Assembly for mounting optical filters

Abstract

There is provided an assembly for mounting a plurality of optical filters, the assembly comprising: a frame comprising a first portion for securing a first optical filter and a second portion for securing a second optical filter; a first shaft connected to the frame, wherein the first shaft is configured to receive a torque from a motor and rotate the frame about a rotation axis of the first shaft; an alignment member coupled to the first shaft, wherein the alignment member comprises at least one alignment surface configured to align with at least one reference surface of an optical bench; and a clamping member configured to clamp the alignment member against the at least one reference surface. There is also provided a system comprising the assembly, a method for mounting the assembly onto an optical bench, and a method of using the assembly

Description

100-182300WC)ASSEMBLY FOR MOUNTING OPTICAL FILTERSCROSS-REFERENCE TO RELATED APPLICATIONS.

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 723,065, filed November 20, 2024, which is incorporated by reference herein in its entirety, and is hereby expressly made a part of this specification.FIELD

[0002] The disclosure relates to an assembly for mounting a plurality of optical filters, a system comprising the assembly, and methods of using the assembly and system to control the alignment of the optical filters in an optics module such as a microscope.BACKGROUND

[0003] In situ detection and analysis methods are emerging from the rapidly developing field of spatial transcriptomics. The key objectives in spatial transcriptomics are to detect, quantify, and map gene activity to specific regions in a tissue sample at cellular or sub-cellular resolution. These techniques allow one to study the subcellular distribution of gene activity (as evidenced, e.g., by expressed gene transcripts), and have the potential to provide crucial insights in the fields of developmental biology, oncology, immunology, histology, etc.

[0004] Fluorescence microscopes are widely used tools that illuminate fluorescently-tagged or stained targets within a sample to image those targets with the sample. In fluorescence microscopy, fluorophores are excited by excitation light (also referred to herein as illumination light) having a fluorophore-dependent excitation spectrum and then emit a fluorescence emission light having a fluorophore-dependent emission spectrum. Images of the fluorescence can be detected by a camera. Fluorescence microscopes are particularly useful in biological fields because they allow researchers to collect high-resolution images without damaging sensitive samples.

[0005] Epifluorescence microscopy, in which both the excitation light and the emission light travels through the same light path (e.g., through the same objective lens), is one implementation of a microscope used for fluorescence imaging. Transillumination microscopy, in which the excitation light illuminates the sample from the opposite side of the objective lens, is another implementation of a microscope used for fluorescence imaging.

[0006] Some fluorescence microscopes are designed to illuminate, and thereby excite, multiple fluorophores using two or more different wavelengths (or wavelength spectra) of illumination10XG / 1823PC 1light. In this case, each fluorophore respectively requires excitation with fluorescence excitation light of a different fluorophore-dependent excitation wavelength or spectrum. In such systems, components that are wavelength-specific, e.g., optical filters, are swapped in and out of the illumination pathway so that the right filter is present for the illumination wavelength being used at a given time. For example, a first optical filter is required for illuminating with a first wavelength and transmitting the corresponding returned excitation light and a second optical filter is required illuminating with a second wavelength and transmitting the corresponding excitation light. However, introducing replaceable or moveable parts will introduce alignment errors between optical components, or otherwise require lengthy calibration steps between illumination with different wavelengths causing reduced throughput.SUMMARY

[0007] One or more aspects of an invention are set out in the claims. In accordance with the appended independent claims, there is provided an assembly for mounting a plurality of optical filters, a system comprising the assembly, a method for mounting the assembly onto an optical bench, and a method of using the assembly.BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Following drawings are appended to facilitate the understanding of the invention. The drawings show embodiments, which will now be described by way of example only, where:

[0009] FIG. 1 depicts an overview of a volumetric sample imaging system and illustrates a Field of View (FOV) grid bounding the sample (e.g., hydrogel, tissue section, one or more cells, etc.) as projected onto the surface of a solid substrate supporting the sample.

[0010] FIG. 2 depicts the XZ cross-sectional view and illustrates tissue non-uniformity in the Z dimension, where the full (non-reduced) imaging volume is oversampled in the Z dimension.

[0011] FIG. 3 is an example workflow of analysis of a biological sample (e.g., a cell or tissue sample) using an opto-fluidic instrument, according to various embodiments.

[0012] FIGS. 4A-4B illustrate cross-sectional views of an optics module in an imaging system.

[0013] FIG. 5 depicts a computing node according to some embodiments disclosed herein.

[0014] FIG. 6A depicts an example of a microscope including a single-source illumination device.10XG / 1823PC 2

[0015] FIG. 6B depicts an example of a microscope including a dual-source illumination device according to embodiments.

[0016] FIG. 7 depicts an assembly for mounting a plurality of optical filters.

[0017] FIG. 8 depicts an assembly for mounting a plurality of optical filters mounted onto an optical bench.

[0018] FIG. 9 depicts an isometric view of an assembly for mounting a plurality of optical filters.

[0019] FIG. 10A depicts a side view of a portion of an assembly for mounting a plurality of optical filters.

[0020] FIG. 10B depicts an isometric view of an assembly for mounting a plurality of optical filters.

[0021] FIG. 11 depicts an optical system including an assembly for mounting a plurality of optical filters.

[0022] FIG. 12 depicts a portion of an assembly for mounting a plurality of optical filters.

[0023] FIG. 13A depicts a front view of a frame for securing two optical filters according to a first example.

[0024] FIG. 13B depicts a rear view of the frame in FIG. 13A.

[0025] FIG. 14A depicts a front view of a frame for securing two optical filters according to a second example.

[0026] FIG. 14B depicts a rear view of the frame in FIG. 14A.

[0027] FIG. 15 depicts a method for mounting an assembly onto an optical bench.

[0028] FIG. 16 depicts a method of using an assembly.

[0029] FIG. 17 depicts assembly rotational positions relative to active illumination color channel for a five-color channel illumination system.

[0030] FIGS. 18A-18B depict a clamping member according to a first example.

[0031] FIGS. 19A-19B depict a clamping member according to a second example. FIGS. 19C-19D depict the clamping member according to the second example within an assembly.

[0032] FIG. 20A depicts a clamping member according to a third example. FIGS. 20B-20E depict the clamping member according to the third example within an assembly.10XG / 1823PC 3

[0033] FIGS. 21A-21C depict a clamping member according to a fourth example.

[0034] In the figures, elements and steps having the same or similar reference numeral have the same or similar attributes or description, unless explicitly stated otherwise.

[0035] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.DETAILED DESCRIPTION

[0036] The following overview is provided to introduce in simplified form a selection of concepts that are further described herein. The overview is not intended to identify only key or essential features of the invention.

[0037] In brief, the present disclosure relates to an assembly for mounting a plurality of optical filters onto an optical bench or other component. The plurality of optical filters are mounted onto a frame that is rotated about a rotational axis defined by a first shaft. The first shaft is aligned directly to an optical bench or other component thereby also aligning the optical filters relative to a predetermined optical axis. Directly aligning the first shaft with a highly accurate reference surface provides accurate angular alignment of the optical filters relative to the rest of the optical system. For example, the optical bench may include one or more principal reference surfaces configured to receive an optical component, e.g., a tube lens, and define the optical axis to which all other optical components (including the first shaft and optical filters) will be aligned. Accurate alignment of optical filters is particularly important in optical systems where illumination light is reflected by optical filters, e.g., dichroic filters used in epifluorescence microscopy, in the infinity space (and towards the objective lens) because any angular error in filter alignment will result in double the angular error in the reflected illumination beam. The alignment of the first shaft to the rest of the optical system e.g., to an optical axis defined by another optical component, such as a tube lens) is provided by an alignment member coupled to the first shaft and a clamping member that is configured to clamp the alignment member against at least one reference surface of the optical system (e.g., one or more machined surfaces in an optical bench ). By aligning the first shaft directly with the optical bench, the entire filter assembly does not require precision tolerances on, for example, a motor housing and / or a motor shaft configured to rotate the frame (and optical filters). The direct alignment allows for a simplified alignment process where the tolerances of the motor (e.g., flatness of the motor face, alignment of the motor shaft, etc.) can be effectively ignored10XG / 1823PC 4by removing the tolerance stack-up between the motor housing and the first shaft. For example, error in the flatness of the motor housing may stack with error in the rotational alignment of the motor shaft, and the combined mechanical alignment error between these components causes alignment error of the rotating dichroic filters. The alignment member, e.g., a cylindrical bearing, and reference surface (or surfaces) on an optical bench can be manufactured with high precision (e.g., by machining surfaces without ever removing the part from the machining tool), providing a high overall precision for the filter alignment in the optical system. As the reference surface on the optical bench can be machined in the same machining process as reference positions for other components in the optical system, a precise alignment between the reference surface and other components (e.g., a tube lens, fold mirrors, field lens, illumination assembly, etc.) can be achieved. For example, the reference surface can be made using the same machining tool and without moving the part in the machine tool securement (e.g., vice), providing a precise angle between the reference surface all of the other alignment features in the optical bench, such as the alignment features for the tube lens, fold mirrors, field lens, illumination assembly, etc.

[0038] In view of the above, described herein are methods to align optical filters within an optical system that is precise, fast, reliable, and repeatable, even when switching between modular assemblies (e.g., for servicing purposes).

[0039] In the following, embodiments will be discussed in more detail with reference to the appended drawings. It should be understood, however, that the drawings are not intended to limit the present disclosure to the subject-matter depicted in the drawings. The embodiments described with reference to the drawings can be understood in isolation from, as well as in the context of, the concepts set out in the claims, summary and / or overview of the present disclosure.

[0040] In volumetric sample imaging systems (e.g., an optofluidic instrument), a z-stack of images is obtained for each Field of View (FOV) of the objective (FIG. 1). The objective lens focal point is positioned to acquire an image at every Z-slice in a Z-stack (FIG. 2). An XZ image of signal distribution (bottom of FIG. 2) demonstrates a non-uniform distribution of detected signal within the imaging volume. For such automated, high-throughput tissue imaging applications, automatically identifying relevant regions - those regions that contain target molecules such as nucleic acids or proteins - can be challenging as distribution of tissue is non-uniform in many biological samples (FIG. 2). FIG. 2 depicts the XZ cross-sectional view and illustrates tissue non-uniformity in the Z dimension in a tissue section 306, where the10XG / 1823PC 5100-182300WC) full (non-reduced) imaging volume 301 is oversampled in the Z dimension. The objective lens focal point 302 is positioned to acquire an image at every Z-slice 303 in a Z-stack 304. An XZ image of signal distribution 305 (bottom) demonstrates a non-uniform distribution of detected signal within the imaging volume. The data extracted from the detection and analysis methods disclosed herein (e.g., in situ detection and analysis of target analytes, such as SBS, SBL, SBH; and in situ hybridization techniques, such as smFISH and MERFISH) include the relative coordinates within a field of view (FOV) and provides intricate information regarding tissue organization.

[0041] In general, the systems and methods described herein use any suitable method to generate contrast of a sample against a background (e.g., illumination of a sample via bright field imaging, illumination of a sample via fluorescent imaging, inducing autofluorescence within the sample, adding contrast to the sample with one or more stains, etc.)

[0042] FIG. 3 shows an example workflow of analysis of a biological sample 110 (e.g., cell or tissue sample) using an opto-fluidic instrument 120, according to various embodiments. In various embodiments, the sample 110 can be a biological sample (e.g., a tissue) that includes molecules such as DNA, RNA, proteins, antibodies, etc. For example, the sample 110 can be a sectioned tissue that is treated to access the RNA thereof for labelling with circularizable DNA probes. Ligation of the probes may generate a circular DNA probe which can be enzymatically amplified and bound with fluorescent oligonucleotides, which can create bright signal that is convenient to image and has a high signal-to-noise ratio.

[0043] In various embodiments, the sample 110 may be placed in the opto-fluidic instrument 120 for analysis and detection of the molecules in the sample 110. In various embodiments, the opto-fluidic instrument 120 can be a system configured to facilitate the experimental conditions conducive for the detection of the target molecules. For example, the opto-fluidic instrument 120 can include a fluidics module 140, an optics module 150, a sample module 160, and an ancillary module 170, and these modules may be operated by a system controller 130 to create the experimental conditions for the probing of the molecules in the sample 110 by selected probes e.g., circularizable DNA probes), as well as to facilitate the imaging of the probed sample (e.g., by an imaging system of the optics module 150). In various embodiments, the various modules of the opto-fluidic instrument 120 may be separate components in communication with each other, or at least some of them may be integrated together.10XG / 1823PC 6

[0044] In various embodiments, the sample module 160 may be configured to receive the sample 110 into the opto-fluidic instrument 120. For instance, the sample module 160 may include a sample interface module (SIM) that is configured to receive a sample device (e.g., cassette) onto which the sample 110 can be deposited. That is, the sample 110 may be placed in the opto-fluidic instrument 120 by depositing the sample 110 (e.g., the sectioned tissue) on a sample device that is then inserted into the SIM of the sample module 160. In some instances, the sample module 160 may also include an X-Y stage onto which the SIM is mounted. The X-Y stage may be configured to move the SIM mounted thereon (e.g., and as such the sample device containing the sample 110 inserted therein) in perpendicular directions along the two- dimensional (2D) plane of the opto-fluidic instrument 120.

[0045] The experimental conditions that are conducive for the detection of the molecules in the sample 110 may depend on the target molecule detection technique that is employed by the opto-fluidic instrument 120. For example, in various embodiments, the opto-fluidic instrument 120 can be a system that is configured to detect molecules in the sample 110 via hybridization of probes. In such cases, the experimental conditions can include molecule hybridization conditions that result in the intensity of hybridization of the target molecule (e.g., nucleic acid) to a probe (e.g., oligonucleotide) being significantly higher when the probe sequence is complementary to the target molecule than when there is a single-base mismatch. The hybridization conditions include the preparation of the sample 110 using reagents such as washing / stripping reagents, hybridizing reagents, etc., and such reagents may be provided by the fluidics module 140.

[0046] In various embodiments, the fluidics module 140 may include one or more components that may be used for storing the reagents, as well as for transporting said reagents to and from the sample device containing the sample 110. For example, the fluidics module 140 may include reservoirs configured to store the reagents, as well as a waste container configured for collecting the reagents (e.g., and other waste) after use by the opto-fluidic instrument 120 to analyze and detect the molecules of the sample 110. Further, the fluidics module 140 may also include pumps, tubes, pipettes, etc., that are configured to facilitate the transport of the reagent to the sample device (e.g., and as such the sample 110). For instance, the fluidics module 140 may include pumps (“reagent pumps”) that are configured to pump washing / stripping reagents to the sample device for use in washing / stripping the sample 110 (e.g., as well as other washing functions such as washing an objective lens of the imaging system of the optics module 150).10XG / 1823PC 7100-182300WC)

[0047] In various embodiments, the ancillary module 170 can be a cooling system of the optofluidic instrument 120, and the cooling system may include a network of coolant-carrying tubes that are configured to transport coolants to various modules of the opto-fluidic instrument 120 for regulating the temperatures thereof. In such cases, the fluidics module 140 may include coolant reservoirs for storing the coolants and pumps (e.g., “coolant pumps”) for generating a pressure differential, thereby forcing the coolants to flow from the reservoirs to the various modules of the opto-fluidic instrument 120 via the coolant-carrying tubes. In some instances, the fluidics module 140 may include returning coolant reservoirs that may be configured to receive and store returning coolants, i.e., heated coolants flowing back into the returning coolant reservoirs after absorbing heat discharged by the various modules of the opto-fluidic instrument 120. In such cases, the fluidics module 140 may also include cooling fans that are configured to force air (e.g., cool and / or ambient air) into the returning coolant reservoirs to cool the heated coolants stored therein. In some instances, the fluidics module 140 may also include cooling fans that are configured to force air directly into a component of the opto- fluidic instrument 120 so as to cool said component. For example, the fluidics module 140 may include cooling fans that are configured to direct cool or ambient air into the system controller 130 to cool the same.

[0048] As discussed above, the opto-fluidic instrument 120 may include an optics module 150 which include the various optical components of the opto-fluidic instrument 120, such as but not limited to a camera, an illumination module (e.g. any of the illumination devices described in the present disclosure including two or more light sources), filters, an objective lens, and / or the like. The optics module 150 may include a fluorescence imaging system that is configured to image the fluorescence emitted by the probes (e.g., oligonucleotides) in the sample 110 after the probes are excited by light from the illumination module of the optics module 150.

[0049] In some instances, the optics module 150 may also include an optical frame onto which the camera, the illumination module, and / or the X-Y stage of the sample module 160 may be mounted.

[0050] In various embodiments, the system controller 130 may be configured to control the operations of the opto-fluidic instrument 120 (e.g., and the operations of one or more modules thereof). In some instances, the system controller 130 may take various forms, including a processor, a single computer (or computer system), or multiple computers in communication with each other. In various embodiments, the system controller 130 may be communicatively coupled with data storage, set of input devices, display system, or a combination thereof. In10XG / 1823PC 8100-182300WC) some cases, some or all of these components may be considered to be part of or otherwise integrated with the system controller 130, may be separate components in communication with each other, or may be integrated together. In other examples, the system controller 130 can be, or may be in communication with, a cloud computing platform.

[0051] In various embodiments, the opto-fluidic instrument 120 may analyze the sample 110 and may generate the output 190 that includes indications of the presence of the target molecules in the sample 110. For instance, with respect to the example embodiment discussed above where the opto-fluidic instrument 120 employs a hybridization technique for detecting molecules, the opto-fluidic instrument 120 may cause the sample 110 to undergo successive rounds of fluorescent probe hybridization (using two or more sets of fluorescent probes, where each set of fluorescent probes is excited by a different color channel) and be imaged to detect target molecules in the probed sample 110. In such cases, the output 190 may include optical signatures (e.g., a codeword) specific to each gene, which allow the identification of the target molecules.

[0052] In some instances, an assembly for transilluminating a substrate can include a sample carrier device (e.g., a microfluidic chip or glass slide), a thermal control module configured to control the temperature of the sample carrier device (e.g., a thermoelectric module), and a light source configured to illuminate the sample carrier device. In some instances, the assembly includes a heat exchanger (e.g., a fluid block having a cooling fluid flowing therethrough). In some instances, an assembly for transilluminating can include sample carrier device (e.g., a sample substrate), an optically transparent substrate, a light source configured to illuminate the optically transparent substrate, a light scattering layer configured to scatter light from the light source, and / or a thermal control module configured to control the temperature of the sample carrier device and / or optically transparent substrate.

[0053] In some embodiments, the sample carrier device (e.g., a cassette) can be configured to receive a sample. In some embodiments, the sample carrier device can include one or more microfluidic channels, e.g., sample chambers or microfluidic channels etched into a planar substrate or chambers within a flow cell or microfluidic device.

[0054] A sample carrier device for the systems disclosed herein can include, but is not limited to, a substrate configured to receive a sample, a microscope slide and / or an adapter configured to mount microscope slides (with or without coverslips) on a microscope stage or automated stage (e.g., an automated translation or rotational stage), a substrate, and / or an adapter10XG / 1823PC 9configured to mount slides on a microscope stage or automated stage, a substrate comprising etched sample containment chambers (e.g., chambers open to the environment) and / or an adapter configured to mount such substrates on a microscope stage or automated stage, a flow cell and / or an adapter configured to mount flow cells on a microscope stage or automated stage, or a microfluidic device and / or an adapter configured to mount microfluidic devices on a microscope stage or automated stage. In some embodiments, the sample carrier device further includes a cassette configured to secure a substrate (e.g., a glass slide). In some embodiments, the cassette includes two or more components (e.g., a top half and a bottom half) into which the substrate is secured.

[0055] In some instances, the one or more sample carrier devices can be designed for performing a variety of chemical analysis, biochemical analysis, nucleic acid analysis, cell analysis, or tissue analysis applications. In some instances, for example, the sample carrier device (e.g., flow cells and microfluidic devices) may comprise a sample, e.g., a tissue sample. In some instances, the sample carrier device (e.g., flow cells and microfluidic devices) may comprise a sample, e.g., a tissue sample, placed in contact with, e.g., a substrate (e.g., a surface of the flow cell or microfluidic device).

[0056] The sample carrier devices for the disclosed systems (e.g., microscope slides, substrates comprising one or more etched microfluidic channel, flow cells or microfluidic devices comprising one or more microfluidic channels, etc.) can be fabricated from any of a variety of materials known to those of skill in the art including, but not limited to, glass (e.g., borosilicate glass, soda lime glass, etc.), fused silica (quartz), silicon, polymer (e.g., polystyrene (PS), macroporous polystyrene (MPPS), polymethylmethacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high density polyethylene (HDPE), cyclic olefin polymers (COP), cyclic olefin copolymers (COC), polyethylene terephthalate (PET), polydimethylsiloxane (PDMS), etc.), polyetherimide (PEI) and perfluoroelastomer (FFKM) as more chemically inert alternatives, or any combination thereof. FFKM is also known as Kalrez.

[0057] The one or more materials used to fabricate sample carrier devices for the disclosed systems (e.g., substrates configured to receive a sample, microscope slides, substrates comprising one or more etched microfluidic channels, flow cells or microfluidic devices comprising one or more microfluidic channels or sample chambers, etc.) can be optically transparent to facilitate use with spectroscopic or imaging-based detection techniques. In some instances, the entire sample carrier device can be optically transparent. Alternatively, in some10XG / 1823PC 10instances, only a portion of the sample carrier device (e.g., an optically transparent “window”) can be optically transparent.

[0058] The sample carrier devices for the disclosed systems (e.g., substrates configured to receive a sample, microscope slides, substrates comprising one or more etched microfluidic channels, flow cells or microfluidic devices comprising one or more microfluidic channels or sample chambers, etc.) can be fabricated using any of a variety of techniques known to those of skill in the art, where the choice of fabrication technique is often dependent on the choice of material used, and vice versa. Examples of suitable sample carrier device fabrication techniques include, but are not limited to, extrusion, drawing, precision computer numerical control (CNC) machining and boring, laser photoablation, photolithography in combination with wet chemical etching, deep reactive ion etching (DRIE), micro-molding, embossing, 3D- printing, thermal bonding, adhesive bonding, anodic bonding, and the like (see, e.g., Gale, et al. (2018), “A Review of Current Methods in Microfluidic Device Fabrication and Future Commercialization Prospects”, Inventions 3, 60, 1 - 25, which is hereby incorporated by reference in its entirety).

[0059] FIG. 4A illustrates a cross-sectional view of an optics module 200 in a comparative imaging system. One or more illumination sources 210, e.g., one or more light emitting diodes (LEDs), provides light through one or more optical components and an objective lens 220 to thereby illuminate a sample 230 in a sample holder 250. The one or more illumination sources 210 may be two or more illumination sources forming part of any of the illumination devices described in the present disclosure including those described with reference to FIGs 6B. In various embodiments, the optical components include a collimator 211. In various embodiments, the optical components include a field stop 212. In various embodiments, the optical components include one or more excitation filters 213. In various embodiments, the one or more excitation filters 213 are configured to filter light from the illumination source(s) 210 for a predetermined range of wavelengths (e.g., each filter has one or more blocking band(s) and / or transmission band(s) that may be different or may overlap at least in part) and each excitation filter 213 is aligned with appropriate illumination sources (e.g., blue LEDs, green LEDs, yellow LEDs, red LEDs, ultraviolet LEDs, etc.). In various embodiments, the optical components include a condenser 214. In various embodiments, the optical components include a beam splitter 215, e.g., a dichroic filter. An optical axis 251 is illustrated extending through the center of the optical surfaces in the objective lens 220 and its path includes an image plane, a focal plane, and input / output pupils (illustrated in FIG. 4B - also showing a comparative10XG / 1823PC 11imaging system 200 comprising an image plane 401, an object plane 402, a pupil 403, a 1.0 NA 20x objective 404, a 26.5mm FN tube lens 405 and a small pixel, large sensor, fast readout camera 406).

