assay preparation for HTS and will provide a dataset for establishing hit compound selection criteria, as this library is likely to contain active compounds. The compound library will be prepared in barcoded 384-well plates in 100% DMSO.

[0168] The method involves the production and banking of reagents for HTS. To ensure homogeneous cell preparation, large batches of 300 million iPSC-derived NGN2 neurons, 100 million primary rodent glial cells, and lentiviruses encoding Optopatch components can be generated, divided, and frozen, each sufficient for 1.5 screenings. Automated cell culture processes will be applied throughout the HTS activity to improve efficiency and homogeneity.

[0169] The method may include HTS screening and hit compound confirmation. Compounds will be screened in a 384-well format (n=1) at selected screening concentrations, with 32 wells in each plate reserved for the control group. The scanning time per plate will depend on the assay protocol and is expected to be approximately 90 minutes, which will allow screening of >5,000 compounds per week on a single Firefly instrument with 3 days of screening per week. Plates with excessive variability (Z'<0.3), few active cells, or heterogeneous plating will be flagged for repetition. Hit compound selection and confirmation will be performed following HTS.

[0170] Figure 50 shows a method for high-performance screening.

[0171] Hit compounds will initially be selected based on the reversal of multi-parameter phenotypic scores and adverse event scores. Based on statistical criteria, hit compounds will be defined as compounds exhibiting a >3 SD change from the in-plate control value.

[0172] The activity of up to 200 selected hit compounds is first double-confirmed at 1x and 0.3x screening concentrations, with the two concentrations helping to identify compounds with non-monotonic concentration responses. Confirmed hit compounds will be tested in 11-point concentration responses to quantitatively characterize phenotypic reversal and side effects. The results will confirm platform performance and serve as the basis for hit-to-lead compound activity through grants or collaborations with pharmaceutical partners. The apparatus and methods of this disclosure provide rapid and affordable phenotypic identification and phenotypic drug screening.

Claims

1. It is a microscope, A stage configured to hold a multiwell plate, A light source for emitting a beam of light, which is mounted inside the microscope, An optical system for directing the beam from below toward the stage, wherein the optical system comprises a homogenizer for spatially homogenizing the beam. A microscope comprising, wherein the homogenizer comprises at least two microlens arrays.

2. The microscope according to claim 1, wherein the stage comprises an electrically powered xy parallel movement stage.

3. The microscope according to claim 2, further comprising a control system having a memory connected to a processor capable of moving the translation stage and positioning the individual wells of the multiwell plate within the beam path.

4. The microscope according to claim 1, wherein the optical system includes a prism directly below the stage, so that the beam is incident on the side of the prism and passes through the well of the plate.

5. The microscope according to claim 4, wherein when the wells of the plate containing the aqueous sample are positioned above the prism, the prism directs the beam into the sample at an angle that avoids all internal reflections within the bottom of the plate.

6. The microscope according to claim 4, wherein when the wells of the plate containing the aqueous sample are positioned above the prism, the prism directs the beam into the aqueous sample at an angle of refraction that restricts the light to the bottom of the wells to about 10 microns.

7. The microscope according to claim 1, comprising at least three light sources for emitting three beams at three distinctly different wavelengths, wherein the optical system comprises one or more dichroic mirrors for merging the three beams in space and passing the three beams through the homogenizer.

8. The microscope according to claim 1, wherein the homogenizer forms the beam into substantially uniform illumination of a rectangular region.

9. The microscope according to claim 1, wherein the optical system comprises an opaque screen having a plurality of apertures, the screen being positioned so that the beam passes through one of the apertures.

10. The microscope according to claim 9, wherein the homogenizer comprises two microlens arrays, and the optical system comprises a plurality of microlens array position stoppers at predetermined intervals, thereby fixing the distance between the two microlens arrays, and thereby shaping the beam to match each of the apertures.

11. The microscope according to claim 1, further comprising a stimulating light source that emits a stimulating beam, wherein the optical system comprises a digital micromirror device (DMD), the stimulating beam being reflected from the DMD and illuminating the bottom of the wells of the plate using a pattern defined by the DMD.

12. The microscope according to claim 11, wherein the beam is at the excitation wavelength of the phosphor, and the stimulating beam is at a second wavelength.

13. The microscope according to claim 1, further comprising an imaging lens below the stage for directing light from a sample in the wells of the plate onto an image sensor mounted in the microscope.

14. The microscope according to claim 13, wherein the optical system includes a prism directly below the stage, thereby causing the beam to be incident on the side of the prism, and the prism directs the beam into the aqueous sample in the wells of the plate at an angle of refraction that restricts the light to the bottom of the wells at approximately 10 microns; the microscope further comprises a stimulating light source that emits a stimulating beam; and the optical system comprises a digital micromirror device (DMD), the stimulating beam being reflected from the DMD, and illuminating the bottom of the wells of the plate using a pattern defined by the DMD.