[0060] A sensor array 260 (e.g., CMOS sensor) receives light signals from the sample 250. In various embodiments, the optical components include one or more emission filters 265. In various embodiments, the one or more emission filters 265 are configured to filter light from the sample (e.g., emitted from one or more fluorophores, autofluorescence, etc.) for a predetermined range of wavelengths (e.g., each filter has one or more blocking band(s) and / or transmission band(s) that may be different or may overlap at least in part). In various embodiments, the emission filters 265 align (e.g., via motorized translation) with optics and / or the sensor array. In various embodiments, the sample 230 is probed with fluorescent probes configured to bind to a target (e.g., DNA or RNA) that, when illuminated with a particular wavelength (or range of wavelengths) of light, emit light signals that can be detected by the sensor array 260. In various embodiments, the sample 230 is repeatedly probed with two or more (e.g., two, three, four, five, six, etc.) different sets of probes. In various embodiments, each set of probes corresponds to a specific color (e.g., blue, green, yellow, or red) such that, when illuminated by that color, probes bound to a target emit light signals. In some embodiments, the sensor array 260 is aligned with the optical axis 251 of the objective lens 220 (i.e., the optical axis of the camera is coincident with and parallel to the optical axis of the objective lens 220). In various embodiments, the sensor array 260 is positioned perpendicularly to the objective lens 220 (i.e., the optical axis of the camera is perpendicular to and intersects the optical axis of the objective lens 220). In various embodiments, a tube lens 261 is mounted in the optical path to focus light on the sensor array 260 thereby allowing for image formation with infinity-corrected objectives. Descriptions of optical modules and illumination assemblies for use in opto-fluidic instruments can be found in U.S. provisional patent application no. 63 / 427,282, filed on November 22, 2022, titled “Systems and Methods for Illuminating a Sample” and U.S. provisional patent application no. 63 / 427,360, file on November 22, 2022, titled “Systems and Methods for Imaging Samples,” each of which is incorporated by reference in its entirety.

[0061] In various embodiments, the sample is illuminated with one or more wavelengths configured to induce fluorescence in the sample. In various embodiments, the sample is probed during one or more probing cycles with one or more fluorescent probes configured to bind to one or more target analytes. In various embodiments, the one or more wavelengths are selected10XG / 1823PC 12100-182300WC) to induce fluorescence in a subset of the one or more fluorescent probes. In various embodiments, each probing cycle includes illumination with two or more (e.g., four) colors of light. In various embodiments, the sample is treated with a fluorescent stain configured to illuminate one or more structures within the sample. In various embodiments, the sample is contacted with a nuclear stain. In various embodiments, the sample is contacted with 4', 6- diamidino-2-phenylindole (“DAPI”) configured to bind to adenine-thymine-rich regions in DNA. In various embodiments, illumination of the sample causes autofluorescence of the sample. In various embodiments, autofluorescence is the natural emission of light by biological structures when they have absorbed light, and may be used to distinguish the light originating from artificially added fluorescent markers. In various embodiments, fluorescence of the sample through fluorescent probes, autofluorescence, and / or a fluorescent stain can be used with the methods described herein to determine one or more focus metrics of a tissue sample.

[0062] In various embodiments, the sample is illuminated via edge lighting or transillumination along one or more edges of the sample and / or sample substrate. In various embodiments, the edge lighting provides dark-field illumination of the sample. In various embodiments, edge lighting is provided by one or more light sources positioned to provide light substantially perpendicular to a normal of the substrate surface on which the sample is disposed. In various embodiments, the substrate is a glass slide. In various embodiments, the substrate is configured as a wave guide to thereby guide light emitted from the edge lighting towards the sample. In various embodiments, illumination of the sample via edge lighting can be used with the methods described herein to determine one or more focus metrics of a tissue sample.

[0063] Referring now to FIG. 5, a schematic of an example of a computing node is shown. Computing node 10 is only one example of a suitable computing node and is not intended to suggest any limitation as to the scope of use or functionality of embodiments described herein. Regardless, computing node 10 is capable of being implemented and / or performing any of the functionality set forth hereinabove.

[0064] In computing node 10 there is a computer system / server 12, which is operational with numerous other general purpose or special purpose computing system environments or configurations. Examples of well-known computing systems, environments, and / or configurations that may be suitable for use with computer system / server 12 include, but are not limited to, personal computer systems, server computer systems, thin clients, thick clients, handheld or laptop devices, multiprocessor systems, microprocessor-based systems, set top10XG / 1823PC 13boxes, programmable consumer electronics, network PCs, minicomputer systems, mainframe computer systems, and distributed cloud computing environments that include any of the above systems or devices, and the like.

[0065] Computer system / server 12 may be described in the general context of computer system-executable instructions, such as program modules, being executed by a computer system. Generally, program modules may include routines, programs, objects, components, logic, data structures, and so on that perform particular tasks or implement particular abstract data types. Computer system / server 12 may be practiced in distributed cloud computing environments where tasks are performed by remote processing devices that are linked through a communications network. In a distributed cloud computing environment, program modules may be located in both local and remote computer system storage media including memory storage devices.

[0066] As shown in FIG. 5, computer system / server 12 in computing node 10 is shown in the form of a general-purpose computing device. The components of computer system / server 12 may include, but are not limited to, one or more processors or processing units 16, a system memory 28, and a bus 18 that couples various system components including system memory 28 to processor 16.

[0067] Bus 18 represents one or more of any of several types of bus structures, including a memory bus or memory controller, a peripheral bus, an accelerated graphics port, and a processor or local bus using any of a variety of bus architectures. By way of example, and not limitation, such architectures include Industry Standard Architecture (ISA) bus, Micro Channel Architecture (MCA) bus, Enhanced ISA (EISA) bus, Video Electronics Standards Association (VESA) local bus, and Peripheral Component Interconnect (PCI) bus.

[0068] Computer system / server 12 typically includes a variety of computer system readable media. Such media may be any available media that is accessible by computer system / server 12, and it includes both volatile and non-volatile media, removable and non-removable media.

[0069] System memory 28 can include computer system readable media in the form of volatile memory, such as random-access memory (RAM) 30 and / or cache memory 32. Computer system / server 12 may further include other removable / non-removable, volatile / non-volatile computer system storage media. By way of example only, storage system 34 can be provided for reading from and writing to a non-removable, non-volatile magnetic media (not shown and typically called a “hard drive”). Although not shown, a magnetic disk drive for reading from10XG / 1823PC 14and writing to a removable, non-volatile magnetic disk (e.g., a “floppy disk”), and an optical disk drive for reading from or writing to a removable, non-volatile optical disk such as a CD- ROM, DVD-ROM or other optical media can be provided. In such instances, each can be connected to bus 18 by one or more data media interfaces. As will be further depicted and described below, memory 28 may include at least one program product having a set (e.g., at least one) of program modules that are configured to carry out the functions of embodiments described herein.

[0070] Program / utility 40, having a set (at least one) of program modules 42, may be stored in memory 28 by way of example, and not limitation, as well as an operating system, one or more application programs, other program modules, and program data. Each of the operating system, one or more application programs, other program modules, and program data or some combination thereof, may include an implementation of a networking environment. Program modules 42 generally carry out the functions and / or methodologies of embodiments described herein.

[0071] Computer system / server 12 may also communicate with one or more external devices 14 such as a keyboard, a pointing device, a display 24, etc.; one or more devices that enable a user to interact with computer system / server 12; and / or any devices (e.g., network card, modem, etc.) that enable computer system / server 12 to communicate with one or more other computing devices. Such communication can occur via Input / Output (VO) interfaces 22. Still yet, computer system / server 12 can communicate with one or more networks such as a local area network (LAN), a general wide area network (WAN), and / or a public network (e.g., the Internet) via network adapter 20. As depicted, network adapter 20 communicates with the other components of computer system / server 12 via bus 18. It should be understood that although not shown, other hardware and / or software components could be used in conjunction with computer system / server 12. Examples, include, but are not limited to microcode, device drivers, redundant processing units, external disk drive arrays, RAID systems, tape drives, and data archival storage systems, etc.

[0072] The present disclosure includes systems, methods, and / or computer program products. The computer program product may include a computer readable storage medium (or media) having computer readable program instructions thereon for causing a processor to carry out aspects of the present disclosure.10XG / 1823PC 15

[0073] The computer readable storage medium can be a tangible device that can retain and store instructions for use by an instruction execution device. The computer readable storage medium may be, for example, but is not limited to, an electronic storage device, a magnetic storage device, an optical storage device, an electromagnetic storage device, a semiconductor storage device, or any suitable combination of the foregoing. A non-exhaustive list of more specific examples of the computer readable storage medium includes the following: a portable computer diskette, a hard disk, a random access memory (RAM), a read-only memory (ROM), an erasable programmable read-only memory (EPROM or Flash memory), a static random access memory (SRAM), a portable compact disc read-only memory (CD-ROM), a digital versatile disk (DVD), a memory stick, a floppy disk, a mechanically encoded device such as punch-cards or raised structures in a groove having instructions recorded thereon, and any suitable combination of the foregoing. A computer readable storage medium, as used herein, is not to be construed as being transitory signals per se, such as radio waves or other freely propagating electromagnetic waves, electromagnetic waves propagating through a waveguide or other transmission media (e.g., light pulses passing through a fiber-optic cable), or electrical signals transmitted through a wire.

[0074] Computer readable program instructions described herein can be downloaded to respective computing / processing devices from a computer readable storage medium or to an external computer or external storage device via a network, for example, the Internet, a local area network, a wide area network and / or a wireless network. The network may comprise copper transmission cables, optical transmission fibers, wireless transmission, routers, firewalls, switches, gateway computers and / or edge servers. A network adapter card or network interface in each computing / processing device receives computer readable program instructions from the network and forwards the computer readable program instructions for storage in a computer readable storage medium within the respective computing / processing device.

[0075] Computer readable program instructions for carrying out operations of the present disclosure may be assembler instructions, instruction-set-architecture (ISA) instructions, machine instructions, machine dependent instructions, microcode, firmware instructions, statesetting data, or either source code or object code written in any combination of one or more programming languages, including an object oriented programming language such as Smalltalk, C++ or the like, and conventional procedural programming languages, such as the “C” programming language or similar programming languages. The computer readable10XG / 1823PC 16program instructions may execute entirely on the user's computer, partly on the user's computer, as a stand-alone software package, partly on the user's computer and partly on a remote computer or entirely on the remote computer or server. In the latter scenario, the remote computer may be connected to the user's computer through any type of network, including a local area network (LAN) or a wide area network (WAN), or the connection may be made to an external computer (for example, through the Internet using an Internet Service Provider). In some embodiments, electronic circuitry including, for example, programmable logic circuitry, field- programmable gate arrays (FPGA), or programmable logic arrays (PLA) may execute the computer readable program instructions by utilizing state information of the computer readable program instructions to personalize the electronic circuitry, in order to perform aspects of the present disclosure.

[0076] Aspects of the present disclosure are described herein with reference to flowchart illustrations and / or block diagrams of methods, apparatus (systems), and computer program products according to embodiments of the disclosure. It will be understood that each block of the flowchart illustrations and / or block diagrams, and combinations of blocks in the flowchart illustrations and / or block diagrams, can be implemented by computer readable program instructions.

[0077] These computer readable program instructions may be provided to a processor of a general purpose computer, special purpose computer, or other programmable data processing apparatus to produce a machine, such that the instructions, which execute via the processor of the computer or other programmable data processing apparatus, create means for implementing the functions / acts specified in the flowchart and / or block diagram block or blocks. These computer readable program instructions may also be stored in a computer readable storage medium that can direct a computer, a programmable data processing apparatus, and / or other devices to function in a particular manner, such that the computer readable storage medium having instructions stored therein comprises an article of manufacture including instructions which implement aspects of the function / act specified in the flowchart and / or block diagram block or blocks.

[0078] The computer readable program instructions may also be loaded onto a computer, other programmable data processing apparatus, or other device to cause a series of operational steps to be performed on the computer, other programmable apparatus or other device to produce a computer implemented process, such that the instructions which execute on the computer, other10XG / 1823PC 17programmable apparatus, or other device implement the functions / acts specified in the flowchart and / or block diagram block or blocks.

[0079] FIG. 6A illustrates an optics module 600A. The optics module 600A can be as described below and optionally can be based on (and include any or all features of) the optics module and / or the imaging system and / or the computer node described with reference to FIG. 1 to FIG. 5. For convenience of explanation, where the optics module can be used as a microscope, the optics module 600A may alternatively be referred to as a microscope. The optics module 600A includes an objective 602, a dichroic filter 604, a tube lens 606, an image sensor 608, and an illumination device 620A. In some examples, the image sensor 608 is a camera, CCD or CMOS image sensor. In this setup, the objective 602 is an infinity corrected objective arranged to focus the illumination light at a focal plane and arranged to collect emission light from a sample 610 (e.g., emission light from the focal plane of the objective 602 or in the vicinity thereof) and collimate the emission light in the pupil conjugate. The collimated emission light is transmitted from the objective 602 to the tube lenses 606a, 606b through the infinity space 612. In some embodiments, a z-distance between the objective and sample is adjusted one or more times to thereby image additional focal planes (i.e., z-slices) within a field of view (FOV) of the objective 602. In some embodiments, a plurality of z-slices of a FOV form a z-stack of images representing an image volume. Where the term ‘between’ is used to describe the relative position of components, it may be understood to mean ‘between’ along the optical path, rather than strictly spatially between. Where the term “lens” is used throughout the present disclosure, this can be replaced with any other type of optic having the same function. Examples include diffraction plates (such as zone plates or spatial light modulators) and reflectors (such as mirrors).

[0080] The dichroic filter 604 (also known as a dichroic mirror or plate and which may also be described herein as an illumination dichroic) is positioned to intersect the illumination light path in the infinity space 612. The dichroic filter 604 has wavelength dependent reflectivity such that it reflects the illumination light from the illumination device 620A to fold the optical path of the illumination light 622 toward the objective 602. The dichroic filter 604 is also arranged to transmit emission light 615 from the sample to allow it to pass substantially undeviated from the objective 602 to the tube lens 606.

[0081] Accordingly, the optics module 600A of FIG. 6A finds use in illuminating a sample 610 having one or more target analytes each tagged with a fluorophore (coloured, e.g., red, yellow, green, blue). In some embodiments, each fluorophore is sequentially excited by10XG / 1823PC 18illumination light from the illumination device. The excitation light for each fluorophore includes at least a portion of the respective fluorophore-dependent excitation wavelengths for exciting each fluorophore in the sample. In response, the fluorophores emit emission light, which is captured through the objective, which then directs collected emission light 615 through the infinity space 612, the dichroic filter 604 and the tube lens 606 to the image sensor 608. The image of the emission light is then captured by the image sensor 608.

[0082] In an alternative configuration, one or more of the image sensors may be replaced by an eyepiece allowing a user to view the sample directly, through the optics module. In some embodiments, the image sensors 608a, 608b include any suitable image sensors for detecting the emission light, such as at least one photodiode array, at least one CCD sensor or camera, and / or at least one CMOS sensor or camera. The emission light is focused (i.e., rays are converged) by the tube lens 606 onto the image sensors.

[0083] The illumination device 620A comprises an illumination unit 621 including an illumination source 623, a collector lens 625, a field stop 627, and a field lens 629. The illumination device 620 is arranged to provide illumination light and introduce it into the infinity space 612 between the objective 602 and the tube lens 606 for onwards reflection to the objective 602 via the dichroic filter 604, and thereby to the sample at or near the focal plane of the objective 602.

[0084] The illumination unit 621 houses the illumination source 623 and may include other components which support the operation of the illumination source, such as thermal management components and driving circuitry. The illumination source 623 comprises, for example, an LED die, lamp, laser, or other light emitting structure and is arranged to emit illumination light 622 at a suitable wavelength for the application of the microscope. The wavelength is, in the example of a fluorescence microscope, an excitation wavelength of a fluorophore in the sample 610.

[0085] The collector lens 625 is arranged in the path of the illumination light 622 such that it collects a substantial proportion of the illumination light. The collector lens 625 is arranged with its optical axis parallel to or collinear with a normal of the center (or central area) of the illumination source 623. The collector lens 625 is of positive optical power.

[0086] The field stop 627 is arranged on the opposite side of the collector lens 625 to the illumination source 623 and comprises an aperture arranged to provide an aperture stop for the illumination light 622 collimated or focussed by the collector lens 625.10XG / 1823PC 19

[0087] The field lens 629 is arranged on the opposite side of the field stop 627 from the collector lens 625 and is arranged to form an image of the field stop between the field lens 629 and the objective 602 (i.e. in the infinity space, or between the dichroic filter 604 and the field lens 629).

[0088] FIG. 6B shows an example of the path of illumination (e.g. excitation) light and the path of sample luminance (e.g. emission light from the sample) in an optics module (which may also be described where convenient as a microscope) 600B which includes a dual source illumination device. The optics module 600B takes the structure and function of the optics module 600A described with reference to FIG. 6A, except where stated otherwise. The components having the same or similar reference numerals take the structure and function of any component (or its variants) described with reference to FIG. 6A, except where stated otherwise.

[0089] As in the optics module 600A of FIG. 6A, the optics module 600B of FIG. 6B includes an objective (not shown), dichroic filter 604, tube lens (not shown), and an illumination device 620B arranged relative to each other in the same way as described with reference to FIG. 6A. The optics module 600B of FIG. 6B is also arranged to illuminate and image a sample 610 as described with reference to FIG. 6A. The optics module 600B of FIG. 6B also includes an emission dichroic 611, and includes a first image sensor 608a and a second image sensor 608b in place of the image sensor 608 of FIG. 6A. The optics module 600B of FIG. 6B includes a dual-source illumination device 620B in place of the single-source illumination device 620A described with reference to FIG. 6A.

[0090] The dual source illumination device 620B includes a first illumination unit 621a, a first collector lens 625a, field stop 627 and field lens 629, which take the structure and function as described with reference to the same components in FIG. 6A (or any of the variants described) and are arranged in the same configuration. The dual-source illumination device 620B additionally includes a second illumination unit 621b, a second collector lens 625b, and a first and second filter 626a, 626b associated with the collector lens 625 and second collector lens 625b, respectively, as well as a wavelength-dependent reflector plate 628b.

[0091] The second illumination unit 621b is substantially identical in structure and function as the first illumination unit 621a except that it houses a second illumination source 623b different from the first illumination source 623a. The second illumination source 623b may have a different luminance uniformity than that of the first illumination source 623a. The second10XG / 1823PC 20illumination source 623b may have a different emission spectrum (for example including a different peak wavelength) than that of the first illumination source 623a. However, the arrangement in FIG. 6B is not limited thereto and, depending on application, the second illumination source 623b may have another difference in light emitting characteristics to the first illumination source in addition to a difference in uniformity (e.g., brightness or polarisation).

[0092] The second collector lens 625b is arranged in the path of second illumination light 622b emitted by the second illumination source 623b such that the second collector lens collects a substantial proportion of the second illumination light. The second collector lens 625b is arranged with its optical axis parallel to or collinear with a normal of the center (or central area) of the second illumination source. The second collector lens 625b is of positive optical power.

[0093] In implementations requiring different wavelengths of illumination light at the sample, a first bandpass filter 626a is optionally included between the first collector lens 625a and the wavelength-dependent reflector plate 628b and a second band pass filter 626b is optionally included between the second collector lens 625b and the wavelength-dependent reflector plate 628b. The first and second bandpass filter are arranged to pass a portion of the spectrum of the first and second illumination light, respectively. The pass band of the first bandpass filter is different from the passband of the second bandpass filter so that the wavelength spectrum of the first illumination light 622a reaching the sample is different from that of the second illumination light 622b.

[0094] The wavelength-dependent reflector plate 628b is positioned in the optical path of the first illumination light 622a between the first collector lens 625a and the field stop 627 as well as in the optical path of the second illumination light 622b on the other side of the second collection lens 625b from the second illumination unit 621b. The wavelength-dependent reflector plate 628b is, for example, a dichroic filter arranged at 45 degrees to the optical axis of the first and second collector lenses 625a, 625b. The wavelength-dependent reflector plate 628b is arranged to transmit the first illumination light 622a and to reflect the second illumination light 622b through 90 degrees such that the first illumination light 622a and second illumination light 622b emerge from the wavelength-dependent reflector plate 628b overlapping with their optical axes substantially colinear with each other.

[0095] The field stop 627 is placed in the path of the overlapping beams of first illumination light 622a and second illumination light 622b. The first and second illumination light follow a10XG / 1823PC 21path to the sample via the dichroic filter 604 and the objective as described with reference to FIG. 6A. Emission light 615a, 615b emanating from the sample as a result of the illumination light 622a, 622b then follows the same path as described with reference to FIG. 6A through the objective, dichroic filter 604 and tube lens to a respective image sensor 608a, 608b. In the example of fluorescence microscopy, first emission light 615a and second emission light 615b is emitted from the sample due to excitation of fluorophores in the sample by the first illumination light 622a and second illumination light 622b, respectively. However, other applications may result in light emanating from the sample as a result of irradiation by illumination light via mechanisms other than fluorescence, for example via simple reflection from the surface.

[0096] A difference between the optics module 600A of FIG. 6A and the optics module 600B of FIG. 6B is the arrangement of image sensors. As in the optics module 600A of FIG. 6A, the optics module 600B of FIG. 6B includes an image sensor arranged downstream of the tube lens to capture the emission light. However, the setup of FIG. 6B includes a first image sensor 608a arranged to capture the first emission light 615a and a second image sensor 608b arranged to capture the second emission light 615b. The first image sensor 608a is arranged downstream of an imaging dichroic filter 611 which is positioned in the optical path of the first emission light 615a between the tube lens and the first image sensor 608a. The second image sensor 608b is arranged downstream of the imaging dichroic filter 611 which is positioned in the optical path of the second emission light 615b between the tube lens and the second image sensor 608b. The imaging dichroic filter 611 is positioned at, e.g., 45 degrees to the optical axis of the tube lens and is arranged to reflect the first emission light 615a through, e.g., 90 degrees toward the first image sensor 608a and to transmit the second emission light 615b to allow it to pass to the second image sensor 608b.

[0097] The arrangement of FIG. 6B is provided to show how two illumination sources can be used in a device according to the present disclosure. However, the present disclosure is not limited thereto and devices having more than two illumination sources are also envisaged. Furthermore, more than two image sensors are also envisaged, for example one per illumination source, or even just a single image sensor arranged to detect all emission light (e.g., both the first and second emission light 615a, 615b). Further, the arrangement of the illumination sources and collection lenses relative to each other can be different from that illustrated in FIG. 6B. In other implementations, no image sensor is provided and an eyepiece for viewing the sample is provided instead.10XG / 1823PC 22

[0098] For example, in another implementation, instead of being positioned as shown in FIG. 6B, the first illumination source 623a and first collection lens 615a are positioned in the same orientation as, and to one side of, the second illumination source 623b and second collection lens 615b, respectively. In this case, a reflector (e.g., a second wavelength-dependent reflector plate 628a) is provided in the same orientation, and to the left (with respect to FIG. 6B) of the wavelength-dependent reflector plate 628b and in the path of the first illumination light (and the optical axis of the first collector lens). The path of first illumination light 622a is thereby turned through 90 degrees by the reflector so that it follows the same path to the field stop as the second illumination light 622a shown in FIG. 6B. The second illumination light 622b passes through the second wavelength-dependent reflector plate 628a so that it emerges therefrom parallel to the path of the first illumination light 622a towards the field stop 627. In this arrangement, the illumination sources are placed side by side in a row.

[0099] In some implementations, such as those where each component of the illumination light corresponds to excitation of respective fluorophores, each component of illumination light includes a respective unique wavelength or spectral range. For instance, the first component of illumination light (e.g., corresponding to both a first illumination source and excitation of a first fluorophore in the sample) has a first peak wavelength and the second illumination light (e.g., corresponding to both a second illumination source and excitation of a second fluorophore in the sample) has a second peak wavelength. In some embodiments, the first peak wavelength corresponds to a wavelength of excitation of the first fluorophore and the second peak wavelength corresponds to a wavelength of excitation of the second fluorophore.

[0100] With reference to FIG. 7, an assembly 700 for mounting a plurality of optical filters, for example, the optical filter(s) fulfilling the role of the dichroic filter(s) 604 in an optics module as described above with reference to FIG. 6, includes a frame 702, a first shaft 710, an alignment member 712, and a clamping member 720. The frame 702 has a first portion 704 for securing a first optical filter and a second portion 706 for securing a second optical filter. The first portion 704 and the second portion 706 each define respective apertures 705, 707. In various implementations, each aperture 705, 707 has a substantially rectangular shape. In various implementations, each aperture 705, 707 has filleted (rounded) corners. In various implementations, each rounded corner has the same radius. In various implementations, the rounded comers have different radii. In various implementations, two opposing corners have a first radius and two opposing comers have a second radius that is larger than the first radius. In various implementations, each aperture 705, 707 has a perimeter, and extending around each10XG / 1823PC 23perimeter is a flange configured to provide a location for an optical filter to be secured thereon in a fixed alignment in relation to the front face 703 of the frame 702. The apertures 705, 707 of the frame 702 allow light to be incident on, and pass through, the optical filters held by the frame 702. The shape of the frame 702 is rotationally symmetric about the axis of rotation of the first shaft 710. Therefore, any given position that the first portion 704 of the frame 702 can be in can be reached by the second portion 706 by rotating the frame, thereby switching or ‘flipping’ the filters mounted thereon.

[0101] The first shaft 710 is connected to the frame 702 such that the first shaft 710 can impart a torque to the frame. In various implementations, the first shaft 710 is shrunk fit to the frame, for example, by freezing a coupling end of the first shaft 710 and / or heating the corresponding coupling portion of the frame 702 configured to receive the coupling end of the first shaft 710. When the first shaft 710 and the frame 702 return to the same temperature, a secure connection is achieved. In various implementations, the coupling end of the first shaft 710 includes a keylike protrusion configured to engage with a corresponding notch in the coupling portion of the frame 702. In various implementations, the coupling end of the first shaft 710 includes a D- shape configured to engage a corresponding D- shape notch in the coupling portion of the frame 702. In various implementations, the connection between the first shaft 710 and the frame 702 is a fixed connection, e.g., screws, bolts, pins, interlocking complementary faces (such as those described above), or any other suitable type of connection, so that the first shaft 710 determines the rotational position of the frame 702 and the shaft and frame rotate in sync. The first shaft 710 receives torque to drive the frame 702 from a motor 730 via a second shaft 732 that is powered by the motor. The motor 730 has power and / or control signal connections through which it can receive power to drive the motor and / or instructions how and when to rotate the second shaft 732, e.g., start time or duration information, rotation speed, rotation speed profile over time, etc.

[0102] The first shaft 710 and the second shaft 732 are connected via a flexible coupling 734 (e.g., a universal joint coupling) which can transfer torque between shafts without exact alignment of the rotational axes of the shafts. Therefore, the exact alignment of the first shaft 710 can be at least partially independent of the second shaft 732.

[0103] The alignment member 712 provides an alignment surface for aligning against a reference surface to provide a precise orientation of optical filters mounted on the frame with respect to the reference surface (e.g., a precision-machined surface of an optical bench). As such, the alignment surface has a predetermined and constant alignment with respect to the first10XG / 1823PC 24shaft 710, e.g., the alignment surface is parallel to the axis of rotation of the first shaft 710. With reference to FIG. 7, the alignment member 712 is a cylindrical bearing with an inner portion coupled to the first shaft 710 and an outer portion arranged concentrically around the inner portion such that the inner portion can rotate freely with respect to the outer portion (e.g., the bearing includes a plurality of spherical bearings between the inner and outer portions). In this example, the alignment surface is the cylindrical outer surface of the outer portion of the cylindrical bearing. In various implementations the assembly 700 includes two or more alignment members.

[0104] The clamping member 720 is configured to clamp the alignment member against the reference surface to align the first shaft 710, and thereby also align the frame and optical filters, with the reference surface. With reference to FIG. 7, the clamping action is provided by a clamp body having a clamping surface and one or more attachments for attaching the clamp body to the reference surface (or another part of the component that comprises the reference surface, e.g., an optical bench). The attachments comprise through holes 722 extending through the clamp body that align with corresponding threaded holes on the reference surface (or another part of the component that comprises the reference surface). When screws are inserted through the through holes 722 and screwed into the corresponding threaded holes and tightened, the clamping member 720 exerts a force on the alignment member 712 to cause the alignment member to press against the reference surface thereby aligning the first shaft 710 to the reference surface. It may be understood that any other suitable fixation member as is known in the art may alternatively be used (with or without corresponding through holes and threaded holes) to cause the clamping member 720 to exert said force on the alignment member. In various implementations, the clamping surface is arranged facing and parallel to the reference surface. In various implementations, the clamping surface is arranged at an angle to the reference surface. In various implementations, if the reference surface has more than one face forming a corner between the faces (which may alternatively be referred to as two perpendicular reference surfaces), the clamping surface is arranged facing the corner, i.e., perpendicular to a line bisecting the angle made by the corner. In various implementations, the clamping surface is a curved surface (e.g., a curved surface that matches a curved surface of the alignment member 712).

[0105] With reference to FIG. 8, a system 800 includes an assembly, as described above with reference to FIG. 7, and an optical bench 810 having the reference surface 714 to which the alignment member 712 is clamped. Accordingly, the frame 702, and any optical filters mounted10XG / 1823PC 25100-182300WC) thereon, has a precise alignment with respect to the optical bench 810. In the implementation shown in FIG. 8, the frame 702 is aligned perpendicularly to the reference surface 714 of the optical bench 810. The optical bench 810 defines a recess 830, comprising a first recess portion 832 configured to receive the alignment member 712, first shaft 710, and clamping member 720 of the assembly 700. The recess 830 also comprises a second recess portion 834 for receiving the motor 730. Positioning the assembly in a recess within the optical bench 810 lowers the center of mass of the assembly 700 with respect to the optical bench 810, which reduces vibrations as the frame rotates back and forth. The first recess portion 832 comprises attachment points for receiving screws used to attach the clamping member 720 to the optical bench 810. In various implementations, as shown in FIG. 8, the first recess portion 832 has a square or rectangular cross-sectional shape, e.g., having a recess base which is flat and parallel to the top surface 812 of the optical bench and recess side walls arranged perpendicular to the recess base and extending between the recess base and the top surface of the optical bench. In alternative implementations, the first recess portion has a different cross-sectional shape, e.g., semi-circular, U-shaped, V-shaped, obround, etc. The clamping member 720 exerts a force on the one or more alignment members e.g., alignment member 712) to cause the alignment member(s) to press against the reference surface (or reference surfaces) of the first recess portion to stably fix the alignment member(s) and securely align the first shaft with the reference surface(s).

[0106] A component of the force applied by the clamping member acts on the one or more alignment members in a direction perpendicular (i.e. normal) to each reference surface. Each reference surface provides an equal and opposite force on the one or more alignment members. The force provided by the clamping member and the reactive force(s) applied by each surface are together in equilibrium. For example, in the case of a first and second reference surface, a first component of the force applied by the clamping member to the one or more alignment members is provided in a first direction perpendicular to the first reference surface. Meanwhile, a second component of the force applied by the clamping member to the one or more alignment members is provided in a second direction perpendicular to the second reference surface. The first and second reference surfaces provide an equal and opposite force to the first and second component of the force applied by the clamping member. The first reference surface is not parallel with the second reference surface, and therefore the first direction is different from the second direction. The force applied to the at least one alignment member by the clamping member and the reactive forces applied to the alignment member by the first and second10XG / 1823PC 26reference surfaces are in equilibrium. Therefore, each of the at least one alignment members is a three-force member and is therefore in equilibrium. This allows the first shaft to be retained reliably in the same position relative to the optical bench, thus achieving accurate and repeatable alignment between the first shaft and other components coupled to the optical bench.

[0107] The optical bench 810 defines a slot 840 for receiving the frame to permit rotation of the frame around the rotation axis. Having a slot 840 for the frame allows the axis of rotation of the first shaft 710 and frame 702 to be below the top surface 812 of the optical bench 810, which increases the stability of the assembly 700. In various implementations, the slot 840 does not extend through the entire thickness of the optical bench 810 (e.g., the frame rotates within a recess in the optical bench 810). A slot 840 extending through the entire thickness of the optical bench 810, as shown in FIG. 8, is used if the depth needed for the slot is greater than the depth of the optical bench.

[0108] With reference to FIG. 9, which is a rear isometric view of an assembly 700, the assembly includes two alignment members 712, 716 in the form of cylindrical bearings. The clamping member 720 has a clamping surface 721 arranged to clamp both alignment members 712, 716 against the reference surface(s). The clamping surface 721 is angled at approximately 45 degrees with respect to the rest of the clamp body (and with respect to a horizon) to provide a force on the alignment members 712, 716 and press the alignment members 712, 716 against reference surface(s) with a horizontal component and a vertical component. With a 45 degree angle clamping surface, the horizontal component of force and vertical component of force on the alignment members 712, 716 will be substantially equal. In various implementations, the clamping surface 721 is configured with any suitable angle, such as 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 degrees. In various implementations, a single clamping member 720 and a single set of attachments can be used to align the first shaft 710 in multiple directions (e.g., the directions analogous to yaw and pitch, or defined by a polar angle from vertical and an azimuthal angle from a reference optical axis). In various implementations, the clamping member 721 includes two or more independent pieces, where each piece is configured to independently provide a force on a respective alignment member 712, 716. By using two alignment members 712, 716, a more stable and accurate alignment with the reference surface(s) is provided.

[0109] In some implementations, such as shown in FIG. 9, the assembly 700 includes a communication module 910 for receiving control signals from a controller. The communication may be wireless or wired.10XG / 1823PC 27

[0110] In some implementations, such as shown in FIG. 9 and FIG. 10, the clamping member 720 includes an end plate 724 between the frame 702 and the alignment member 712 closest to the frame 702. With reference to FIG. 10A, the assembly 700 includes an O-ring 1010 positioned between the end plate 724 and the alignment member 712. The end plate 724 provides a force against the alignment member 712 (e.g., the outer portion of alignment member 712) via the O-ring 1010. The force (left arrow) on the alignment member 712 causes a reaction force (right arrow) at the other alignment member 716 to secure the alignment members and support the shaft (e.g., mitigate bending due to the mass of the dichroic filters secured within the frame). This arrangement of the end plate 724 and O-ring provides the clamping member with a more secure grip on the alignment members 712, 716, precise alignment of the alignment members against the reference surface(s) and reduces vibrations that could be transmitted from the motor and alignment members to the frame 702 and filters thereon. In various implementations, the O-ring 1010 is made from rubber or any other suitable durable and flexible material. In various implementations, the assembly 700 includes a second O-ring between a motor housing and the alignment member 716 closest to the motor.

[0111] With reference to FIG. 10B, in some implementations the clamping member 720 includes a pad 728 configured to contact the alignment member 712 and / or the alignment member 716. In various implementations, the pad 728 matches the shape of the clamping surface 721. For example, where the clamping surface 721 includes two perpendicular surfaces, the pad 728 may be shaped to match the two perpendicular surfaces. In various implementations, the pad 728 includes two or more pads. In various implementations, the pad 728 is L-shaped and configured to push against the alignment members 712, 716 in two perpendicular directions to securely clamp the alignment members against the reference surface(s). As the clamp screws 726 are tightened, the pads 728 are forced against the alignment surface(s) of the alignment members 712, 716, thereby causing the alignment surface to be flush against the reference surface(s). Accordingly, the pads 728 may comprise, or be referred to, as the clamping surface. The pads 728 can be made from rubber or any other suitable resilient and flexible material.

[0112] Although exemplary implementations of the assembly 700 and system 800 are described above with reference to FIG. 7 to FIG. 10B, alternative arrangements are also suitable for implementing the present disclosure, some of which are explained further below.

[0113] In some implementations, a frame for securing optical filters has a different shape from the frame 702 described above. For example, the frame may comprise more than two portions10XG / 1823PC 28for securing optical filters, e.g., the frame may be configured to hold three, four, five, or six optical filters. The portions for securing optical filters may be arranged at an equal radial distance from the axis of rotation of the first shaft 710. The frame may position the optical filters with an equal angle between adjacent optical filters, e.g., 180 degrees apart for two optical filters; 120 degrees apart for three optical filters; 90 degrees apart for four optical filters; 72 degrees apart for give optical filters; 60 degrees apart for six optical filters, etc. Alternatively, the frame may position optical filters unevenly spaced around the axis of rotation. In some implementations, portions for securing optical filters have a different shape of aperture from the rounded rectangular shape of FIG. 7 to FIG. 9, e.g., circular, square, rectangular, etc. In various implementations, the thickness profile of the frame may change along the radial length. For example, the frame may have a first thickness at a first end close to the axis of rotation (near the connection to the first shaft) and taper to a smaller, second thickness at a second end (e.g., further radial end away from the axis of rotation). One skilled in the art will recognize that the shape of the frame and thickness of the frame can be modified to optimize moment of inertia (e.g., require less motor torque to flip between dichroic filters).

[0114] In some implementations, the first shaft is configured to receive a torque from a motor via one or more intermediate drive mechanisms, such as a combination of belts, chains, gears, transmissions, etc., for transferring the torque. Using an intermediate drive mechanism, the second shaft of the motor does not need to be colinear with the first shaft or parallel with the first shaft. In some implementations, positioning the second shaft substantially colinear and substantially parallel with the first shaft can reduce vibrations. If an intermediate drive mechanism is used, an output shaft of the intermediate drive mechanism may be coupled to the first shaft via flexible coupling 734, as described above with reference to FIG. 7. An alternative, for any implementation using a flexible coupling 734, to the flexible coupling 734 is a universal joint, or any other suitable device that is configured to transfer a torque without requiring a rigid connection between its input and output shafts, thereby isolating the first shaft from the source of the torque. In some implementations, the motor 730 is a servo motor, or any other suitable motor for providing a controlled torque. In some implementations, the motor has an internal power source, e.g., a battery.

[0115] In some implementations, alignment members 712, 716 have a different outer profile than those described above with reference to FIG. 7 and FIG. 8 and below with reference to FIG. 12. For example, the alignment members may have an outer portion having a shape other than cylindrical, e.g., having a square or rectangular cross-sectional shape, having a cylindrical10XG / 1823PC 29shape with a flat portion of the surface, etc. In particular, the outer surface of the bearing (the alignment surface) may have a complementary shape to the reference surface. For any external form of the alignment surface, the internal structure of the alignment member may have an inner component and an outer component arranged concentrically and allowing rotational motion between the inner and outer components. In some implementations, the bearing of the alignment member comprises one or more, or a combination of: ball bearings, fluid bearings, electromagnetic bearings, or bushings. In another example, where the alignment member is a cylindrical bearing, the cylindrical bearing can be fit into a housing having any suitable external profile (e.g., square, rectangular, elliptical, etc.). In some implementations, the alignment member is a material or coating between the first shaft and the reference surface that allows the shaft to rotate freely (reducing friction) and supports and aligns the shaft mechanically against the reference surface. As such, the alignment member can be any suitable part that is used to align the shaft to a reference surface.

[0116] In some implementations, the clamping member 720 has a different form from the examples described above with reference to FIG. 7 to FIG. 10. For example, the clamping member 720 may have a clamping surface at an angle of greater or less than 45 degrees, or a curved clamping surface having a radius of curvature. The clamping surface may have a complementary shape to the alignment members to stably press against the alignment members. Instead of a single clamp body, separate clamp bodies or a second clamping member may be provided to perform the role of a clamping member as described above for separate alignment members. In some implementations, the fixing of the clamping member with respect to the reference surface (possibly via one or more intermediate components) is non-mechanical, e.g., using one or more of suction, a vacuum, permanent magnets, electromagnets, etc. to hold the clamping member against the alignment member to align the alignment member with the reference surface.

[0117] In some implementations, depending on the alignment required for the optical filters mounted on the assembly, the reference surface 714 includes a wall extending above the top surface 812 of the optical bench instead of, or in addition to, a base or wall of a recess, or the reference surface may be the top surface 812 itself. As used herein, the optical bench may be any component that the optical components of a system are configured to attach to, thereby setting the relative positions and angles between components. For example, the optical bench may be a machined plate with attachment positions, e.g., through holes and / or threaded holes for attaching optical components. Accordingly, the precise alignment of the first shaft with the10XG / 1823PC 30reference surface of the optical bench positions and angles the optical filters mounted on the assembly sets the required angle between the optical filters for receiving, reflecting, and / or transmitting light for functioning as part of the optical system. In some implementations, there are a plurality of reference surfaces to align the alignment members in more than one angular direction, e.g., according to a polar angle from vertical and an azimuthal angle from a horizontal reference direction such as the optical axis. The plurality of reference surfaces may be continuous with each other or discontinuous.

[0118] With reference to FIG. 11, in some implementations, a system 800 having an assembly 700 for mounting a plurality of optical filters and an optical bench 810 is used for a microscopy system, e.g., for epifluorescence microscopy. The optical bench 810 has a top surface 812 and a primary optical axis 814. In various implementations, the primary optical axis 814 is defined as the axis running through the center of the imaging components, e.g., the tube lens 1130, objective (not shown), and imaging sensor, and is used to define the orientation of the optical components of the system.

[0119] The system includes a field lens 1112 for receiving illumination light 1110 from one or more of a plurality of light sources (typically via a collector lens for each light source and a field stop to create a beam size and shape that will produce an image size and shape at the imaging equipment to match the field of view of the imaging equipment). The field lens 1112 focuses the illumination light onto the back focal plane of an objective (not shown). The objective is positioned to direct the illumination light to a sample (not shown) and receive emission light from the sample. An illumination pathway of the illumination light 1110 follows from the field lens 1112, reflection off of an optical filter held in the frame 702, reflection off a fold mirror 1122 e.g., 50-50 beamsplitter), and into the objective below the fold mirror for illuminating the sample. An emission pathway of emission light follows from the sample and the objective, reflection off the fold mirror 1122, transmission through the optical filter held in the frame 702, focusing by a tube lens 1130 (optionally after transmission through an emission filter), and recording the sample image at the imaging equipment 1140.

[0120] The components of the system 800 are attached to the optical bench 810, e.g., using screws, bolts, clamps, or any suitable attachment mechanism, and calibrated to achieve precise alignment between components to direct the illumination and emission pathways to and from the sample. Some components are positioned in recesses in the top planar surface 812 of the optical bench 810. For example, the assembly 700 for mounting a plurality of optical filters is positioned in a recess 830, and the frame 702 partially sits in a slot in the optical bench, as10XG / 1823PC 31described above with reference to FIG. 8. The tube lens 1130 is positioned in a tube lens recess 1132. The use of machined recesses in this way provides a reference surface 714 on the walls of the recess for the assembly 700 to align with, and also means that the primary optical axis 814 is lower with respect to the top planar surface 812 compared to all the components being mounted on the top planar surface 812. In various implementations, a lower primary optical axis can improve compactness of the optical subsystem.

[0121] The assembly 700 for mounting a plurality of optical filters is aligned with the reference surface 714, being a side wall of a recess 830, to precisely align the first shaft with respect to the primary optical axis 814. Thus, the recess 830 determines the angle that optical filters mounted in the frame 702 intersect the optical axis and the incoming direction of illumination light 1110. The precision of this angle is important as the illumination light is reflected off the optical filter towards the fold mirror 1122 and therefore any angular error in the optical filter positioning is doubled at the fold mirror 1122. The motor of the assembly 700 is controlled and powered via connectors (shown unconnected) to a power source and controller. By setting the positioning and orientation of the optical filters, via the first shaft, with reference to the optical bench itself (in particular, to the reference surface), there are fewer sources of angular error compared to relying on the alignment of the motor housing being attached to the optical bench. The frame 702 is rotatable by the first shaft to position each of a first optical filter and a second optical filter, mounted in the first and second portions of the frame, respectively, in the illumination pathway and emission pathway. Therefore, the assembly 700 can switch (or ‘flip’) between the two optical filters while maintaining precise alignment with the primary optical axis 814. In various implementations, one or more light sources (e.g., a plurality of light sources) can be controlled to provide different illumination bands (e.g., each light source producing a different wavelength / frequency bandwidth of light), and the assembly 700 will rotate such that the optimal optical filter having the appropriate filter spectrum (including transmission band(s) and reflection band(s)) for that illumination light) is positioned in the illumination pathway. In various implementations, the optical filters have a single reflection band and a single transmission band. In various implementations, the optical filters are multiband and have two or more (e.g., 2, 3, etc.) reflection bands and two or more (e.g., 2, 3, etc.) transmission bands. The rotation of the frame 702 and optical filters is performed while the clamping member 720 maintains the first shaft aligned with the reference surface 714. Therefore, the optical filters can be switched back and forth while maintaining the precise alignment with primary optical axis 812 and the illumination light. Maintaining the precise10XG / 1823PC 32alignment provides a microscopy system that can image multiple fluorophores, responsive to different wavelengths of light, while using a single tube lens and single imaging sensor for emission detection (to reduce overall cost of the system). The system 800 described with reference to FIG. 11 has the reference surface 714 machined to form an angle of 30 degrees with the primary optical axis 814. In various implementations, the reference surface 714 is machined into the optical bench at any suitable angle to the primary optical axis 814, such as, for example, 5 degrees, 10 degrees, 15 degrees, 20 degrees, 25 degrees, 30 degrees, 35 degrees, 40 degrees, 45 degrees, 50 degrees, 55 degrees, 60 degrees, 65 degrees, 70 degrees, 75 degrees, 80 degrees, 85 degrees, or any suitable value therebetween. Accordingly, the optical filters mounted on the frame 702 intersect the primary optical axis at 60 degrees. In other implementations, this angle is different according to the design of the system and the required illumination pathway.

[0122] In some implementations, the system 800 includes two, three, four, five, or more light sources. In an exemplary system having two light sources (such as the system shown in FIG. 6B), the assembly 700 holds two optical filters such that the first optical filter reflects light at the wavelength of illumination light from the first light source and transmits emission light at the wavelength emitted by a first fluorophore that is excited by the first light source, and the second optical filter reflects light at the wavelength of light from the second light source and transmits emission light at the wavelength emitted by a second fluorophore that is excited by the second light source. The same principles apply to systems with three, four, five, or more light sources. In other examples, each optical filter may be usable for more than one light source, by having more than one reflection band and more than one transmission band, each corresponding to one of the sources of light and a corresponding fluorophore in the sample. In an example having five light sources, the light sources are referred to by their approximate colors, namely, red, yellow, green, blue and near ultraviolet light sources. In various implementations, a first optical filter mounted in the frame 702 has: a first reflection band (near ultraviolet) between about 300nm and about 425nm; a first transmission band between about 425nm and about 500nm; a second reflection band between about 510nm and about 550nm (green); a second transmission band between about 550nm and about 600nm; a third reflection band between about 600nm and about 650nm (red); and a third transmission band between about 650nm and about 750nm; and a second optical filter mounted in the frame 702 has: a first reflection band between about 425nm and about 500nm (blue); a first transmission band between about 500nm and about 550nm; a second reflection band between about 550nm and10XG / 1823PC 33about 600nm (yellow); and a second transmission band between about 600nm and about 675nm. In alternative implementations, a different set of wavelength ranges are used for the reflection and transmission bands. In some implementations, the light sources are lasers, LEDs, etc. or a combination thereof. In some implementations, the optical filters are dichroic filters.