15. A method for imaging a sample, wherein the method is The method involves positioning a multiwell plate on a microscope stage, wherein the microscope includes an optical system comprising a stage configured to hold the multiwell plate, a light source for emitting a beam of light, and a homogenizer having at least two microlens arrays, and the plate has at least one viable cell on the bottom surface of each well. To obtain an image of the aforementioned cells, The process involves processing the aforementioned image to create a spatial mask that identifies the area of ​​the base occupied by the cells and the area not occupied by the cells, Using the aforementioned spatial mask, the micromirrors of the digital micromirror device (DMD) are selectively activated relative to the cells. By irradiating the DMD with light, the light is specifically reflected onto the area of ​​the bottom surface occupied by the cells, while no light is reflected onto the area not occupied by the cells. Methods that include...

16. Creating a spatial mask for the cells in each of the multiple wells of the aforementioned multiwell plate, The spatial mask is stored in memory, Using the aforementioned spatial mask and DMD, selective illumination of the cells in the plurality of wells in a continuous manner. The method according to claim 15, further comprising:

17. The method according to claim 16, wherein the DMD is controlled by a computer comprising a process coupled to a non-transient memory system, the memory system having the spatial mask stored therein.

18. The method according to claim 15, wherein the light is a stimulating light at a wavelength that excites the phosphor in the cell.

19. The method according to claim 15, wherein the light is activation light at a wavelength that activates the photo-aperture ion channels in the cell.

20. The method according to claim 19, wherein the light-aperture ion channel is located in a presynaptic neuron connected to a non-selective cell via a synapse.

21. The method according to claim 20, wherein the non-selected cells are equipped with an optical reporter for synaptic activity.

22. The method according to claim 17, wherein the stage comprises an electric xy translation stage, and the computer instructs the stage to sequentially position each of the plurality of wells across the imaging lens of the microscope and to activate the micromirrors of the DMD according to a spatial mask that is uniquely created with respect to the cells in the well and stored in the memory system.

23. The method according to claim 22, wherein the cells include nerve cells, and selective illumination of the cells in the plurality of wells initiates electroactivation in the illuminated cells.

24. The method according to claim 23, wherein the computer uses an imaging sensor positioned below the imaging lens to record a video for each well and stores the resulting plurality of videos in the memory system.

25. The method according to claim 24, wherein the microscope includes a light source and an optical system for inducing an excitation beam of light into each well from below at an angle that restricts the light to the bottom of the well by about 10 microns.

26. The method according to claim 25, wherein the well contains nerve cells expressing an electroactive optical reporter, and the nerve cells emit fluorescence when they fire an action potential.

27. The method according to claim 26, wherein the video depicts the fluorescent activity indicating the firing of the nerve cells.

28. The method according to claim 14, wherein the cells express a fluorescent protein, and a computer system analyzes the signal from the fluorescent protein and automatically creates the spatial mask.

29. The method according to claim 28, wherein the computer system can automatically create a spatial mask relating to the labeled cells in each of the multiple wells and store the spatial mask in memory, while the microscope uses the DMD and the individual spatial masks to illuminate each well sequentially and create spatially patterned illumination specific to the cells in that well.

30. A method for imaging a sample, wherein the method is The method involves positioning a multiwell plate on a microscope stage, wherein the plate has at least one cell surviving on the bottom surface of each well, and the microscope includes a stage configured to hold the multiwell plate, a light source for emitting a beam of light, an optical system comprising a homogenizer with at least two microlens arrays, and an autofocus system. Acquiring images of the multi-well plate from multiple fields of view (FOV), Using the aforementioned autofocus system, the position of each field of view is determined, Using a subset of the FOV, generate a map of the curvature of the well, By interpolating the position of the FOV between subsets of the FOV, measurements can be obtained from the cells surviving at the bottom of the well. Methods that include...

31. The method according to claim 30, wherein the autofocus system is a laser-based autofocus system.

32. The method according to claim 31, further comprising moving the microscope stage to a first position, irradiating the well with a laser beam using the laser of the laser-based autofocus system, and determining the position of the field of view (FOV) based on the displacement of the laser beam reflected from the sample using the autofocus system.

33. The method according to claim 32, wherein the well containing the sample is moved and the laser beam is detected for each FOV.

34. The method according to claim 30, wherein the autofocus system is an image-based autofocus system.

35. The method according to claim 34, further comprising: recording the X,Y coordinates of n wells traversing the microplate from a subset of the FOV; measuring an image quality metric in the n wells across a series of Z steps; and finding the Z step that provides the highest image quality metric across the n wells.

Images