[0123] With reference to FIG. 12, an exemplary frame 702, first shaft 710, and alignment member 712, 716 of an assembly is illustrated. The frame 702 has a first optical filter 740 secured to a first portion 704 thereof and a second optical filter 742 secured to a second portion 706 thereof. The optical filters 740, 742 are mounted by insertion from rear face 709 of the frame and secured parallel to the front face against a flange running around the aperture of the respective first and second portions. Accordingly, the front surface of the optical filters (i.e., the surface from which illumination light is reflected) is aligned perpendicular to the first shaft 710 (defining the rotational axis of the frame 702), which provides alignment with respect to a reference surface. The first and second optical filters 740, 742 may be securely directly to the frame 702, or may be attached to a substrate that is attached to the frame 702. In various implementations, the optical filters are secured by friction due to a snug fit within the respective first and second portions, or secured by adhesive, clips, tabs, etc. or any other suitable attachment means. In various implementations, the optical filters are secured within the respective portions via a mechanical fitting (e.g., a flange allows for an optical filter to slide into an operable position and a fixation mechanism, such as a screw attached to a securing member / plate, can be used to mechanically secure the optical filter within the frame). In various implementations, the optical filters are secured within the respective portions via an adhesive (e.g., an epoxy suitable for securing optical components).

[0124] The first alignment member 712, in the form of a cylindrical bearing, has an inner portion 743 coupled to the first shaft 710 and an outer portion 744 arranged concentrically around the inner portion and that can rotate freely with respect to the inner portion. A plurality of rolling components, such as ball bearings, is disposed between the inner portion 743 and the outer portion 744. In some embodiments, the plurality of rolling components is made of a metal (e.g., high carbon chrome steel, stainless steel, etc.). In some embodiments, the plurality of rolling components is made of a ceramic (e.g., silicon nitride, zirconia oxide, alumina oxide, silicon carbide, etc.). In some embodiments, a lubricant (e.g., grease) is dispensed between the inner portion 743 and the outer portion 744, such that the plurality of rolling components is lubricated by the lubricant. In some embodiments, the cylindrical bearing includes shielding on one or both sides configured to protect the inner rolling components. In some embodiments,10XG / 1823PC 34the shielding is made of a metal (e.g., high carbon chrome steel, stainless steel, etc.). In some embodiments, the shielding is made of a polymer e.g., nitrile rubber, fluoroelastomer, etc.). In some embodiments, where shielding on both sides forms a sealed chamber in which the roller components are contained, a lubricant is included in the sealed chamber between the inner portion 743 and the outer portion 744. For example, a friction-reducing powder lubricant (e.g., graphite, PTFE, molybdenum disulfide, boron nitride, etc.) may be included within the sealed chamber. Accordingly, the outer portion 744 can be static pressed against a reference surface by a clamping member (not shown) which the inner portion 743, first shaft 710, and frame 702 rotate.

[0125] With reference to FIG. 13A, a view of the front face of the frame 1302 shows the arrangement of the first and second portions 1304, 1306 for securing the first and second optical filters, respectively. The first portion 1304 has a first aperture 1305 and the second portion 1306 has a second aperture 1307, where each aperture 1305, 1307 forms a rounded rectangular shape. With reference to FIG. 13B, a view of the rear face of the frame 1302 shows the flanges running around the perimeter of the apertures for attaching the optical filters thereto. The frame 1302 comprises a shaft connection 1310 at its center for connecting the frame to the first shaft, e.g., using a threaded portion at the distal end of the first shaft, a shrink fit between the shaft and frame, and / or a positive shape (e.g., a key-like protrusion) or negative shape (e.g., D-shape cutout). The positioning of the optical filters in the first and second portions 1304, 1306 is such that the optical filters are as close to the center of the frame as is permitted by the shaft connection 1310. In other words, the first and second filters are positioned adjacent the rotation axis of the first shaft. The filters being positioned closer to the rotation axis reduces the rotational moment of inertia, which increases acceleration and deceleration and reduces the time need to switch positions of the first and second optical filters. Positioning the optical filters adjacent the rotational axis also reduces the maximum radius of the frame 1302, providing a more compact arrangement.

[0126] Each of the first optical filter and the second optical filter have a first dimension 1352 and a second dimension 1354 (i.e., in a direction perpendicular to the first dimension), wherein both the first and second dimensions are in a plane perpendicular to the first axis. Accordingly, the first and second dimensions define the approximate aspect ratio and area of the front surface of the optical filters from which illumination light is reflected. As the illumination light is incident on the optical filters at an angle, an illumination light beam with a circular or square cross-section (e.g., as produced by a field stop prior to reaching field lens 1112) will project10XG / 1823PC 35100-182300WC) onto an area of the optical filters that is greater in the first dimension 1352 than in the second dimension 1354. Accordingly, the optical filters and / or the apertures of the frame have a greater first dimension 1352 than the second dimension 1354. For example, the first dimension may be 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% greater, approximately 15% greater, about 10% to about 20% greater, about 10% to about 30% greater, about 10% to about 40% greater, about 10% to about 50% greater, or about 5% to about 30% greater. In general, the relationship between the first and second dimensions can be defined as d2 = di . Cos (a), wherein di is the first dimension, d2 is the second dimension, and a is the angle of incidence of light onto the optical filter (which corresponds to the angle between reference surface 714 and the primary optical axis 812 in the system as described above with reference to FIG. 11). Accordingly, for an angle a of 30 degrees, di is approximately 1 - (1 / Cos 30) = 15.5 % greater than d2. In an example, each optical filter has a first dimension 1352 of 70 mm to 75 mm, a second dimension 1354 of 60 mm to 65 mm, and a thickness of 2 mm to 6 mm.

[0127] With reference to FIG. 14A and FIG. 14B, an alternative frame design is provided that is capable of securing the same size and shape of optical filter as the frame described above with reference to FIG. 13A and FIG. 13B. FIG. 14A shows a view of a front face of the frame 1402 and FIG. 14B shows a view of a rear face of the frame 1402. The means for securing the optical filters to the first portion 1404 and the second portion 1406, and securing the frame 1402 to the first shaft via the shaft connection 1410 is substantially the same as in the examples described above. Similar to the above, the first portion 1404 includes a first aperture 1405 and the second portion includes a second aperture 1407. In contrast to the frames as described in FIGS. 12 to 13B, the optical filters 740, 742 are mounted by insertion from the front face of the frame 1402 and secured parallel to the rear face against the flange running around the aperture of the respective first and second portions. Securing parallel to the rear face allows the optical filters to be positioned closer to the center of the frame because the shaft connection is on the opposite side of the frame to the optical filters. Accordingly, the optical filters are positioned closer to the rotational axis of the first shaft, thereby providing a frame with a smaller radius, lower rotational moment of inertia, and associated increased rotational acceleration and deceleration. The first and second portions, to which the first and second optical filters are secured, include a first pair of arms and a second pair of arms 1404, 1406, respectively. Using pairs of arms reduces the maximum radius of the frame 1402 and also reduces the rotational moment of inertia. The apertures of the first and second portions are10XG / 1823PC 36partially, but not fully, enclosed by the pairs of arms. In various implementations, the frame 1402 in configured with flanges such that the optical filters are mountable from the rear.

[0128] With reference to FIG. 15, a method for mounting an assembly as described above with reference to any of FIGS. 7 to 14B, or variations thereof, onto an optical bench includes securing 1502 the assembly to the optical bench to thereby contact the alignment surface of the alignment member against the at least one reference surface. The contact between the alignment surface and the at least one reference surface provides precise positioning and alignment of the first shaft, the frame and the optical filters, with respect to the optical bench and other components mounted on the optical bench. The securing may include clamping the alignment member against the reference surface using the clamping member, e.g., tightening screws, bolts, or other attachments means through the clamping member and into the optical bench. The method may also comprise securing a motor housing, that comprises a motor configured to provide torque, to the optical bench, although the precise alignment of the alignment members does not depend on the securing of the motor housing because the alignment member abuts the reference surface directly. The method may further comprise providing power and control connection to the motor for controlling the rotation of the frame.

[0129] With reference to FIG. 16, a method of using an assembly as described above with reference to any of FIGS. 7 to 14, or variations thereof, comprises providing 1602 a torque from a motor and rotating 1604 the first shaft using the torque thereby rotating the frame. The alignment surface of the alignment member contacts the reference surface throughout the method. Therefore, the first shaft, frame and optical filters maintain a precise alignment with respect to the reference surface while changing the positions of the optical filters. For example, a first optical filter may be positioned in an illumination pathway, such as described above with reference to FIG. 11, during imaging using a first illumination band of light. The first optical filter is rotated out of the illumination pathway and the second optical filter is rotated into the illumination pathway for imaging to occur using a second illumination band of light at different wavelengths (e.g., illumination light having a different spectrum than the first illumination band of light). Thus, imaging methods are provided in which a sequence of imaging requiring different optical filters can be performed while using a single tube lens and single imaging sensor for emission detection (to reduce overall cost of the system).

[0130] In some implementations, the method of using the assembly comprises rotating the frame such that the first optical filter is positioned in the illumination pathway to receive first illumination light and energizing a first light source of a plurality of light sources to direct first10XG / 1823PC 37illumination light to the first optical filter, wherein the first illumination light has a first wavelength and the first optical filter has a first reflectance band that includes the first wavelength. In a system as described above with reference to FIG. 11, the first illumination light then travels to the fold mirror, objective and sample, exciting fluorophores that are responsive to at least some of the wavelengths of the first illumination light. The emission light produced by the fluorophores passes back through the objective and fold mirror, through the first optical filter (z.e., is transmitted through the first optical filter), through the tube lens, optionally through an emission filter, and to the imaging sensor for imaging the sample. In some implementations, the method comprises then rotating the frame such that the second optical filter is positioned in the illumination path to receive second illumination light and energizing a second light source of the plurality of light sources to direct second illumination light to the second optical filter, wherein the second illumination light has a second wavelength and the second optical filter has a second reflectance band that includes the second wavelength. The sample is imaged accordingly using the second light source in the same manner as described above, except using the second optical filter and a different fluorophore. When energizing the second light source, the first light source may be de-energized (switched off), and vice versa.

[0131] The same principles apply to implementations having three, four, five, or more light sources. In some implementations, each of the first and second optical filters has a plurality of reflectance bands. As an example, the first optical filter is positioned in the illumination pathway during energizing of the first, third, and fifth light sources and reflects the illumination light for imaging the sample using each of these light sources, whereas the second optical filter is positioned in the illumination pathway during energizing of the second and fourth light sources and reflects the illumination light for imaging the sample using either of these light sources. In this way, a composite image of the sample using multiple wavelengths of light, e.g., exciting multiple different fluorophores, can be provided. The imaging may be performed by energizing each of the first to fifth light sources in sequence, and therefore rotating the frame between the positions that have the first and second optical filters in the illumination pathway between each energizing. Alternatively, all the light sources that require the first optical filter to be in the illumination pathway (e.g., first, third, and fifth light sources) can be energized before rotating the frame to position the second optical filter in the illumination pathway for energizing the second and fourth light sources. In some implementations, the first light source produces red illumination light (e.g. between 600 and 650 nm), the second light source10XG / 1823PC 38100-182300WC) produces yellow illumination light (e.g., between 550 and 600 nm), the third light source produces green illumination light (e.g., between 510 and 550 nm), the fourth light source produces blue illumination light (e.g., between 425 and 500 nm), and the fifth light source produces near ultraviolet light source (e.g. between 300 and 425 nm). In other implementations, other combinations of light sources can be used. The order of energizing the light sources can be starting from shortest wavelength to longest wavelength, longest to shortest, or a different sequence.

[0132] FIG. 17 depicts assembly rotational positions relative to active illumination color channel for a five-color channel illumination system. As shown in FIG. 17, for yellow (YEL) and blue (BLU) illumination channels, the assembly is rotated to the first position (Pl). For red (RED), green (GRN), and near ultraviolet (NUV) illumination color channels, the assembly is rotated to the second position (P2). In various implementations, the dichroic filter that is in the illumination pathway in the first position (Pl) has reflection bands corresponding to spectra of the yellow and blue illumination color channels. In various implementations, the dichroic filter that is in the illumination pathway in the second position (P2) has reflection bands corresponding to spectra of the red, green, and near ultraviolet illumination color channels. In various implementations, the assembly starts in the second position (P2) to while the red color channel is imaged, rotates to the first position (Pl) while the yellow color channel is imaged, rotates to the second position (P2) to while the green color channel is imaged, rotates to the first position (Pl) while the blue color channel is imaged, and, optionally, rotates to the second position (P2) to while the near ultraviolet color channel is imaged. In various implementations, the order is reversed such that the assembly starts in the second position (P2) to while the near ultraviolet color channel is imaged, rotates to the first position (Pl) while the blue color channel is imaged, rotates to the second position (P2) to while the green color channel is imaged, rotates to the first position (Pl) while the yellow color channel is imaged, and rotates to the second position (P2) to while the red color channel is imaged. In various implementations, to minimize the amount of rotations (flipping), a specific color channel order is illuminated and imaged. In various implementations, the blue and yellow color channels are imaged (in any permutation of order) first while the assembly remains in the first position (Pl) and then the assembly rotates to the second position (P2) where the red, green, and / or near ultraviolet color channels are imaged (in any permutation of order). In various implementations, the red, green, and / or near ultraviolet color channels are imaged (in any permutation of order) first while the assembly remains in the second position (P2) and then the assembly rotates to the first position (Pl)10XG / 1823PC 39where the blue and yellow color channels are imaged (in any permutation of order). In this way, the assembly only needs to rotate once for the optical system to complete a full cycle of imaging all color channels (e.g., all 5 color channels).

[0133] FIGS. 18A-18B depict a clamping member 1820 according to a first example. The clamping member 1820 is similar to the clamping member 720 shown in FIGS. 7-11 and can be used in any of the assemblies, or with any of the methods, described herein to clamp one or more alignment members (configured to hold a shaft) against one or more reference surfaces to thereby align a rotational axis of the shaft with the reference surface. The clamping member 1820 includes one or more (e.g., one) through hole 722 configured to receive a fixation member (e.g., a shoulder screw) to secure the clamping member 1820 to an optical bench (e.g., optical bench 810). The clamping member 1820 includes a recess 750 configured to receive the end plate 724. In various embodiments, a depth of the recess 750 is about equal to a thickness of the end plate 724. In some embodiments, the depth of the recess 750 is greater than a thickness of the end plate 724. In some embodiments, the depth of the recess 750 is less than a thickness of the end plate 724. The clamping member 1820 includes one or more (e.g., one, two, three, etc.) holes 752 configured to receive a fixation member (e.g., a screw) configured to secure the end plate 724 to the clamping member 1820. For example, a screw may be inserted into a through hole in the end plate, into the hole 752, and secured by engaging the threads of the screw to counterthreads in the hole 752. As shown in FIG. 18B, the clamping member 1820 includes a clamping surface 721 that is integrally formed with the clamping member 1820. For example, the clamping surface 721 can be machined by removing material from the clamping member 1820 using a machine tool. As described above, the clamping surface 721 can have any suitable angle relative to the horizontal, such as 45 degrees.

[0134] FIGS. 19A-19B depict a clamping member according to a second example. The clamping member 1920 is similar to the clamping member 720 shown in FIGS. 7-11 and clamping member 1820 shown in FIGS. 18A-18B and can be used in any of the assemblies, or with any of the methods, described herein to clamp one or more alignment members (configured to hold a shaft) against one or more reference surfaces to thereby align a rotational axis of the shaft with the reference surface. The clamping member 1920 includes one or more through holes 722 configured to receive a fixation member (e.g., a shoulder screw) to secure the clamping member 1920 to an optical bench (e.g., optical bench 810). The clamping member 1920 includes a recess 750 configured to receive the end plate 724. In various embodiments, a depth of the recess 750 is about equal to a thickness of the end plate 724. In10XG / 1823PC 40some embodiments, the depth of the recess 750 is greater than a thickness of the end plate 724. In some embodiments, the depth of the recess 750 is less than a thickness of the end plate 724. The clamping member 1920 includes one or more (e.g., one, two, three, etc.) holes 752 configured to receive a fixation member (e.g., a screw) configured to secure the end plate 724 to the clamping member 1920. For example, a screw may be inserted into a through hole in the end plate, into the hole 752, and secured by engaging the threads of the screw to counterthreads in the hole 752. As shown in FIG. 19B, the clamping member 1920 includes a clamping surface 721 that is integrally formed with the clamping member 1920. For example, the clamping surface 721 can be machined by removing material from the clamping member 1920 using a machine tool. As described above, the clamping surface 721 can have any suitable angle relative to the horizontal, such as 45 degrees. As shown in FIG. 19B, the clamping surface 721 is formed between a first cutout section 725a and a second cutout section 725b of the clamping member 1920.

[0135] FIGS. 19C-19D depict the clamping member 1920 according to the second example within an assembly. When assembled together with a motor 730, second shaft 732, flexible coupling 734, first shaft 710, alignment members 712, 716, the clamping member 1920 presses against the alignment members 712, 716 against the reference surfaces of the optical bench (e.g., optical bench 810) thereby aligning the first shaft 710 to the reference surfaces.

[0136] FIG. 20A depicts a clamping member 2020 according to a third example. The clamping member 2020 is similar to the clamping member 720 shown in FIGS. 7-11 and clamping members 1820, 1920 shown in FIGS. 18A-19D and can be used in any of the assemblies, or with any of the methods, described herein to clamp one or more alignment members (configured to hold a shaft) against one or more reference surfaces to thereby align a rotational axis of the shaft with the reference surface. The clamping member 2020 includes one or more (e.g., one) through hole 722 configured to receive a fixation member (e.g., a shoulder screw) to secure the clamping member 2020 to an optical bench (e.g., optical bench 810). The clamping member 2020 includes a recess 750 configured to receive the end plate 724. In various embodiments, a depth of the recess 750 is about equal to a thickness of the end plate 724. In some embodiments, the depth of the recess 750 is greater than a thickness of the end plate 724. In some embodiments, the depth of the recess 750 is less than a thickness of the end plate 724. The clamping member 2020 may include one or more (e.g., one, two, three, etc.) holes (e.g., holes 752) configured to receive a fixation member (e.g., a screw) configured to secure the end plate 724 to the clamping member 2020. In FIG. 20A, the clamping surface10XG / 1823PC 41is formed (e.g., machined) to include two discrete clamping surfaces (e.g., to minimize material and reduce component weight compared to a single continuous clamping surface): a first clamping surface 721a configured to contact the first alignment member 712 and a second clamping surface 721b configured to contact the second alignment member 716. As described above, the clamping surfaces 721a, 721b can have any suitable angle relative to the horizontal, such as 45 degrees.

[0137] FIGS. 20B-20E depict the clamping member 2020 according to the third example within an assembly. When assembled together with a motor (not shown), second shaft (not shown), flexible coupling (not shown), first shaft 710, and alignment members 712, 716, the clamping member 2020 presses against the alignment members 712, 716 against the reference surfaces of the optical bench e.g., optical bench 810) thereby aligning the first shaft 710 to the reference surfaces 714a, 714b (collectively, reference surface 714). In some embodiments, the reference surfaces 714a, 714b, may be formed as a single continuous reference surface 714 or as one or more discrete reference surfaces 714a, 714b (e.g., having an abrupt change of angle therebetween).

[0138] FIGS. 21A-21C depict a clamping member 2120 according to a fourth example. The clamping member 2120 is similar to the clamping member 720 shown in FIGS. 7-11 and 18A- 20E and can be used in any of the assemblies, or with any of the methods, described herein to clamp one or more alignment members (configured to hold a shaft) against one or more reference surfaces to thereby align a rotational axis of the shaft with the reference surface. The clamping member 2020 includes one or more (e.g., one) through hole 722 configured to receive a fixation member (e.g., a shoulder screw) to secure the clamping member 2120 to an optical bench (e.g., optical bench 810). The clamping member 2120 includes a recess 750 configured to receive the end plate 724. In various embodiments, a depth of the recess 750 is about equal to a thickness of the end plate 724. In some embodiments, the depth of the recess 750 is greater than a thickness of the end plate 724. In some embodiments, the depth of the recess 750 is less than a thickness of the end plate 724. The clamping member 2020 may include one or more (e.g., one, two, three, etc.) holes (e.g., holes 752) configured to receive a fixation member (e.g., a screw) configured to secure the end plate 724 to the clamping member 2120.

[0139] In FIGS. 21A-21C, the clamping surface is formed as a separate clamping component 723 that is secured to the clamping member 2120 (e.g., disposed within a recess formed into the clamping member 2120). The clamping component 723 includes a movable clamping base 723a, a fixation member 723b (e.g., shoulder screw), and a spring member 723c. The spring10XG / 1823PC 42member 723c (e.g., compression spring) is positioned on the fixation member 723b (and may be preloaded prior to subsequent steps) and the fixation member 723b is secured to the clamping base 723a e.g., inserted through a through hole in the clamping base 723a and secured in a threaded hole of the clamping member 2120) such that the spring member 723c provides a biasing force against the clamping base 723a. A first end of the spring is secured against the fixation member 723b, e.g., an inner diameter of the spring member 723c is less than a diameter of a head of the fixation member and the inner diameter of the spring member 723c is greater than a diameter of a body of the fixation member such that the spring member 723c can fit over the body. If the spring member 723c is preloaded, the spring member 723c may have a first length when resting, and a second length when preloaded that is shorter than the first length. The spring member 723c may be preloaded by pushing the clamping base 723a against the spring member 723c. When a force having an upward vertical component is applied to the clamping base 723a (e.g., when the clamping base presses against alignment members 712, 716), an end of the spring member 723c is displaced and the spring member 723c provides a reaction force on the clamping base 723a that is proportional to the displacement (Hookes Law, F=-kx). In this way, the clamping member 2120 can adapt to different sizes of alignment members 712, 716 while also providing a clamping force configured to maintain the first shaft 710 in alignment with the reference surface(s) of the optics bench. Similar to the clamping surfaces 721a, 721b in FIG. 20A, the clamping surfaces 721a, 721b on the clamping base 723a are formed (e.g., machined) as two or more discrete clamping surfaces (e.g., to minimize material and reduce component weight compared to a single continuous clamping surface): a first clamping surface 721a configured to contact the first alignment member 712 and a second clamping surface 721b configured to contact the second alignment member 716. In some embodiments, the clamping surface of the clamping base 723a can be a single continuous clamping surface. As described above, the clamping surfaces 721a, 721b can have any suitable angle relative to the horizontal, such as 45 degrees.General terminology:

[0140] Specific terminology is used throughout this disclosure to explain various aspects of the methods, systems, and compositions that are described. Unless otherwise defined, all of the technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art in the field to which this disclosure belongs.

[0141] It is to be understood that certain terminology is used in the preceding description for convenience and is not limiting. The terms “a”, “an” and “the” should be read as meaning “at10XG / 1823PC 43least one” unless otherwise specified. The term “comprising” will be understood to mean “including but not limited to” such that systems or method comprising a particular feature or step are not limited to only those features or steps listed but may also comprise features or steps not listed. Similarly, any features included as examples are not to be construed as limiting to the disclosure. Additionally, any combination of features from one implementation with features from one or more other example(s) is to be understood as within the present disclosure. Equally, terms such as “after”, “before”, “in front”, “behind”, “downstream”, “upstream” and so on are used for convenience in interpreting the drawings and are not necessarily to be construed as limiting in absolute terms. Additionally, any method steps which are depicted in the figures as carried out sequentially, without causal connection, may alternatively be carried out in series in any order. Further, any method steps which are depicted as dashed or dotted flowchart boxes are to be understood as being optional.

[0142] As used herein, the term “amplitude” refers to a signed value (e.g., +1, -1, +0.1, -0.1, +0.01, -0.01, etc.) representing direction of movement of a pixel in an image. In a first example, the amplitude indicates a direction of movement (e.g., towards an attraction basin) using single, discrete values for a positive direction, a negative direction, and no movement along a given dimension (e.g., x-dimension, y-dimension, and / or z-dimension). In some embodiments, the amplitude is a whole integer selected from a set of {-1, 0, +1 } that indicates a direction of motion. In this case, a positive 1 indicates motion in a first direction (e.g., up / +y) along the given dimension (e.g., the vertical dimension / y). A negative value of the amplitude indicates motion in a second, opposite, direction (e.g., down / — y) along the given dimension (e.g., the vertical dimension / y). A zero value indicates no motion in the given dimension. In other embodiments, the amplitude is a signed probability value. In particular, an amplitude on the interval [-1,1] is provided corresponding to a given dimension (e.g., x-dimension, y-dimension, or z-dimension) of an image. In this case, a positive value of the amplitude indicates the probability of movement in a first direction (e.g., up / +y) along the given dimension (e.g., the vertical dimension / y). A negative value of the amplitude indicates the probability of movement in a second, opposite, direction (e.g., down / -y) along the given dimension (e.g., the vertical dimension / y). A zero value indicates no motion in the given dimension. The magnitude of an amplitude refers to the absolute value of the amplitude, that is the magnitude is without direction. For ease of reference, a pixel having a zero amplitude or an amplitude of low magnitude (i.e., below a given threshold) is referred to as stationary. A pixel having magnitude exceeding that threshold are referred to as moving.10XG / 1823PC 44

[0143] As used herein, the term “flow” as applied to pixels refers to the piecewise path from a pixel through zero or more intermediate pixels to a basin conforming to the amplitudes of those pixels. For example, a pixel that is adjacent to a basin pixel and has an amplitude indicating movement towards the basin pixel has length one flow to the basin. A piecewise path may be constructed from pixel to pixel according to the movement indicated by each pixel’s amplitude until arrival at a basin.

[0144] With reference to pixels of an image, adjacent pixels are those that share an edge or a corner.

[0145] As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. For example, "a" or "an" means "at least one" or "one or more". Any reference to “or” herein is intended to encompass “and / or” unless otherwise stated.

[0146] As used herein, the terms "comprising" (and any form or variant of comprising, such as "comprise" and "comprises"), "having" (and any form or variant of having, such as "have" and "has"), "including" (and any form or variant of including, such as "includes" and "include"), or "containing" (and any form or variant of containing, such as "contains" and "contain"), are inclusive or open-ended and do not exclude additional, un-recited additives, components, integers, elements or method steps.

[0147] As used herein, the term “about” a number refers to that number plus or minus 10% of that number. The term ‘about’ when used in the context of a range refers to that range minus 10% of its lowest value and plus 10% of its greatest value.

[0148] Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits,10XG / 1823PC 45ranges excluding either or both of those included limits are also included in the claimed subject matter. This applies regardless of the breadth of the range.

[0149] Use of ordinal terms such as “first”, “second”, “third”, etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements. Similarly, use of a), b), etc., or i), ii), etc. does not by itself connote any priority, precedence, or order of steps in the claims. Similarly, the use of these terms in the specification does not by itself connote any required priority, precedence, or order.

[0150] The term “platform” (or “system”) may refer to an ensemble of: (i) instruments (e.g., imaging instruments, fluid controllers, temperature controllers, motion controllers and translation stages, etc.), (ii) devices (e.g., specimen slides, substrates, flow cells, microfluidic devices, etc., which may comprise fixed and / or removable or disposable components of the platform), (iii) reagents and / or reagent kits, and (iv) software, or any combination thereof, which allows a user to perform one or more bioassay methods (e.g., analyte detection, in situ detection or sequencing, and / or nucleic acid detection or sequencing) depending on the particular combination of instruments, devices, reagents, reagent kits, and / or software utilized.

[0151] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.Barcoding and decoding terminology:

[0152] A “barcode” is a label, or identifier, that conveys or is capable of conveying information (e.g., information about an analyte in a sample, a cell, a bead, a location, a sample, and / or a capture probe). The term “barcode” may refer either to a physical barcode molecule (e.g., a nucleic acid barcode molecule) or to its representation in a computer-readable, digital format (e.g., as a string of characters representing the sequence of bases in a nucleic acid barcode molecule).

[0153] The phrase “barcode diversity” refers to the total number of unique barcode sequences that may be represented by a given set of barcodes.

[0154] A physical barcode molecule (e.g., a nucleic acid barcode molecule) that forms a label or identifier as described above. In some instances, a barcode can be part of an analyte, can be independent of an analyte, can be attached to an analyte, or can be attached to or part of a probe10XG / 1823PC 46that targets the analyte. In some instances, a particular barcode can be unique relative to other barcodes.

[0155] Physical barcodes can have a variety of different formats. For example, barcodes can include polynucleotide barcodes, random nucleic acid and / or amino acid sequences, and synthetic nucleic acid and / or amino acid sequences. A physical barcode can be attached to an analyte, or to another moiety or structure, in a reversible or irreversible manner. A physical barcode can be added to, for example, a fragment of a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sample before or during sequencing of the sample. In some instances, barcodes can allow for identification and / or quantification of individual sequencing-reads in sequencing-based methods (e.g., a barcode can be or can include a unique molecular identifier or “UMI”). Barcodes can be used to detect and spatially-resolve molecular components found in biological samples, for example, at single-cell resolution (e.g., a barcode can be, or can include, a molecular barcode, a spatial barcode, a unique molecular identifier (UMI), etc.).

[0156] In some instances, barcodes may comprise a series of two or more segments or subbarcodes (e.g., corresponding to “letters” or “code words” in a decoded barcode), each of which may comprise one or more of the subunits or building blocks used to synthesize the physical (e.g., nucleic acid) barcode molecules. For example, a nucleic acid barcode molecule may comprise two or more barcode segments, each of which comprises one or more nucleotides. In some instances, a barcode may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 segments. In some instances, each segment of a barcode molecule may comprise at least 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, or more than 20 subunits or building blocks. For example, each segment of a nucleic acid barcode molecule may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, or more than 20 nucleotides. In some instances, two or more of the segments of a barcode may be separated by non-barcode segments, i.e., the segments of a barcode molecule need not be contiguous.

[0157] A “digital barcode” (or “digital barcode sequence”) is a representation of a corresponding physical barcode (or target analyte sequence) in a computer-readable, digital format as described above. A digital barcode may comprise one or more “letters” (e.g., 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, or more than 20 letters) or one or more “code words” (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 code words), where a “code word” comprises, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, or more than 20 letters. In some instances, the sequence of letters or code words in a digital barcode sequence may correspond directly with the sequence of building blocks (e.g., nucleotides) in a physical barcode. In some10XG / 1823PC 47100-182300WC) instances, the sequence of letters or code words in a digital barcode sequence may not correspond directly with the sequence of building blocks in a physical barcode, but rather may comprise, e.g., arbitrary code words that each correspond to a segment of a physical barcode. For example, in some instances, the disclosed methods for decoding and error correction may be applied directly to detecting target analyte sequences e.g., mRNA sequences) as opposed to detecting target barcodes, and the barcode probes used to detect the target analyte sequences may correspond to letters or code words that have been assigned to specific target analyte sequences but that do not directly correspond to the target analyte sequences.

[0158] A “designed barcode” (or “designed barcode sequence”) is a barcode (or its digital equivalent; in some instances a designed barcode may comprise a series of code words that can be assigned to gene transcripts and subsequently decoded into a decoded barcode) that meets a specified set of design criteria as required for a specific application. In some instances, a set of designed barcodes may comprise at least 2, at least 5, at least 10, at least 20, at least 40, at least 60, at least 80, at least 100, at least 200, at least 400, at least 600, at least 800, at least 1,000, at least 2,000, at least 4,000, at least 6,000, at least 8,000, at least 10,000, at least 20,000, at least 40,000, at least 60,000, at least 80,000, at least 100,000, at least 200,000, at least 400,000, at least 600,000, at least 800,000, at least 1,000,000, at least 2 x 106, at least 3 x 106, at least 4 x106, at least 5 x 106, at least 6 x 106, at least 7 x 106, at least 8 x 106, at least 9 x 106, at least107, at least 108, at least 109, or more than 109unique barcodes. In some instances, a set of designed barcodes may comprise any number of designed barcodes within the range of values in this paragraph, e.g., 1,225 unique barcodes or 2.38 x 106unique barcodes. As noted above for barcodes in general, in some instances designed barcodes may comprise two or more segments (corresponding to two or more code words in a decode barcode). In those cases, the specified set of design criteria may be applied to the designed barcodes as a whole, or to one or more segments (or positions) within the designed barcodes.

[0159] A “decoded barcode” (or “decoded barcode sequence”) is a digital barcode sequence generated via a decoding process that ideally matches a designed barcode sequence, but that may include errors arising from noise in the synthesis process used to create barcodes and / or noise in the decoding process itself. As noted above, in some instances, the disclosed methods for decoding and error correction may be applied directly to detecting target analytes (e.g., mRNA sequences) as opposed to detecting target barcodes, and the barcode probes used to detect the target analytes may correspond to letters or code words that have been assigned to specific target analytes but that do not directly correspond to the target analytes. In these10XG / 1823PC 48instances, a decoded barcode (z.e., a series of letters or code words) may serve as a proxy for the target analyte.

[0160] A “corrected barcode” (or “corrected barcode sequence”) is a digital barcode sequence derived from a decoded barcode sequence by applying one or more error correction methods.Probe terminology:

[0161] The term “probe” may refer either to a physical probe molecule (e.g., a nucleic acid probe molecule) or to its representation in a computer-readable, digital format (e.g., as a string of characters representing the sequence of bases in a nucleic acid probe molecule). A “probe” may be, for example, a molecule designed to recognize (and bind or hybridize to) another molecule, e.g., a target analyte, another probe molecule, etc.

[0162] In some instances, a physical probe molecule may comprise one or more of the following: (i) a target recognition element (e.g., an antibody capable of recognizing and binding to a target peptide, protein, or small molecule; an oligonucleotide sequence that is complementary to a target gene sequence or gene transcript; or a poly-T oligonucleotide sequence that is complementary to the poly-A tails on messenger RNA molecules), (ii) a barcode element (e.g., a molecular barcode, a cell barcode, a spatial barcode, and / or a unique molecular identifier (UMI)), (iii) an amplification and / or sequencing primer binding site, (iv) one or more linker regions, (v) one or more detectable tags (e.g., fluorophores), or any combination thereof. In some instances, each component of a probe molecule may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, or more than 20 subunits or building blocks. For example, in some instances, each component of a nucleic acid probe molecule may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, or more than 20 nucleotides.

[0163] In some instances, physical probes may bind or hybridize directly to their target. In some instances, physical probes may bind or hybridize indirectly to their target. For example, in some instances, a secondary probe may bind or hybridize to a primary probe, where the primary probe binds or hybridizes directly to the target analyte. In some instances, a tertiary probe may bind or hybridize to a secondary probe, where the secondary probe binds or hybridizes to a primary probe, and where the primary probe binds or hybridizes directly to the target analyte.

[0164] Examples of “probes” and their applications include, but are not limited to, primary probes (e.g., molecules designed to recognize and bind or hybridize to target analyte), intermediate probes (e.g., molecules designed to recognize and bind or hybridize to another10XG / 1823PC 49molecule and provide a hybridization or binding site for another probe (e.g., a detection probe), detection probes e.g., molecules designed to recognize and bind or hybridize to another molecule, detection probes may be labeled with a fluorophore or other detectable tag). In some instances, a probe may be designed to recognize and bind (or hybridize) to a physical barcode sequence (or segments thereof). In some instances, a probe may be used to detect and decode a barcode, e.g., a nucleic acid barcode. In some instances, a probe may bind or hybridize directly to a target barcode. In some instances, a probe may bind or hybridize indirectly to a target barcode (e.g., by binding or hybridizing to other probe molecules which itself is bound or hybridized to the target barcode).Nucleic acid molecule and nucleotide terminology:

[0165] The terms “nucleic acid” (or “nucleic acid molecule”) and “nucleotide” are intended to be consistent with their use in the art and to include naturally- occurring species or functional analogs thereof. Particularly useful functional analogs of nucleic acids are capable of hybridizing to a nucleic acid in a sequence- specific fashion (e.g., capable of hybridizing to two nucleic acids such that ligation can occur between the two hybridized nucleic acids) or are capable of being used as a template for replication of a particular nucleotide sequence. Naturally-occurring nucleic acids generally have a backbone containing phosphodiester bonds. An analog structure can have an alternate backbone linkage including any of a variety of those known in the art. Naturally-occurring nucleic acids generally have a deoxyribose sugar (e.g., found in deoxyribonucleic acid (DNA)) or a ribose sugar (e.g. found in ribonucleic acid (RNA)).

[0166] A nucleic acid can contain nucleotides having any of a variety of analogs of these sugar moieties that are known in the art. A nucleic acid can include natural or non-natural nucleotides. In this regard, a naturally- occurring deoxyribonucleic acid can have one or more bases selected from the group consisting of adenine (A), thymine (T), cytosine (C), or guanine (G), and a ribonucleic acid can have one or more bases selected from the group consisting of uracil (U), adenine (A), cytosine (C), or guanine (G). Useful non-natural bases that can be included in a nucleic acid or nucleotide are known in the art. See, for example, Appella (2009), “Non-Natural Nucleic Acids for Synthetic Biology”, Curr Opin Chem Biol. 13(5-6): 687-696; and Duffy, et al. (2020), “Modified Nucleic Acids: Replication, Evolution, and Next-Generation Therapeutics”, BMC Biology 18:112.Samples:10XG / 1823PC 50

[0167] A sample disclosed herein can be or derived from any biological sample. Methods and compositions disclosed herein may be used for analyzing a biological sample, which may be obtained from a subject using any of a variety of techniques including, but not limited to, biopsy, surgery, and laser capture microscopy (LCM), and generally includes cells and / or other biological material from the subject. In addition to the subjects described above, a biological sample can be obtained from a prokaryote such as a bacterium, an archaea, a virus, or a viroid. A biological sample can also be obtained from non-mammalian organisms (e.g., a plant, an insect, an arachnid, a nematode, a fungus, or an amphibian). A biological sample can also be obtained from a eukaryote, such as a tissue sample, a patient derived organoid (PDO) or patient derived xenograft (PDX). A biological sample from an organism may comprise one or more other organisms or components therefrom. For example, a mammalian tissue section may comprise a prion, a viroid, a virus, a bacterium, a fungus, or components from other organisms, in addition to mammalian cells and non-cellular tissue components. Subjects from which biological samples can be obtained can be healthy or asymptomatic individuals, individuals that have or are suspected of having a disease (e.g., a patient with a disease such as cancer) or a pre-disposition to a disease, and / or individuals in need of therapy or suspected of needing therapy.

[0168] The biological sample can include any number of macromolecules, for example, cellular macromolecules and organelles (e.g., mitochondria and nuclei). The biological sample can be a nucleic acid sample and / or protein sample. The biological sample can be a carbohydrate sample or a lipid sample. The biological sample can be obtained as a tissue sample, such as a tissue section, biopsy, a core biopsy, needle aspirate, or fine needle aspirate. The sample can be a fluid sample, such as a blood sample, urine sample, or saliva sample. The sample can be a skin sample, a colon sample, a cheek swab, a histology sample, a histopathology sample, a plasma or serum sample, a tumor sample, living cells, cultured cells, a clinical sample such as, for example, whole blood or blood-derived products, blood cells, or cultured tissues or cells, including cell suspensions. In some instances, the biological sample may comprise cells which are deposited on a surface.

[0169] Cell-free biological samples can include extracellular macromolecules, e.g., polynucleotides. Extracellular polynucleotides can be isolated from a bodily sample, e.g., blood, plasma, serum, urine, saliva, mucosal excretions, sputum, stool, and tears.10XG / 1823PC 51

[0170] Biological samples can be derived from a homogeneous culture or population of the subjects or organisms mentioned herein or alternatively from a collection of several different organisms, for example, in a community or ecosystem.

[0171] Biological samples can include one or more diseased cells. A diseased cell can have altered metabolic properties, gene expression, protein expression, and / or morphologic features. Examples of diseases include inflammatory disorders, metabolic disorders, nervous system disorders, and cancer. Cancer cells can be derived from solid tumors, hematological malignancies, cell lines, or obtained as circulating tumor cells. Biological samples can also include fetal cells and immune cells.

[0172] In some instances, a substrate herein can be any support that is insoluble in aqueous liquid and which allows for positioning of biological samples, analytes, features, and / or reagents (e.g., probes) on the support. In some instances, a biological sample can be attached to a substrate. Attachment of the biological sample can be irreversible or reversible, depending upon the nature of the sample and subsequent steps in the analytical method. In certain instances, the sample can be attached to the substrate reversibly by applying a suitable polymer coating to the substrate, and contacting the sample to the polymer coating. The sample can then be detached from the substrate, e.g., using an organic solvent that at least partially dissolves the polymer coating. In some instances, the substrate can be coated or functionalized with one or more substances to facilitate attachment of the sample to the substrate. Suitable substances that can be used to coat or functionalize the substrate include, but are not limited to, lectins, poly-lysine, antibodies, and polysaccharides.

[0173] A variety of steps can be performed to prepare or process a biological sample for and / or during an assay. Except where indicated otherwise, the preparative or processing steps described below can generally be combined in any manner and in any order to appropriately prepare or process a particular sample for and / or analysis.Endogenous analytes:

[0174] In some instances, an analyte herein is endogenous to a biological sample and can include nucleic acid analytes and non-nucleic acid analytes. Methods and compositions disclosed herein can be used to analyze nucleic acid analytes (e.g., using a nucleic acid probe or probe set that directly or indirectly hybridizes to a nucleic acid analyte) and / or non-nucleic acid analytes (e.g., using a labelling agent that comprises a reporter oligonucleotide and binds directly or indirectly to a non-nucleic acid analyte) in any suitable combination.10XG / 1823PC 52

[0175] Examples of non-nucleic acid analytes include, but are not limited to, lipids, carbohydrates, peptides, proteins, glycoproteins (N-linked or O-linked), lipoproteins, phosphoproteins, specific phosphorylated or acetylated variants of proteins, amidation variants of proteins, hydroxylation variants of proteins, methylation variants of proteins, ubiquitylation variants of proteins, sulfation variants of proteins, viral coat proteins, extracellular and intracellular proteins, antibodies, and antigen binding fragments. In some instances, the analyte is inside a cell or on a cell surface, such as a transmembrane analyte or one that is attached to the cell membrane. In some instances, the analyte can be an organelle (e.g., nuclei or mitochondria). In some instances, the analyte is an extracellular analyte, such as a secreted analyte. Exemplary analytes include, but are not limited to, a receptor, an antigen, a surface protein, a transmembrane protein, a cluster of differentiation protein, a protein channel, a protein pump, a carrier protein, a phospholipid, a glycoprotein, a glycolipid, a cell-cell interaction protein complex, an antigen-presenting complex, a major histocompatibility complex, an engineered T-cell receptor, a T-cell receptor, a B-cell receptor, a chimeric antigen receptor, an extracellular matrix protein, a posttranslational modification (e.g., phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation or lipidation) state of a cell surface protein, a gap junction, and an adherens junction.

[0176] Examples of nucleic acid analytes include DNA analytes such as single-stranded DNA (ssDNA), double- stranded DNA (dsDNA), genomic DNA, methylated DNA, specific methylated DNA sequences, fragmented DNA, mitochondrial DNA, in situ synthesized PCR products, and RNA / DNA hybrids. The DNA analyte can be a transcript of another nucleic acid molecule (e.g., DNA or RNA such as mRNA) present in a tissue sample.

[0177] Examples of nucleic acid analytes also include RNA analytes such as various types of coding and non-coding RNA. Examples of the different types of RNA analytes include messenger RNA (mRNA), including a nascent RNA, a pre-mRNA, a primary-transcript RNA, and a processed RNA, such as a capped mRNA (e.g., with a 5’ 7-methyl guanosine cap), a polyadenylated mRNA (poly-A tail at the 3’ end), and a spliced mRNA in which one or more introns have been removed. Also included in the analytes disclosed herein are non-capped mRNA, a non-polyadenylated mRNA, and a non-spliced mRNA. The RNA analyte can be a transcript of another nucleic acid molecule (e.g., DNA or RNA such as viral RNA) present in a tissue sample. Examples of a non-coding RNAs (ncRNA) that is not translated into a protein include transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs), as well as small non-coding RNAs such as microRNA (miRNA), small interfering RNA (siRNA), Piwi-interacting RNA10XG / 1823PC 53(piRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), extracellular RNA (exRNA), small Cajal body-specific RNAs (scaRNAs), and the long ncRNAs such as Xist and HOTAIR. The RNA can be small (e.g., less than 200 nucleic acid bases in length) or large (e.g., RNA greater than 200 nucleic acid bases in length). Examples of small RNAs include 5.8S ribosomal RNA (rRNA), 5S rRNA, tRNA, miRNA, siRNA, snoRNAs, piRNA, tRNA-derived small RNA (tsRNA), and small rDNA-derived RNA (srRNA). The RNA can be double-stranded RNA or single-stranded RNA. The RNA can be circular RNA. The RNA can be a bacterial rRNA (e.g., 16s rRNA or 23s rRNA).

[0178] In some instances described herein, an analyte may be a denatured nucleic acid, wherein the resulting denatured nucleic acid is single- stranded. The nucleic acid may be denatured, for example, optionally using formamide, heat, or both formamide and heat. In some instances, the nucleic acid is not denatured for use in a method disclosed herein.

[0179] In certain instances, an analyte can be extracted from a live cell. Processing conditions can be adjusted to ensure that a biological sample remains live during analysis, and analytes are extracted from (or released from) live cells of the sample. Live cell-derived analytes can be obtained only once from the sample, or can be obtained at intervals from a sample that continues to remain in viable condition.

[0180] Methods and compositions disclosed herein can be used to analyze any number of analytes. For example, the number of analytes that are analyzed can be at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 20, at least about 25, at least about 30, at least about 40, at least about 50, at least about 100, at least about 1,000, at least about 10,000, at least about 100,000 or more different analytes present in a region of the sample or within an individual feature of the substrate.

[0181] In any implementation described herein, the analyte comprises a target sequence. In some instances, the target sequence may be endogenous to the sample, generated in the sample, added to the sample, or associated with an analyte in the sample. In some instances, the target sequence is a single- stranded target sequence e.g., a sequence in a rolling circle amplification product). In some instances, the analytes comprise one or more single- stranded target sequences. In one aspect, a first single- stranded target sequence is not identical to a second single-stranded target sequence. In another aspect, a first single-stranded target sequence is10XG / 1823PC 54identical to one or more second single- stranded target sequence. In some instances, the one or more second single-stranded target sequence is comprised in the same analyte (e.g., nucleic acid) as the first single-stranded target sequence. Alternatively, the one or more second singlestranded target sequence is comprised in a different analyte (e.g., nucleic acid) from the first single- stranded target sequence.Labelling agents:

[0182] In some instances, provided herein are methods and compositions for analyzing endogenous analytes (e.g., RNA, ssDNA, and cell surface or intracellular proteins and / or metabolites) in a sample using one or more labelling agents. In some instances, an analyte labelling agent may include an agent that interacts with an analyte (e.g., an endogenous analyte in a sample). In some instances, the labelling agents can comprise a reporter oligonucleotide that is indicative of the analyte or portion thereof interacting with the labelling agent. For example, the reporter oligonucleotide may comprise a barcode sequence that permits identification of the labelling agent. In some cases, the sample contacted by the labelling agent can be further contacted with a probe (e.g., a single- stranded probe sequence), that hybridizes to a reporter oligonucleotide of the labelling agent, in order to identify the analyte associated with the labelling agent. In some instances, the analyte labelling agent comprises an analyte binding moiety and a labelling agent barcode domain comprising one or more barcode sequences, e.g., a barcode sequence that corresponds to the analyte binding moiety and / or the analyte. An analyte binding moiety barcode includes to a barcode that is associated with or otherwise identifies the analyte binding moiety. In some instances, by identifying an analyte binding moiety by identifying its associated analyte binding moiety barcode, the analyte to which the analyte binding moiety binds can also be identified. An analyte binding moiety barcode can be a nucleic acid sequence of a given length and / or sequence that is associated with the analyte binding moiety. An analyte binding moiety barcode can generally include any of the variety of aspects of barcodes described herein.

[0183] In some instances, the method comprises one or more post-fixing (also referred to as post-fixation) steps after contacting the sample with one or more labelling agents.

[0184] In the methods and systems described herein, one or more labelling agents capable of binding to or otherwise coupling to one or more features may be used to characterize analytes, cells and / or cell features. In some instances, cell features include cell surface features. Analytes may include, but are not limited to, a protein, a receptor, an antigen, a surface protein, a10XG / 1823PC 55transmembrane protein, a cluster of differentiation protein, a protein channel, a protein pump, a carrier protein, a phospholipid, a glycoprotein, a glycolipid, a cell-cell interaction protein complex, an antigen-presenting complex, a major histocompatibility complex, an engineered T-cell receptor, a T-cell receptor, a B-cell receptor, a chimeric antigen receptor, a gap junction, an adherens junction, or any combination thereof. In some instances, cell features may include intracellular analytes, such as proteins, protein modifications (e.g., phosphorylation status or other post-translational modifications), nuclear proteins, nuclear membrane proteins, or any combination thereof.

[0185] In some instances, an analyte binding moiety may include any molecule or moiety capable of binding to an analyte (e.g., a biological analyte, e.g., a macromolecular constituent). A labelling agent may include, but is not limited to, a protein, a peptide, an antibody (or an epitope binding fragment thereof), a lipophilic moiety (such as cholesterol), a cell surface receptor binding molecule, a receptor ligand, a small molecule, a bi- specific antibody, a bispecific T-cell engager, a T-cell receptor engager, a B-cell receptor engager, a pro-body, an aptamer, a monobody, an affimer, a darpin, and a protein scaffold, or any combination thereof. The labelling agents can include e.g., are attached to) a reporter oligonucleotide that is indicative of the cell surface feature to which the binding group binds. For example, the reporter oligonucleotide may comprise a barcode sequence that permits identification of the labelling agent. For example, a labelling agent that is specific to one type of cell feature (e.g., a first cell surface feature) may have coupled thereto a first reporter oligonucleotide, while a labelling agent that is specific to a different cell feature (e.g., a second cell surface feature) may have a different reporter oligonucleotide coupled thereto.

[0186] Accordingly, terms such as “stain”, “staining”, “labeling”, and the like, may be used interchangeably to refer to elements, complexes, and macromolecules that allow a substance, structure, organelle, and / or component in a sample to be more easily detected than if said substance, structure, organelle, and / or component had not been stained or stained. For example, a tissue sample treated with a DNA dye such as DAPI (4',6-diamidino-2-phenylindole) makes the nucleus of a cell more visible and makes detection or quantification of such cells easier than if they were not stained. Without being bound by theory or methodology, the labeling described herein may be used to mark a cell, structure, particle, or other target, and may be useful in discovering, determining expression, localization, confirmation, quantification, or measuring properties within a sample. Without limitation, labeling agents disclosed herein include stains, dyes, ligands, antibodies, particles, and other substances that may bind to or be10XG / 1823PC 56localized at certain specific objects or locations. “Labels” or “labeling agents” may also refer to compounds or compositions which are conjugated or fused directly or indirectly to a reagent such as an oligonucleotide as disclosed herein or an antibody, and facilitates detection of the reagent to which it is conjugated or fused. The label may itself be detectable (e.g., radioisotope labels or fluorescent labels) or may catalyze chemical alteration of a substrate compound or composition which is detectable, e.g., an enzymatic label.

[0187] As provided by the invention disclosed herein, one or more features are derived by detecting nuclei, cell membrane, and / or cytoplasm of cells within the input image and / or by extracting features from the detected nuclei, cell membrane, and / or cytoplasm (depending upon the labeling agent(s) utilized within the input image). In some embodiments, features are derived by analyzing cell membrane staining, cell cytoplasm staining, and / or cell nucleus staining. Without being bound by theory or methodology “cytoplasmic staining” may describe a group of pixels arranged in a pattern bearing the morphological characteristics of a cytoplasmic region of a cell. Similarly “membrane staining” may refer to a group of pixels arranged in a pattern bearing the morphological characteristics of a cell membrane, preferably the plasma membrane separating the intracellular environment from the extracellular space; and “nucleus staining” may refer to a group of pixels with strong localized intensity in a pattern bearing the morphological characteristics of a nucleus of the cell. Those of skill in the art will appreciate that the nucleus, cytoplasm, and membrane of a cell have different characteristics and that differently stained tissue samples may reveal different biological features. For example, those of skill would understand that certain cell surface elements and receptors can have staining patterns localized to the membrane or localized to the cytoplasm. Thus, a “membrane” staining pattern may be analytically distinct from a “cytoplasmic” staining pattern. Likewise, a “cytoplasmic” staining pattern and a “nuclear” staining pattern may be analytically distinct.

[0188] In some such embodiments, labels or labelling comprises tissue and / or cell surface staining. Surface stains may include general lipid stains, fluorescent lipid analogues, sugar- binding lectins, label-conjugated protein-specific antibodies, and plasma membrane-specific dyes, stains, and label-conjugated antibodies. Those of skill in the art will appreciate and understand that a biological sample may be stained for different types of and / or cell membrane structures / components. Stains and dyes that label cell nuclei may include hematoxylin dyes, cyanine dyes, Draq dyes, and DAPI stain. Stains and dyes that label the cytoplasm of cells may include eosin dyes, fluorescein dyes, and the like. Alternatively, binding moieties e.g., ligands,10XG / 1823PC 57antibodies, and or peptides) directed / localizing to a cell membrane (e.g., the plasma membrane), the cytoplasm, the nucleus, or other structure / organelle of the cell may be conjugated to a labeling moiety described herein, thereby providing a detectable signal that identifies said membrane, cytoplasm, and / or nucleus. Such labeling can be used individually or in combination to aid in visualization, identification, and quantification of cells.

[0189] In some embodiments of the invention, the labelling described herein may be cell specific e.g., cell-type specific), thus providing the detection of different cell types within a sample. In some embodiments, the invention disclosed herein, or elements thereof, incorporate identification of cell polarity and / or morphology. Cell polarity may refer to an asymmetry in molecular composition or structure between two sides, thus defining a polarity axis along which cellular processes will be differentially regulated. In some such embodiments, the invention incorporates identifying cellular symmetry, including the distribution of structures and / or organelles within the cells. For example and without limitation, the radial symmetry of labeled structures or organelles relative to other stains, e.g., plasma membrane, cytoplasmic and / or nuclear labels, such as the radial staining pattern of cytoskeletal structures or mitochondria relative to nuclear, cytoplasmic, and / or plasma membrane stains / labels in fibroblastic cell types. Similarly, the polarization of structures or organelles relative to other stains, e.g., plasma membrane, cytoplasmic and / or nuclear stains / labels, such as those polarized structures observed in the axonal projections of neuronal cells or the apical / basal polarity of epithelial cells.

[0190] Exemplary methods for staining tissue structures and guidance in the choice of stains appropriate for various purposes are known in the art and are discussed, for example, in “Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)” and “Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley-Intersciences (1987),” the disclosures of which are incorporated herein by reference. For a description of exemplary labelling agents, reporter oligonucleotides, and methods of use, see, e.g., U.S. Pat. 10,550,429; U.S. Pat. Pub. 20190177800; and U.S. Pat. Pub. 20190367969, which are each incorporated by reference herein in their entirety.

[0191] In some instances, an analyte binding moiety includes one or more antibodies or antigen binding fragments thereof. The antibodies or antigen binding fragments including the analyte binding moiety can specifically bind to a target analyte. In some instances, the analyte is a protein (e.g., a protein on a surface of the biological sample (e.g., a cell) or an intracellular protein). In some instances, a plurality of analyte labelling agents comprising a plurality of10XG / 1823PC 58analyte binding moieties bind a plurality of analytes present in a biological sample. In some instances, the plurality of analytes includes a single species of analyte (e.g., a single species of polypeptide). In some instances in which the plurality of analytes includes a single species of analyte, the analyte binding moieties of the plurality of analyte labelling agents are the same. In some instances in which the plurality of analytes includes a single species of analyte, the analyte binding moieties of the plurality of analyte labelling agents are the different (e.g., members of the plurality of analyte labelling agents can have two or more species of analyte binding moieties, wherein each of the two or more species of analyte binding moieties binds a single species of analyte, e.g., at different binding sites). In some instances, the plurality of analytes includes multiple different species of analyte e.g., multiple different species of polypeptides).

[0192] In other instances, e.g., to facilitate sample multiplexing, a labelling agent that is specific to a particular cell feature may have a first plurality of the labelling agent (e.g., an antibody or lipophilic moiety) coupled to a first reporter oligonucleotide and a second plurality of the labelling agent coupled to a second reporter oligonucleotide.

[0193] In some aspects, these reporter oligonucleotides may comprise nucleic acid barcode sequences that permit identification of the labelling agent which the reporter oligonucleotide is coupled to. The selection of oligonucleotides as the reporter may provide advantages of being able to generate significant diversity in terms of sequence, while also being readily attachable to most biomolecules, e.g., antibodies, etc., as well as being readily detected.

[0194] Attachment (coupling) of the reporter oligonucleotides to the labelling agents may be achieved through any of a variety of direct or indirect, covalent or non-covalent associations or attachments. For example, oligonucleotides may be covalently attached to a portion of a labelling agent (such a protein, e.g., an antibody or antibody fragment) using chemical conjugation techniques (e.g., Lightning-Link® antibody labelling kits available from Innova Biosciences), as well as other non-covalent attachment mechanisms, e.g., using biotinylated antibodies and oligonucleotides (or beads that include one or more biotinylated linker, coupled to oligonucleotides) with an avidin or streptavidin linker. Antibody and oligonucleotide biotinylation techniques are available. See, e.g., Fang, et al., “Fluoride-Cleavable Biotinylation Phosphoramidite for 5 '-end- Labelling and Affinity Purification of Synthetic Oligonucleotides,” Nucleic Acids Res. Jan. 15, 2003; 31(2):708-715, which is entirely incorporated herein by reference for all purposes. Likewise, protein and peptide biotinylation techniques have been developed and are readily available. See, e.g., U.S. Pat. No. 6,265,552,10XG / 1823PC 59which is entirely incorporated herein by reference for all purposes. Furthermore, click reaction chemistry may be used to couple reporter oligonucleotides to labelling agents. Commercially available kits, such as those from Thunderlink and Abeam, and techniques common in the art may be used to couple reporter oligonucleotides to labelling agents as appropriate. In another example, a labelling agent is indirectly (e.g., via hybridization) coupled to a reporter oligonucleotide comprising a barcode sequence that identifies the label agent. For instance, the labelling agent may be directly coupled (e.g., covalently bound) to a hybridization oligonucleotide that comprises a sequence that hybridizes with a sequence of the reporter oligonucleotide. Hybridization of the hybridization oligonucleotide to the reporter oligonucleotide couples the labelling agent to the reporter oligonucleotide. In some instances, the reporter oligonucleotides are releasable from the labelling agent, such as upon application of a stimulus. For example, the reporter oligonucleotide may be attached to the labelling agent through a labile bond (e.g., chemically labile, photolabile, thermally labile, etc.) as generally described for releasing molecules from supports elsewhere herein. In some instances, the reporter oligonucleotides described herein may include one or more functional sequences that can be used in subsequent processing, such as an adapter sequence, a unique molecular identifier (UMI) sequence, a sequencer specific flow cell attachment sequence (such as an P5, P7, or partial P5 or P7 sequence), a primer or primer binding sequence, a sequencing primer or primer binding sequence (such as an Rl, R2, or partial R1 or R2 sequence).

[0195] In some cases, the labelling agent can comprise a reporter oligonucleotide and a label. A label can be fluorophore, a radioisotope, a molecule capable of a colorimetric reaction, a magnetic particle, or any other suitable molecule or compound capable of detection. The label can be conjugated to a labelling agent (or reporter oligonucleotide) either directly or indirectly (e.g., the label can be conjugated to a molecule that can bind to the labelling agent or reporter oligonucleotide). In some cases, a label is conjugated to a first oligonucleotide that is complementary (e.g., hybridizes) to a sequence of the reporter oligonucleotide.

[0196] In some instances, multiple different species of analytes (e.g., polypeptides) from the biological sample can be subsequently associated with the one or more physical properties of the biological sample. For example, the multiple different species of analytes can be associated with locations of the analytes in the biological sample. Such information (e.g., proteomic information when the analyte binding moiety(ies) recognizes a polypeptide(s)) can be used in association with other spatial information (e.g., genetic information from the biological sample, such as DNA sequence information, transcriptome information (i.e., sequences of transcripts),10XG / 1823PC 60or both). For example, a cell surface protein of a cell can be associated with one or more physical properties of the cell (e.g., a shape, size, activity, or a type of the cell). The one or more physical properties can be characterized by imaging the cell. The cell can be bound by an analyte labelling agent comprising an analyte binding moiety that binds to the cell surface protein and an analyte binding moiety barcode that identifies that analyte binding moiety. Results of protein analysis in a sample (e.g., a tissue sample or a cell) can be associated with DNA and / or RNA analysis in the sample.Assays for in situ detection and analysis:

[0197] Objectives for in situ detection and analysis methods include detecting, quantifying, and / or mapping analytes (e.g., gene activity) to specific regions in a biological sample (e.g., a tissue sample or cells deposited on a surface) at cellular or sub-cellular resolution. Methods for performing in situ studies include a variety of techniques, e.g., in situ hybridization and in situ sequencing techniques. These techniques allow one to study the subcellular distribution of target analytes (e.g., gene activity as evidenced, e.g., by expressed gene transcripts), and have the potential to provide crucial insights in the fields of developmental biology, oncology, immunology, histology, etc.

[0198] Various methods can be used for in situ detection and analysis of target analytes, e.g., sequencing by synthesis (SBS), sequencing by ligation (SBL), sequencing by hybridization (SBH). Non-limiting examples of in situ hybridization techniques include single molecule fluorescence in situ hybridization (smFISH) and multiplexed error-robust fluorescence in situ hybridization (MERFISH). smFISH enables in situ detection and quantification of gene transcripts in tissue samples at the locations where they reside by making use of libraries of multiple short oligonucleotide probes (e.g., approximately 20 base pairs (bp) in length), each labeled with a fluorophore. The probes are sequentially hybridized to gene sequences (e.g., DNA) or gene transcript sequences (e.g., mRNA) sequences, and visualized as diffractionlimited spots by fluorescence microscopy (Levsky, et al. (2003) “Fluorescence In situ Hybridization: Past, Present and Future”, Journal of Cell Science 116(14): 2833-2838; Raj, et al. (2008) “Imaging Individual mRNA Molecules Using Multiple Singly Labeled Probes”, Nat Methods 5(10): 877-879; Moor, et al. (2016), ibid.). Variations on the smFISH method include, for example, the use of combinatorial labelling schemes to improve multiplexing capability (Levsky, et al. (2003), ibid.), the use of smFISH in combination with super-resolution microscopy (Lubeck, et al. (2014) “Single-Cell In situ RNA Profiling by Sequential Hybridization”, Nature Methods 11(4):360— 361).10XG / 1823PC 61

[0199] MERFISH addresses two of the limitations of earlier in situ hybridization approaches, namely the limited number of target sequences that could be simultaneously identified and the robustness of the approach to readout errors caused by the stochastic nature of the hybridization process (Moor, et al. (2016), ibid.). MERFISH utilizes a binary barcoding scheme in which the probed target mRNA sequences are either fluorescence positive or fluorescence negative for any given imaging cycle (Ke, et al. (2016), ibid.; Moffitt, et al. (2016) “RNA Imaging with Multiplexed Error Robust Fluorescence In situ Hybridization”, Methods Enzymol. 572:1-49). The encoding probes that contain a combination of target- specific hybridization sequence regions and barcoded readout sequence regions are first hybridized to the target mRNA sequences. In each imaging cycle, a subset of fluorophore-conjugated readout probes is hybridized to a subset of encoding probes. Target mRNA sequences that fluoresce in a given cycle are assigned a value of “1” and the remaining target mRNA sequences are assigned a value of “0”. Between imaging cycles, the fluorescent probes from the previous cycle are photobleached. After, e.g., 14 or 16 rounds of readout probe hybridization and imaging, unique combinations of the detected fluorescence signals generate a 14-bit or 16-bit code that identifies the different gene transcripts. To address the increased error rate for correctly calling the readout codes increases as the number of hybridization and imaging cycles increases, the method may also entail the use of Hamming distances for barcode design and correction of decoding errors (see., e.g., Buschmann, et al. (2013) “Levenshtein Error-Correcting Barcodes for Multiplexed DNA Sequencing”, Bioinformatics 14:272), thereby resulting in an error- robust barcoding scheme.

[0200] Some in situ sequencing techniques generally comprise both in situ target capture (e.g. , of mRNA sequences) and in situ sequencing. Non-limiting examples of in situ sequencing techniques include in situ sequencing with padlock probes (ISS-PLP), fluorescent in situ sequencing (FISSEQ), barcode in situ targeted sequencing (Barista-Seq), and spatially- resolved transcript amplicon readout mapping (STARmap) (see, e.g., Ke, et al. (2016), ibid., Asp, et al. (2020), ibid.).

[0201] Some methods for in situ detection and analysis of analytes utilize a probe e.g. , padlock or circular probe) that detects specific target analytes. The in situ sequencing using padlock probes (ISS-PLP) method, for example, combines padlock probing to target specific gene transcripts, rolling-circle amplification (RCA), and sequencing by ligation (SBL) chemistry. Within intact tissue sections, reverse transcription primers are hybridized to target sequence (e.g., mRNA sequences) and reverse transcription is performed to create cDNA to which a10XG / 1823PC 62padlock probe (a single- stranded DNA molecule comprising regions that are complementary to the target cDNA) can bind (see, e.g., Asp, et al. (2020), ibid.). In one variation of the method, the padlock probe binds to the cDNA target with a gap remaining between the ends which is then filled in using a DNA polymerization reaction. In another variation of the method, the ends of the bound padlock probe are adjacent to each other. The ends are then ligated to create a circular DNA molecule. Target amplification using rolling-circle amplification (RCA) results in micrometer-sized RCA products (RCPs), containing a plurality of concatenated repeats of the probe sequence. In some examples, RCPs are then subjected to, e.g., sequencing-by-ligation (SBL) or sequencing-by-hybridization (SBH). In some cases, the method allows for a barcode located within the probe to be decoded.

[0202] Products of endogenous analytes and / or labelling agents:

[0203] In some instances, provided herein are methods and compositions for analyzing one or more products of an endogenous analyte and / or a labelling agent in a biological sample. In some instances, an endogenous analyte (e.g., a viral or cellular DNA or RNA) or a product (e.g., a hybridization product, a ligation product, an extension product (e.g., by a DNA or RNA polymerase), a replication product, a transcription / reverse transcription product, and / or an amplification product such as a rolling circle amplification (RCA) product) thereof is analyzed. In some instances, a labelling agent that directly or indirectly binds to an analyte in the biological sample is analyzed. In some instances, a product (e.g., a hybridization product, a ligation product, an extension product (e.g., by a DNA or RNA polymerase), a replication product, a transcription / reverse transcription product, and / or an amplification product such as a rolling circle amplification (RCA) product) of a labelling agent that directly or indirectly binds to an analyte in the biological sample is analyzed.

[0204] In some instances, the analyzing comprises using primary probes which comprise a target binding region (e.g., a region that binds to a target such as RNA transcripts) and the primary probes may contain one or more barcodes (e.g., primary barcode). In some instances, the barcodes are bound by detection primary probes, which do not need to be fluorescent, but that include a target-binding portion (e.g., for hybridizing to one or more primary probes) and one or more barcodes (e.g., secondary barcodes). In some instances, the detection primary probe comprises an overhang that does not hybridize to the target nucleic acid but hybridizes to another probe. In some examples, the overhang comprises the barcode(s). In some instances, the barcodes of the detection primary probes are targeted by detectably labeled detection oligonucleotides, such as fluorescently labeled oligos. In some instances, one or more decoding10XG / 1823PC 63schemes are used to decode the signals, such as fluorescence, for sequence determination. Various probes and probe sets can be used to hybridize to and detect an endogenous analyte and / or a sequence associated with a labelling agent. In some instances, these assays may enable multiplexed detection, signal amplification, combinatorial decoding, and error correction schemes. Exemplary barcoded probes or probe sets may be based on a padlock probe, a gapped padlock probe, a SNAIL (Splint Nucleotide Assisted Intramolecular Ligation) probe set, a PLAYR (Proximity Ligation Assay for RNA) probe set, a PLISH (Proximity Ligation in situ Hybridization) probe set. The specific probe or probe set design can vary.Hybridization and ligation:

[0205] Various probes and probe sets can be hybridized to an endogenous analyte and / or a labelling agent and each probe may comprise one or more barcode sequences. The specific probe or probe set design can vary. In some instances, the hybridization of a primary probe or probe set (e.g., a circularizable probe or probe set) to a target nucleic acid analyte and may lead to the generation of a rolling circle amplification (RCA) template. In some instances, the assay uses or generates a circular nucleic acid molecule which can be the RCA template.

[0206] In some instances, a product of an endogenous analyte and / or a labelling agent is a ligation product. In some instances, the ligation product is formed from circularization of a circularizable probe or probe set upon hybridization to a target sequence. In some instances, the ligation product is formed between two or more endogenous analytes. In some instances, the ligation product is formed between an endogenous analyte and a labelling agent. In some instances, the ligation product is formed between two or more labelling agent. In some instances, the ligation product is an intramolecular ligation of an endogenous analyte. In some instances, the ligation product is an intramolecular ligation of a labelling agent, for example, the circularization of a circularizable probe or probe set upon hybridization to a target sequence. The target sequence can be comprised in an endogenous analyte (e.g., nucleic acid such as a genomic DNA or mRNA) or a product thereof (e.g., cDNA from a cellular mRNA transcript), or in a labelling agent (e.g., the reporter oligonucleotide) or a product thereof.

[0207] In some instances, provided herein is a probe or probe set capable of DNA-templated ligation, such as from a cDNA molecule. See, e.g., U.S. Pat. 8,551,710, which is hereby incorporated by reference in its entirety. In some instances, provided herein is a probe or probe set capable of RNA-templated ligation. See, e.g., U.S. Pat. Pub. 2020 / 0224244 which is hereby incorporated by reference in its entirety. In some instances, the probe set is a SNAIL probe set.10XG / 1823PC 64100-182300WC)See, e.g., U.S. Pat. Pub. 20190055594, which is hereby incorporated by reference in its entirety. In some instances, provided herein is a multiplexed proximity ligation assay. See, e.g., U.S. Pat. Pub. 20140194311 which is hereby incorporated by reference in its entirety. In some instances, provided herein is a probe or probe set capable of proximity ligation, for instance a proximity ligation assay for RNA (e.g., PLAYR) probe set. See, e.g., U.S. Pat. Pub. 20160108458, which is hereby incorporated by reference in its entirety. In some instances, a circular probe can be indirectly hybridized to the target nucleic acid. In some instances, the circular construct is formed from a probe set capable of proximity ligation, for instance a proximity ligation in situ hybridization (PLISH) probe set. See, e.g., U.S. Pat. Pub. 2020 / 0224243 which is hereby incorporated by reference in its entirety.

[0208] In some instances, the ligation involves chemical ligation. In some instances, the ligation involves template dependent ligation. In some instances, the ligation involves template independent ligation. In some instances, the ligation involves enzymatic ligation.

[0209] In some instances, the enzymatic ligation involves use of a ligase. In some aspects, the ligase used herein comprises an enzyme that is commonly used to join polynucleotides together or to join the ends of a single polynucleotide. An RNA ligase, a DNA ligase, or another variety of ligase can be used to ligate two nucleotide sequences together. Ligases comprise ATP- dependent double-strand polynucleotide ligases, NAD-i-dependent double-strand DNA or RNA ligases and single-strand polynucleotide ligases, for example any of the ligases described in EC 6.5.1.1 (ATP-dependent ligases), EC 6.5.1.2 (NAD+-dependent ligases), EC 6.5.1.3 (RNA ligases). Specific examples of ligases comprise bacterial ligases such as E. coli DNA ligase, Tth DNA ligase, Thermococcus sp. (strain 9° N) DNA ligase (9°N™ DNA ligase, New England Biolabs), Taq DNA ligase, Ampligase™ (Epicentre Biotechnologies) and phage ligases such as T3 DNA ligase, T4 DNA ligase and T7 DNA ligase and mutants thereof. In some instances, the ligase is a T4 RNA ligase. In some instances, the ligase is a splintR ligase. In some instances, the ligase is a single stranded DNA ligase. In some instances, the ligase is a T4 DNA ligase. In some instances, the ligase is a ligase that has an DNA-splinted DNA ligase activity. In some instances, the ligase is a ligase that has an RNA-splinted DNA ligase activity.

[0210] In some instances, the ligation herein is a direct ligation. In some instances, the ligation herein is an indirect ligation. "Direct ligation" means that the ends of the polynucleotides hybridize immediately adjacently to one another to form a substrate for a ligase enzyme resulting in their ligation to each other (intramolecular ligation). Alternatively, "indirect" means that the ends of the polynucleotides hybridize non- adjacently to one another, i.e.,10XG / 1823PC 65100-182300WC) separated by one or more intervening nucleotides or "gaps". In some instances, said ends are not ligated directly to each other, but instead occurs either via the intermediacy of one or more intervening (so-called "gap" or "gap-filling" (oligo)nucleotides) or by the extension of the 3' end of a probe to "fill" the "gap" corresponding to said intervening nucleotides (intermolecular ligation). In some cases, the gap of one or more nucleotides between the hybridized ends of the polynucleotides may be "filled" by one or more "gap" (oligo)nucleotide(s) which are complementary to a splint, padlock probe, or target nucleic acid. The gap may be a gap of 1 to 60 nucleotides or a gap of 1 to 40 nucleotides or a gap of 3 to 40 nucleotides. In specific implementations, the gap may be a gap of about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more nucleotides, of any integer (or range of integers) of nucleotides in between the indicated values. In some instances, the gap between said terminal regions may be filled by a gap oligonucleotide or by extending the 3' end of a polynucleotide. In some cases, ligation involves ligating the ends of the probe to at least one gap (oligo)nucleotide, such that the gap (oligo)nucleotide becomes incorporated into the resulting polynucleotide. In some instances, the ligation herein is preceded by gap filling. In other implementations, the ligation herein does not require gap filling.

[0211] In some instances, ligation of the polynucleotides produces polynucleotides with melting temperature higher than that of un-ligated polynucleotides. Thus, in some aspects, ligation stabilizes the hybridization complex containing the ligated polynucleotides prior to subsequent steps, comprising amplification and detection.

[0212] In some aspects, a high fidelity ligase, such as a thermostable DNA ligase (e.g., a Taq DNA ligase), is used. Thermostable DNA ligases are active at elevated temperatures, allowing further discrimination by incubating the ligation at a temperature near the melting temperature (Tm) of the DNA strands. This selectively reduces the concentration of annealed mismatched substrates (expected to have a slightly lower Tm around the mismatch) over annealed fully base-paired substrates. Thus, high-fidelity ligation can be achieved through a combination of the intrinsic selectivity of the ligase active site and balanced conditions to reduce the incidence of annealed mismatched dsDNA.

[0213] In some instances, the ligation herein is a proximity ligation of ligating two (or more) nucleic acid sequences that are in proximity with each other, e.g., through enzymatic means (e.g., a ligase). In some instances, proximity ligation can include a “gap-filling” step that involves incorporation of one or more nucleic acids by a polymerase, based on the nucleic acid sequence of a template nucleic acid molecule, spanning a distance between the two nucleic10XG / 1823PC 66acid molecules of interest (see, e.g., U.S. Patent No. 7,264,929, the entire contents of which are incorporated herein by reference). A wide variety of different methods can be used for proximity ligating nucleic acid molecules, including (but not limited to) “sticky-end” and “blunt-end” ligations. Additionally, single- stranded ligation can be used to perform proximity ligation on a single- stranded nucleic acid molecule. Sticky-end proximity ligations involve the hybridization of complementary single- stranded sequences between the two nucleic acid molecules to be joined, prior to the ligation event itself. Blunt-end proximity ligations generally do not include hybridization of complementary regions from each nucleic acid molecule because both nucleic acid molecules lack a single- stranded overhang at the site of ligation.Primer extension and amplification:

[0214] In some instances, the hybridization of a primary probe or probe set (e.g. a circularizable probe or probe set) to a target analyte and may lead to the generation of an extension or amplification product. In some instances, a product is a primer extension product of an analyte, a labelling agent, a probe or probe set bound to the analyte (e.g., a circularizable probe bound to genomic DNA, mRNA, or cDNA), or a probe or probe set bound to the labelling agent (e.g., a circularizable probe bound to one or more reporter oligonucleotides from the same or different labelling agents.

[0215] A primer is generally a single-stranded nucleic acid sequence having a 3’ end that can be used as a substrate for a nucleic acid polymerase in a nucleic acid extension reaction. RNA primers are formed of RNA nucleotides, and are used in RNA synthesis, while DNA primers are formed of DNA nucleotides and used in DNA synthesis. Primers can also include both RNA nucleotides and DNA nucleotides (e.g., in a random or designed pattern). Primers can also include other natural or synthetic nucleotides described herein that can have additional functionality. In some examples, DNA primers can be used to prime RNA synthesis and vice versa (e.g., RNA primers can be used to prime DNA synthesis). Primers can vary in length. For example, primers can be about 6 bases to about 120 bases. For example, primers can include up to about 25 bases. A primer, may in some cases, refer to a primer binding sequence. A primer extension reaction generally refers to any method where two nucleic acid sequences become linked (e.g., hybridized) by an overlap of their respective terminal complementary nucleic acid sequences (i.e., for example, 3’ termini). Such linking can be followed by nucleic acid extension (e.g., an enzymatic extension) of one, or both termini using the other nucleic acid sequence as a template for extension. Enzymatic extension can be performed by an enzyme including, but not limited to, a polymerase and / or a reverse transcriptase.10XG / 1823PC 67100-182300WC)

[0216] In some instances, a product of an endogenous analyte and / or a labelling agent is an amplification product of one or more polynucleotides, for instance, a circular probe or circularizable probe or probe set. In some instances, the disclosed methods may comprise the use of a rolling circle amplification (RCA) technique to amplify signal. Rolling circle amplification is an isothermal, DNA polymerase-mediated process in which long singlestranded DNA molecules are synthesized on a short circular single- stranded DNA template using a single DNA primer (Zhao, et al. (2008), “Rolling Circle Amplification: Applications in Nanotechnology and Biodetection with Functional Nucleic Acids”, Angew Chem Int Ed Engl. 47(34):6330-6337; Ali, et al. (2014), “Rolling Circle Amplification: A Versatile Tool for Chemical Biology, Materials Science and Medicine”, Chem Soc Rev. 43(10):3324-3341). The RCA product is a concatemer containing tens to hundreds of tandem repeats that are complementary to the circular template, and may be used to develop sensitive techniques for the detection of a variety of targets, including nucleic acids (DNA, RNA), small molecules, proteins, and cells (Ali, et al. (2014), ibid.). In some implementations, a primer that hybridizes to the circular probe or circularized probe is added and used as such for amplification. In some instances, the RCA comprises a linear RCA, a branched RCA, a dendritic RCA, or any combination thereof.

[0217] In some instances, the amplification is performed at a temperature between or between about 20°C and about 60°C. In some instances, the amplification is performed at a temperature between or between about 30°C and about 40°C. In some aspects, the amplification step, such as the rolling circle amplification (RCA) is performed at a temperature between at or about 25°C and at or about 50°C, such as at or about 25°C, 27°C, 29°C, 31 °C, 33°C, 35°C, 37°C, 39°C, 41°C, 43°C, 45°C, 47°C, or 49°C.

[0218] In some instances, upon addition of a DNA polymerase in the presence of appropriate dNTP precursors and other cofactors, a primer is elongated to produce multiple copies of the circular template. This amplification step can utilize isothermal amplification or nonisothermal amplification. In some instances, after the formation of the hybridization complex and association of the amplification probe, the hybridization complex is rolling-circle amplified to generate a cDNA nanoball (z.e., amplicon) containing multiple copies of the cDNA. Techniques for rolling circle amplification (RCA) are known in the art such as linear RCA, a branched RCA, a dendritic RCA, or any combination thereof. (See, e.g., Baner et al, Nucleic Acids Research, 26:5073-5078, 1998; Lizardi et al, Nature Genetics 19:226, 1998; Mohsen et al., Acc Chem Res. 2016 November 15; 49(11): 2540-2550; Schweitzer et al. Proc. Natl Acad.10XG / 1823PC 68Sci. USA 97:101 13- 1 19, 2000; Faruqi et al, BMC Genomics 2:4, 2000; Nallur et al, Nucl. Acids Res. 29:el 18, 2001; Dean et al. Genome Res. 1 1 :1095- 1099, 2001; Schweitzer et al, Nature Biotech. 20:359-365, 2002; U.S. Patent Nos. 6,054,274, 6,291,187, 6,323,009, 6,344,329 and 6,368,801). Exemplary polymerases for use in RCA comprise DNA polymerase such phi29 (cp29) polymerase, Klenow fragment, Bacillus stearothe rmophilus DNA polymerase (BST), T4 DNA polymerase, T7 DNA polymerase, or DNA polymerase I. In some aspects, DNA polymerases that have been engineered or mutated to have desirable characteristics can be employed. In some instances, the polymerase is phi29 DNA polymerase.

[0219] In some aspects, during the amplification step, modified nucleotides can be added to the reaction to incorporate the modified nucleotides in the amplification product (e.g., nanoball). Exemplary of the modified nucleotides comprise amine-modified nucleotides. In some aspects of the methods, for example, for anchoring or cross-linking of the generated amplification product (e.g., nanoball) to a scaffold, to cellular structures and / or to other amplification products (e.g., other nanoballs). In some aspects, the amplification products comprises a modified nucleotide, such as an amine-modified nucleotide. In some instances, the amine-modified nucleotide comprises an acrylic acid N- hydroxy succinimide moiety modification. Examples of other amine-modified nucleotides comprise, but are not limited to, a 5-Aminoallyl-dUTP moiety modification, a 5-Propargylamino-dCTP moiety modification, a N6-6-Aminohexyl-dATP moiety modification, or a 7-Deaza-7-Propargylamino-dATP moiety modification.

[0220] In some instances, the RCA template may comprise the target analyte, or a part thereof, where the target analyte is a nucleic acid, or it may be provided or generated as a proxy, or a marker, for the analyte. In some instances, the RCA template may comprise a sequence of the probes and probe sets hybridized to an endogenous analyte and / or a labelling agent. In some instances, the amplification product can be generated as a proxy, or a marker, for the analyte. As noted above, many assays are known for the detection of numerous different analytes, which use a RCA-based detection system, e.g., where the signal is provided by generating a RCP from a circular RCA template which is provided or generated in the assay, and the RCP is detected to detect the analyte. The RCP may thus be regarded as a reporter which is detected to detect the target analyte. However, the RCA template may also be regarded as a reporter for the target analyte; the RCP is generated based on the RCA template, and comprises complementary copies of the RCA template. The RCA template determines the signal which is detected, and is thus indicative of the target analyte. As will be described in more detail below, the RCA10XG / 1823PC 69template may be a probe, or a part or component of a probe, or may be generated from a probe, or it may be a component of a detection assay (z.e. a reagent in a detection assay), which is used as a reporter for the assay, or a part of a reporter, or signal-generation system. The RCA template used to generate the RCP may thus be a circular (e.g. circularized) reporter nucleic acid molecule, namely from any RCA-based detection assay which uses or generates a circular nucleic acid molecule as a reporter for the assay. Since the RCA template generates the RCP reporter, it may be viewed as part of the reporter system for the assay.

[0221] In some instances, an assay may detect a product herein that includes a molecule or a complex generated in a series of reactions, e.g., hybridization, ligation, extension, replication, transcription / reverse transcription, and / or amplification e.g., rolling circle amplification), in any suitable combination. For example, a product comprising a target sequence for a probe disclosed herein (e.g., a bridge probe or L-probe) may be a hybridization complex formed of a cellular nucleic acid in a sample and an exogenously added nucleic acid probe. The exogenously added nucleic acid probe may comprise an overhang that does not hybridize to the cellular nucleic acid but hybridizes to another probe (e.g., a detection probe). The exogenously added nucleic acid probe may be optionally ligated to a cellular nucleic acid molecule or another exogenous nucleic acid molecule. In other examples, a product comprising a target sequence for a probe disclosed herein (e.g., an anchor probe) may be an RCP of a circularizable probe or probe set which hybridizes to a cellular nucleic acid molecule (e.g., genomic DNA or mRNA) or product thereof (e.g., a transcript such as cDNA, a DNA- templated ligation product of two probes, or an RNA-templated ligation product of two probes). In other examples, a product comprising a target sequence for a probe disclosed herein (e.g., a bridge probe or L-probe) may be a probe hybridizing to an RCP. The probe may comprise an overhang that does not hybridize to the RCP but hybridizes to another probe (e.g., a detection probe).Signal amplification methods:

[0222] In some instances, a method disclosed herein may also comprise one or more signal amplification components and detecting such signals. In some instances, the present disclosure relates to the detection of nucleic acid sequences in situ using probe hybridization and generation of amplified signals associated with the probes. In some instances, the target nucleic acid of a nucleic acid probe comprises multiple target sequences for nucleic acid probe10XG / 1823PC 70hybridization, such that the signal corresponding to a barcode sequence of the nucleic acid probe is amplified by the presence of multiple nucleic acid probes hybridized to the target nucleic acid. For example, multiple sequences can be selected from a target nucleic acid such as an mRNA, such that a group of nucleic acid probes (e.g., 20-50 nucleic acid probes) hybridize to the mRNA in a tiled fashion. In another example, the target nucleic acid can be an amplification product (e.g., an RCA product) comprising multiple copies of a target sequence (e.g., a barcode sequence of the RCA product).

[0223] Alternatively or additionally, amplification of a signal associated with a barcode sequence of a nucleic acid probe can be amplified using one or more signal amplification strategies off of an oligonucleotide probe that hybridizes to the barcode sequence. In some aspects, amplification of the signal associated with the oligonucleotide probe can reduce the number of nucleic acid probes needed to hybridize to the target nucleic acid to obtain a sufficient signal-to-noise ratio. For example, the number of nucleic acid probes to tile a target nucleic acid such as an mRNA can be reduced. In some aspects, reducing the number of nucleic acid probes tiling a target nucleic acid enables detection of shorter target nucleic acids, such as shorter mRNAs. In some instances, no more than one, two, three, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18. 19, or 20 nucleic acid probes may be hybridized to the target nucleic acid. In instances wherein the target nucleic acid is an amplification product, signal amplification off of the oligonucleotide probes may reduce the number of target sequences required for detection (e.g., the length of the RCA product can be reduced).

[0224] Exemplary signal amplification methods include targeted deposition of detectable reactive molecules around the site of probe hybridization, targeted assembly of branched structures (e.g., bDNA or branched assay using locked nucleic acid (LNA)), programmed in situ growth of concatemers by enzymatic rolling circle amplification (RCA) (e.g., as described in US 2019 / 0055594 incorporated herein by reference), hybridization chain reaction, assembly of topologically catenated DNA structures using serial rounds of chemical ligation (clampFISH), signal amplification via hairpin-mediated concatemerization (e.g., as described in US 2020 / 0362398 incorporated herein by reference), e.g., primer exchange reactions such as signal amplification by exchange reaction (SABER) or SABER with DNA-Exchange (Exchange- SABER). In some instances, a non-enzymatic signal amplification method may be used.

[0225] The detectable reactive molecules may comprise tyramide, such as used in tyramide signal amplification (TSA) or multiplexed catalyzed reporter deposition (CARD)-FISH. In10XG / 1823PC 71some instances, the detectable reactive molecule may be releasable and / or cleavable from a detectable label such as a fluorophore. In some instances, a method disclosed herein comprises multiplexed analysis of a biological sample comprising consecutive cycles of probe hybridization, fluorescence imaging, and signal removal, where the signal removal comprises removing the fluorophore from a fluorophore-labeled reactive molecule (e.g., tyramide). Exemplary detectable reactive reagents and methods are described in US 6,828,109, US 2019 / 0376956, WO 2019 / 236841, WO 2020 / 102094, WO 2020 / 163397, and WO 2021 / 067475, all of which are incorporated herein by reference in their entireties.

[0226] In some instances, hybridization chain reaction (HCR) can be used for signal amplification. HCR is an enzyme-free nucleic acid amplification based on a triggered chain of hybridization of nucleic acid molecules starting from HCR monomers, which hybridize to one another to form a nicked nucleic acid polymer. This polymer is the product of the HCR reaction which is ultimately detected in order to indicate the presence of the target analyte. HCR is described in detail in Dirks and Pierce, 2004, PNAS, 101(43), 15275-15278 and in US 7,632,641 and US 7,721,721 (see also US 2006 / 00234261; Chemeris et al, 2008 Doklady Biochemistry and Biophysics, 419, 53-55; Niu et al, 2010, 46, 3089-3091; Choi et al, 2010, Nat. Biotechnol. 28(11), 1208-1212; and Song et al, 2012, Analyst, 137, 1396-1401). HCR monomers typically comprise a hairpin, or other metastable nucleic acid structure. In the simplest form of HCR, two different types of stable hairpin monomer, referred to here as first and second HCR monomers, undergo a chain reaction of hybridization events to form a long nicked double-stranded DNA molecule when an “initiator” nucleic acid molecule is introduced. The HCR monomers have a hairpin structure comprising a double stranded stem region, a loop region connecting the two strands of the stem region, and a single stranded region at one end of the double stranded stem region. The single stranded region which is exposed (and which is thus available for hybridization to another molecule, e.g. initiator or other HCR monomer) when the monomers are in the hairpin structure may be known as the “toehold region” (or “input domain”). The first HCR monomers each further comprise a sequence which is complementary to a sequence in the exposed toehold region of the second HCR monomers. This sequence of complementarity in the first HCR monomers may be known as the “interacting region” (or “output domain”). Similarly, the second HCR monomers each comprise an interacting region (output domain), e.g. a sequence which is complementary to the exposed toehold region (input domain) of the first HCR monomers. In the absence of the HCR initiator, these interacting regions are protected by the secondary structure (e.g. they are not10XG / 1823PC 72exposed), and thus the hairpin monomers are stable or kinetically trapped (also referred to as “metastable”), and remain as monomers (e.g. preventing the system from rapidly equilibrating), because the first and second sets of HCR monomers cannot hybridize to each other. However, once the initiator is introduced, it is able to hybridize to the exposed toehold region of a first HCR monomer, and invade it, causing it to open up. This exposes the interacting region of the first HCR monomer (e.g. the sequence of complementarity to the toehold region of the second HCR monomers), allowing it to hybridize to and invade a second HCR monomer at the toehold region. This hybridization and invasion in turn opens up the second HCR monomer, exposing its interacting region (which is complementary to the toehold region of the first HCR monomers), and allowing it to hybridize to and invade another first HCR monomer. The reaction continues in this manner until all of the HCR monomers are exhausted (e.g. all of the HCR monomers are incorporated into a polymeric chain). Ultimately, this chain reaction leads to the formation of a nicked chain of alternating units of the first and second monomer species. The presence of the HCR initiator is thus required in order to trigger the HCR reaction by hybridization to and invasion of a first HCR monomer. The first and second HCR monomers are designed to hybridize to one another are thus may be defined as cognate to one another. They are also cognate to a given HCR initiator sequence. HCR monomers which interact with one another (hybridize) may be described as a set of HCR monomers or an HCR monomer, or hairpin, system.

[0227] An HCR reaction could be carried out with more than two species or types of HCR monomers. For example, a system involving three HCR monomers could be used. In such a system, each first HCR monomer may comprise an interacting region which binds to the toehold region of a second HCR monomer; each second HCR may comprise an interacting region which binds to the toehold region of a third HCR monomer; and each third HCR monomer may comprise an interacting region which binds to the toehold region of a first HCR monomer. The HCR polymerization reaction would then proceed as described above, except that the resulting product would be a polymer having a repeating unit of first, second and third monomers consecutively. Corresponding systems with larger numbers of sets of HCR monomers could readily be conceived. Branching HCR systems have also been devised and described (see, e.g., WO 2020 / 123742 incorporated herein by reference), and may be used in the methods herein.

[0228] In some instances, similar to HCR reactions that use hairpin monomers, linear oligo hybridization chain reaction (LO-HCR) can also be used for signal amplification. In some10XG / 1823PC 73instances, provided herein is a method of detecting an analyte in a sample comprising: (i) performing a linear oligo hybridization chain reaction (LO-HCR), wherein an initiator is contacted with a plurality of LO-HCR monomers of at least a first and a second species to generate a polymeric LO-HCR product hybridized to a target nucleic acid molecule, wherein the first species comprises a first hybridization region complementary to the initiator and a second hybridization region complementary to the second species, wherein the first species and the second species are linear, single- stranded nucleic acid molecules; wherein the initiator is provided in one or more parts, and hybridizes directly or indirectly to or is comprised in the target nucleic acid molecule; and (ii) detecting the polymeric product, thereby detecting the analyte. In some instances, the first species and / or the second species may not comprise a hairpin structure. In some instances, the plurality of LO-HCR monomers may not comprise a metastable secondary structure. In some instances, the LO-HCR polymer may not comprise a branched structure. In some instances, performing the linear oligo hybridization chain reaction comprises contacting the target nucleic acid molecule with the initiator to provide the initiator hybridized to the target nucleic acid molecule. In any of the instances herein, the target nucleic acid molecule and / or the analyte can be an RCA product.

[0229] In some instances, detection of nucleic acids sequences in situ includes combination of the sequential decoding methods described herein with an assembly for branched signal amplification. In some instances, the assembly complex comprises an amplifier hybridized directly or indirectly (via one or more oligonucleotides) to a sequence of an oligonucleotide probe described herein. In some instances, the assembly includes one or more amplifiers each including an amplifier repeating sequence. In some aspects, the one or more amplifiers is labeled. Described herein is a method of using the aforementioned assembly, including for example, using the assembly in multiplexed error-robust fluorescent in situ hybridization (MERFISH) applications, with branched DNA amplification for signal readout. In some instances, the amplifier repeating sequence is about 5-30 nucleotides, and is repeated N times in the amplifier. In some instances, the amplifier repeating sequence is about 20 nucleotides, and is repeated at least two times in the amplifier. In some aspects, the one or more amplifier repeating sequence is labeled. For exemplary branched signal amplification, see e.g., U.S. Pat. Pub. No. US20200399689A1 and Xia et al., Multiplexed Detection of RNA using MERFISH and branched DNA amplification. Scientific Reports (2019), each of which is fully incorporated by reference herein.10XG / 1823PC 74

[0230] In some instances, an oligonucleotide probe described herein can be associated with an amplified signal by a method that comprises signal amplification by performing a primer exchange reaction (PER). In various instances, a primer with domain on its 3’ end binds to a catalytic hairpin, and is extended with a new domain by a strand displacing polymerase. For example, a primer with domain 1 on its 3’ ends binds to a catalytic hairpin, and is extended with a new domain 1 by a strand displacing polymerase, with repeated cycles generating a concatemer of repeated domain 1 sequences. In various instances, the strand displacing polymerase is Bst. In various instances, the catalytic hairpin includes a stopper which releases the strand displacing polymerase. In various instances, branch migration displaces the extended primer, which can then dissociate. In various instances, the primer undergoes repeated cycles to form a concatemer primer (see e.g., U.S. Pat. Pub. No. US20190106733, which is incorporated herein by reference, for exemplary molecules and PER reaction components).Barcoded analytes and detection:

[0231] A target sequence for a probe disclosed herein may be comprised in any analyte disclose herein, including an endogenous analyte (e.g., a viral or cellular nucleic acid), a labelling agent, or a product generated in the biological sample using an endogenous analyte and / or a labelling agent.

[0232] In some aspects, one or more of the target sequences includes or is associated with one or more barcode(s), e.g., at least two, three, four, five, six, seven, eight, nine, ten, or more barcodes. Barcodes can spatially-resolve molecular components found in biological samples, for example, within a cell or a tissue sample. A barcode can be attached to an analyte or to another moiety or structure in a reversible or irreversible manner. A barcode can be added to, for example, a fragment of a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sample before or during sequencing of the sample. Barcodes can allow for identification and / or quantification of individual sequencing -reads (e.g., a barcode can be or can include a unique molecular identifier or “UMI”). In some aspects, a barcode comprises about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30 nucleotides.

[0233] In some instances, a barcode includes two or more sub-barcodes that together function as a single barcode. For example, a polynucleotide barcode can include two or more polynucleotide sequences (e.g., sub-barcodes) that are separated by one or more non-barcode sequences. In some instances, the one or more barcode(s) can also provide a platform for10XG / 1823PC 75targeting functionalities, such as oligonucleotides, oligonucleotide-antibody conjugates, oligonucleotide-streptavidin conjugates, modified oligonucleotides, affinity purification, detectable moieties, enzymes, enzymes for detection assays or other functionalities, and / or for detection and identification of the polynucleotide.

[0234] In any of the preceding implementations, barcodes (e.g., primary and / or secondary barcode sequences) can be analyzed (e.g., detected or sequenced) using any suitable method or technique, including those described herein, such as sequencing by synthesis (SBS), sequencing by ligation (SBL), or sequencing by hybridization (SBH). In some instances, barcoding schemes and / or barcode detection schemes as described in RNA sequential probing of targets (RNA SPOTs), single-molecule fluorescent in situ hybridization (smFISH), multiplexed error-robust fluorescence in situ hybridization (MERFISH) or sequential fluorescence in situ hybridization (seqFISH+) can be used. In any of the preceding implementations, the methods provided herein can include analyzing the barcodes by sequential hybridization and detection with a plurality of labelled probes (e.g., detection probes (e.g., detection oligos) or barcode probes). In some instances, the barcode detection steps can be performed as described in hybridization-based in situ sequencing (HyblSS). In some instances, probes can be detected and analyzed e.g., detected or sequenced) as performed in fluorescent in situ sequencing (FISSEQ), or as performed in the detection steps of the spatially- resolved transcript amplicon readout mapping (STARmap) method. In some instances, signals associated with an analyte can be detected as performed in sequential fluorescent in situ hybridization (seqFISH).

[0235] In some instances, in a barcode-based detection method, barcode sequences are detected for identification of other molecules including nucleic acid molecules (DNA or RNA) longer than the barcode sequences themselves, as opposed to direct sequencing of the longer nucleic acid molecules. In some instances, a N-mer barcode sequence comprises 4N complexity given a sequencing read of N bases, and a much shorter sequencing read may be required for molecular identification compared to non-barcode sequencing methods such as direct sequencing. For example, 1024 molecular species may be identified using a 5-nucleotide barcode sequence (45=1024), whereas 8 nucleotide barcodes can be used to identify up to 65,536 molecular species, a number greater than the total number of distinct genes in the human genome. In some instances, the barcode sequences contained in the probes or RCPs are detected, rather than endogenous sequences, which can be an efficient read-out in terms of information per cycle of sequencing. Because the barcode sequences are pre-determined, they10XG / 1823PC 76can also be designed to feature error detection and correction mechanisms, see, e.g., U.S. Pat. Pub. 20190055594 and WO2019199579A1, which are hereby incorporated by reference in their entirety.Sequential hybridization:

[0236] In some instances, the present disclosure relates to methods and compositions for encoding and detecting analytes in a temporally sequential manner for in situ analysis of an analyte in a biological sample, e.g., a target nucleic acid in a cell in an intact tissue. In some aspects, provided herein is a method for detecting the detectably-labeled probes, thereby generating a signal signature. In some instances, the signal signature corresponds to an analyte of the plurality of analytes. In some instances, the methods described herein are based, in part, on the development of a multiplexed biological assay and readout, in which a sample is first contacted with a plurality of nucleic acid probes comprising one or more probe types e.g., labelling agent, circularizable probe, circular probe, etc.), allowing the probes to directly or indirectly bind target analytes, which may then be optically detected (e.g., by detectably- labeled probes) in a temp orally- sequential manner. In some instances, the probes or probe sets comprising various probe types may be applied to a sample simultaneously. In some instances, the probes or probe sets comprising various probe types may be applied to a sample sequentially. In some aspects, the method comprises sequential hybridization of labelled probes to create a spatiotemporal signal signature or code that identifies the analyte.

[0237] In some aspects, provided herein is a method involving a multiplexed biological assay and readout, in which a sample is first contacted with a plurality of nucleic acid probes, allowing the probes to directly or indirectly bind target analytes, which may then be optically detected (e.g., by detectably-labeled probes) in a temporally sequential manner. The plurality of nucleic acid probes themselves may be detectably-labeled and detected; in other words, the nucleic acid probes themselves serve as the detection probes. In other implementations, a nucleic acid probe itself is not directly detectably-labeled (e.g., the probe itself is not conjugated to a detectable label); rather, in addition to a target binding sequence (e.g., a sequence binding to a barcode sequence in an RCA product), the nucleic acid probe further comprises a sequence for detection which can be recognized by one or more detectably-labeled detection probes. In some instances, the probes or probe sets comprising various probe types may be applied to a sample simultaneously. In some instances, the probes or probe sets10XG / 1823PC 77comprising various probe types may be applied to a sample sequentially. In some instances, the method comprises detecting a plurality of analytes in a sample.

[0238] In some instances, the method presented herein comprises contacting the sample with a plurality of probes comprising one or more probes having distinct labels and detecting signals from the plurality of probes in a temporally sequential manner, wherein said detection generates signal signatures each comprising a temporal order of signal or absence thereof, and the signal signatures correspond to said plurality of probes that identify the corresponding analytes. In some instances, the temporal order of the signals or absence thereof corresponding to the analytes can be unique for each different analyte of interest in the sample. In some instances, the plurality of probes hybridize to an endogenous molecule in the sample, such as a cellular nucleic acid molecule, e.g., genomic DNA, RNA (e.g., mRNA), or cDNA. In some instances, the plurality of probes hybridize to a product of an endogenous molecule in the sample (e.g., directly or indirectly via an intermediate probe). In some instances, the plurality of probes hybridize to labelling agent that binds directly or indirectly to an endogenous molecule in the sample or a product thereof. In some instances, the plurality of probes hybridize to a product (e.g., an RCA product) of a labelling agent that binds directly or indirectly to an endogenous molecule in the sample or a product thereof.

[0239] In any of the implementations disclosed herein, the detection of signals can be performed sequentially in cycles, one for each distinct label. In any of the implementations disclosed herein, signals or absence thereof from detectably-labeled probes targeting an analyte in a particular location in the sample can be recorded in a first cycle for detecting a first label, and signals or absence thereof from detectably-labeled probes targeting the analyte in the particular location can be recorded in a second cycle for detecting a second label distinct from the first label. In any of the implementations disclosed herein, a unique signal signature can be generated for each analyte of the plurality of analytes. In any of the implementations disclosed herein, one or more molecules comprising the same analyte or a portion thereof can be associated with the same signal signature.

[0240] In some instances, the in situ assays employ strategies for optically encoding the spatial location of target analytes (e.g., mRNAs) in a sample using sequential rounds of fluorescent hybridization. Microcopy may be used to analyze 4 or 5 fluorescent colors indicative of the spatial localization of a target, followed by various rounds of hybridization and stripping, in order to generate a large set of unique optical signal signatures assigned to different analytes. These methods often require a large number of hybridization rounds, and a large number of10XG / 1823PC 78microscope lasers (e.g., detection channels) to detect a large number of fluorophores, resulting in a one to one mapping of the lasers to the fluorophores. Specifically, each detectably-labeled probe comprises one detectable moiety, e.g., a fluorophore.

[0241] In some aspects, provided herein is a method for analyzing a sample using a detectably- labeled set of probes. In some instances, the method comprises contacting the sample with a first plurality of detectably-labeled probes for targeting a plurality of analytes; performing a first detection round comprising detecting signals from the first plurality of detectably-labeled probes; contacting the sample with a second plurality of detectably-labeled probes for targeting the plurality of analytes; performing a second detection round of detecting signals from the second plurality of detectably-labeled probes, thereby generating a signal signature comprising a plurality of signals detected from the first detection round and second detection round, wherein the signal signature corresponds to an analyte of the plurality of analytes.

[0242] In some instances, detection of an optical signal signature comprises several rounds of detectably-labeled probe hybridization (e.g., contacting a sample with detectably-labeled probes), detectably-labeled probe detection, and detectably-labeled probe removal. In some instances, a sample is contacted with plurality first detectably-labeled probes, and said probes are hybridized to a plurality of nucleic acid analytes within the sample in decoding hybridization round 1. In some instances, a first detection round is performed following detectably-labeled probe hybridization. After hybridization and detection of a first plurality of detectably-labeled probes, probes are removed, and a sample may be contacted with a second plurality round of detectably-labeled probes targeting the analytes targeted in decoding hybridization round 1. The second plurality of detectably-labeled probes may hybridize to the same nucleic acid(s) as the first plurality of detectably-labeled probes (e.g., hybridize to an identical or hybridize to new nucleic acid sequence within the same nucleic acid), or the second plurality of detectably-labeled probes may hybridize to different nucleic acid(s) compared to the first plurality of detectably-labeled probes. Following m rounds of contacting a sample with a plurality of detectably-labeled probes, probe detection, and probe removal, ultimately a unique signal signature to each nucleic acid is produced that may be used to identify and quantify said nucleic acids and the corresponding analytes (e.g., if the nucleic acids themselves are not the analytes of interest and each is used as part of a labelling agent for one or more other analytes such as protein analytes and / or other nucleic acid analytes).

[0243] In some instances, after hybridization of a detectably-labeled probes (e.g. , fluorescently labeled oligonucleotide) that detects a sequence (e.g., barcode sequence on a secondary probe10XG / 1823PC 79or a primary probe), and optionally washing away the unbound molecules of the detectably- labeled probe, the sample is imaged and the detection oligonucleotide or detectable label is inactivated and / or removed. In some instances, removal of the signal associated with the hybridization between rounds can be performed by washing, heating, stripping, enzymatic digestion, photo-bleaching, displacement (e.g., displacement of detectably-labeled probes with another reagent or nucleic acid sequence), cleavage, quenching, chemical degradation, bleaching, oxidation, or any combinations thereof.

[0244] In some examples, removal of a probe (e.g., un-hybridizing the entire probe), signal modifications (e.g., quenching, masking, photo-bleaching, signal enhancement (e.g., via FRET), signal amplification, etc.), signal removal (e.g., cleaving off or permanently inactivating a detectable label) can be performed. Inactivation may be caused by removal of the detectable label (e.g., from the sample, or from the probe, etc.), and / or by chemically altering the detectable label in some fashion, e.g., by photobleaching the detectable label, bleaching or chemically altering the structure of the detectable label, e.g., by reduction, etc.). In some instances, the fluorescently labeled oligonucleotide and / or the intermediate probe hybridized to the fluorescently labeled oligonucleotide (e.g., bridge probe or L-probe) can be removed. In some instances, a fluorescent detectable label may be inactivated by chemical or optical techniques such as oxidation, photobleaching, chemically bleaching, stringent washing or enzymatic digestion or reaction by exposure to an enzyme, dissociating the detectable label from other components (e.g., a probe), chemical reaction of the detectable label (e.g., to a reactant able to alter the structure of the detectable label) or the like. For instance, bleaching may occur by exposure to oxygen, reducing agents, or the detectable label could be chemically cleaved from the nucleic acid probe and washed away via fluid flow.

[0245] In some instances, removal of a signal comprises displacement of probes with another reagent (e.g., probe) or nucleic acid sequence. For example, a given probe (e.g., detectably- labeled probes and / or the intermediate probe hybridized to the fluorescently labeled oligonucleotide (e.g., bridge probe or E-probe)) may be displaced by a subsequent probe that hybridizes to an overlapping region shared between the binding sites of the probes. In some cases, a displacement reaction can be very efficient, and thus allows for probes to be switched quickly between cycles, without the need for chemical stripping (or any of the damage to the sample that is associated therewith). In some instances, a sequence for hybridizing the subsequent or displacer probe (i.e. a toehold sequence) may be common across a plurality of probes capable of hybridizing to a given binding site. In some aspects, a single displacement10XG / 1823PC 80probe can be used to simultaneously displace detection probes bound to an equivalent barcode position from all of the RCPs within a given sample simultaneously (with the displacement mediated by the subsequent detection probes). This may further increase efficiency and reduce the cost of the method, as fewer different probes are required.

[0246] After a signal is inactivated and / or removed, then the sample is re-hybridized in a subsequent round with a subsequent fluorescently labeled oligonucleotide, and the oligonucleotide can be labeled with the same color or a different color as the fluorescently labeled oligonucleotide of the previous cycle. In some instances, as the positions of the analytes, probes, and / or products thereof can be fixed (e.g., via fixing and / or crosslinking) in a sample, the fluorescent spot corresponding to an analyte, probe, or product thereof remains in place during multiple rounds of hybridization and can be aligned to read out a string of signals associated with each target analyte.Decoding:

[0247] A “decoding process” is a process comprising a plurality of decoding cycles in which different sets of barcode probes are contacted with target analytes (e.g., mRNA sequences) or target barcodes (e.g., barcodes associated with target analytes) present in a sample, and used to detect the target sequences or associated target barcodes, or segments thereof. In some instances, the decoding process comprises acquiring one or more images (e.g., fluorescence images) for each decoding cycle. Decoded barcode sequences are then inferred based on a set of physical signals (e.g., fluorescence signals) detected in each decoding cycle of a decoding process. In some instances, the set of physical signals (e.g., fluorescence signals) detected in a series of decoding cycles for a given target barcode (or target analyte sequence) may be considered a “signal signature” for the target barcode (or target analyte sequence). In some instances, a decoding process may comprise, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 decoding cycles. In some instances, each decoding cycle may comprise contacting a plurality of target sequences or target barcodes with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 barcode probes (e.g., fluorescently-labeled barcode probes) that are configured to hybridize or bind to specific target sequences or target barcodes, or segments thereof. In some instances, a decoding process may comprise performing a series of in situ barcode probe hybridization steps and acquiring images (e.g., fluorescence images) at each step. Systems and methods for performing multiplexed fluorescence in situ hybridization and imaging are described in, for example, WO 2021 / 127019 Al; U.S. Pat. 11,021,737; and PCT / EP2020 / 065090 (W02020240025A1), each of which is incorporated herein by reference in its entirety.10XG / 1823PC 81Anchor probes:

[0248] In some instances, the present methods may further involve contacting the target analyte, e.g., a nucleic acid molecule, or proxy thereof with an anchor probe. In some instances, the anchor probe comprises a sequence complementary to an anchor probe binding region, which is present in all target nucleic acid molecules e.g., in primary or secondary probes), and a detectable label. The detection of the anchor probe via the detectable label confirms the presence of the target nucleic acid molecule. The target nucleic acid molecule may be contacted with the anchor probe prior to, concurrently with, or after being contacted with the first set of detection probes. In some instances, the target nucleic acid molecule may be contacted with the anchor probe during multiple decoding cycles. In some instances, multiple different anchor probes comprising different sequences and / or different reporters may be used to confirm the presence of multiple different target nucleic acid molecules. The use of multiple anchor probes is particularly useful when detection of a large number of target nucleic acid molecules is required, as it allows for optical crowding to be reduced and thus for detected target nucleic acid molecules to be more clearly resolved.

[0249] Reference will now be made in detail to implementations and embodiments of various aspects and variations of systems and methods described herein. Although several exemplary variations of the systems and methods are described herein, other variations of the systems and methods may include aspects of the systems and methods described herein combined in any suitable manner having combinations of all or some of the aspects described.

[0250] Target molecules (e.g., nucleic acids, proteins, antibodies, etc.) can be detected in biological samples (e.g., one or more cells or a tissue sample) using an instrument having integrated optics and fluidics modules (an “opto-fluidic instrument” or “opto-fluidic system”). In an opto-fluidic instrument, the fluidics module is configured to deliver one or more reagents (e.g., fluorescent probes) to the biological sample and / or remove spent reagents therefrom. Additionally, the optics module is configured to illuminate the biological sample with light having one or more spectral emission curves (over a range of wavelengths) and subsequently capture one or more images of emitted light signals from the biological sample during one or more probing cycles. In various embodiments, the captured images may be processed in real time and / or at a later time to determine the presence of the one or more target molecules in the biological sample, as well as three-dimensional position information associated with each detected target molecule. Additionally, the opto-fluidics instrument includes a sample module configured to receive (and, optionally, secure) one or more biological samples. In some10XG / 1823PC 82instances, the sample module includes an X-Y stage configured to move the biological sample along an X-Y plane (e.g., perpendicular to an objective lens of the optics module).

[0251] In various embodiments, the opto-fluidic instrument is configured to analyze one or more target molecules in their naturally occurring place (i.e., in situ) within the biological sample. For example, an opto-fluidic instrument may be an in-situ analysis system used to analyze a biological sample and detect target molecules including but not limited to DNA, RNA, proteins, antibodies, and / or the like.

[0252] A sample disclosed herein can be or be derived from any biological sample. Biological samples may be obtained from any suitable source using any of a variety of techniques including, but not limited to, biopsy, surgery, and laser capture microscopy (LCM), and generally includes cells, tissues, and / or other biological material from the subject. A biological sample can be obtained from a prokaryote such as a bacterium, an archaea, a virus, or a viroid. A biological sample can also be obtained from non-mammalian organisms (e.g., a plant, an insect, an arachnid, a nematode, a fungus, or an amphibian). A biological sample can also be obtained from a eukaryote, such as a tissue sample from a mammal. A biological sample from an organism may comprise one or more other organisms or components therefrom. For example, a mammalian tissue section may comprise a prion, a viroid, a virus, a bacterium, a fungus, or components from other organisms, in addition to mammalian cells and non-cellular tissue components. Subjects from which biological samples can be obtained can be healthy or asymptomatic subjects, subjects that have or are suspected of having a disease (e.g., an individual with a disease such as cancer) or a pre-disposition to a disease, and / or subjects in need of therapy or suspected of needing therapy.

[0253] The biological sample can include any number of macromolecules, for example, cellular macromolecules and organelles (e.g., mitochondria and nuclei). The biological sample can be obtained as a tissue sample, such as a tissue section, biopsy, a core biopsy, needle aspirate, or fine needle aspirate. The sample can be a fluid sample, such as a blood sample, urine sample, or saliva sample. The sample can be a skin sample, a colon sample, a cheek swab, a histology sample, a histopathology sample, a plasma or serum sample, a tumor sample, living cells, cultured cells, a clinical sample such as, for example, whole blood or blood-derived products, blood cells, or cultured tissues or cells, including cell suspensions.

[0254] In some embodiments, the biological sample may comprise cells or a tissue sample which are deposited on a substrate. As described herein, a substrate can be any support that is10XG / 1823PC 83insoluble in aqueous liquid and allows for positioning of biological samples, analytes, features, and / or reagents on the support. In some embodiments, a biological sample is attached to a substrate. In some embodiments, the substrate is optically transparent to facilitate analysis on the opto-fluidic instruments disclosed herein. For example, in some instances, the substrate is a glass substrate (e.g., a microscopy slide, cover slip, or other glass substrate). Attachment of the biological sample can be irreversible or reversible, depending upon the nature of the sample and subsequent steps in the analytical method. In certain embodiments, the sample can be attached to the substrate reversibly by applying a suitable polymer coating to the substrate and contacting the sample to the polymer coating. The sample can then be detached from the substrate, e.g., using an organic solvent that at least partially dissolves the polymer coating. Hydrogels are examples of polymers that are suitable for this purpose. In some embodiments, the substrate can be coated or functionalized with one or more substances to facilitate attachment of the sample to the substrate. Suitable substances that can be used to coat or functionalize the substrate include, but are not limited to, lectins, poly-lysine, antibodies, and polysaccharides.

[0255] It is to be noted that, although the above discussion relates to an opto-fluidic instrument that can be used for in situ target molecule detection via probe hybridization, the discussion herein equally applies to any opto-fluidic instrument that employs any imaging or target molecule detection technique. That is, for example, an opto-fluidic instrument may include a fluidics module that includes fluids needed for establishing the experimental conditions required for the probing of target molecules in the sample. Further, such an opto-fluidic instrument may also include a sample module configured to receive the sample, and an optics module including an imaging system for illuminating e.g., exciting one or more fluorescent probes within the sample) and / or imaging light signals received from the probed sample. The in-situ analysis system may also include other ancillary modules configured to facilitate the operation of the opto-fluidic instrument, such as, but not limited to, cooling systems, motion calibration systems, etc.

[0256] It is to be understood that the above description is intended to be illustrative, and not restrictive. Many other implementations will be apparent to those of skill in the art upon reading and understanding the above description. Although the present disclosure has been described with reference to specific example implementations, it will be recognized that the disclosure is not limited to the implementations described but can be practiced with modification and alteration within the scope of the appended claims. Accordingly, the specification and drawings10XG / 1823PC 84are to be regarded in an illustrative sense rather than a restrictive sense. The scope of the disclosure should, therefore, be determined with reference to the appended claims, along with the full scope of equivalents to which such claims are entitled.10XG / 1823PC 85

Claims

100-182300WC)CLAIMS1. An assembly for mounting a plurality of optical filters, the assembly comprising: a frame comprising a first portion for securing a first optical filter and a second portion for securing a second optical filter; a first shaft connected to the frame, wherein the first shaft is configured to receive a torque from a motor and rotate the frame about a rotation axis of the first shaft; an alignment member coupled to the first shaft, wherein the alignment member comprises at least one alignment surface configured to align with at least one reference surface of an optical bench; and a clamping member configured to clamp the alignment member against the at least one reference surface.

2. An assembly according to claim 1, wherein the alignment member comprises at least one bearing.

3. An assembly according to claim 2, wherein the at least one bearing comprises a first bearing and a second bearing.

4. An assembly according to claim 3, wherein the first bearing has a first alignment surface configured to contact the at least one reference surface and the second bearing has a second alignment surface configured to contact the at least one reference surface.

5. An assembly according to any preceding claim, wherein the at least one reference surface comprises a first reference surface and a second reference surface that is perpendicular to the first reference surface.

6. An assembly according to any preceding claim, the assembly comprising the motor for providing the torque.

7. An assembly according to claim 6, wherein motor comprises a second shaft, wherein the second shaft is connected to the first shaft via a coupling.

8. An assembly according to claim 7, wherein the coupling is a flexible coupling or a universal joint.10XG / 1823PC 869. An assembly according to claim 7 or 8, wherein the rotation axis of the second shaft is aligned with the rotation axis of the first shaft.

10. An assembly according to any of claims 7 to 9, wherein the second shaft is not integral with the first shaft.

11. An assembly according to any preceding claim, wherein the clamping member comprises one or more holes for receiving bolts or screws to fasten the clamping member in a clamping position with respect to the at least one reference surface.

12. An assembly according to any preceding claim, wherein the clamping member comprises a pad configured to contact the alignment member.

13. An assembly according to any preceding claim, wherein the clamping member comprises an end plate between the alignment member and the frame.

14. An assembly according to claim 13, wherein the assembly comprises an O-ring positioned between the end plate and the alignment member.

15. An assembly according to any preceding claim, wherein the first portion of the frame comprises a first aperture and the second portion of the frame comprises a second aperture.

16. An assembly according to any preceding claim, wherein the first portion of the frame comprises a pair of arms and the second portion of the frame comprises a second pair of arms.

17. An assembly according to any preceding claim, wherein a center of the first portion is positioned 180 degrees opposite a center of the second portion.

18. An assembly according to any preceding claim, wherein frame is rotationally symmetric about the rotation axis of the first shaft.

19. An assembly according to any preceding claim, the assembly comprising: a first optical filter mounted to the first portion; and a second optical filter mounted to the second portion.

20. An assembly according to claim 19, wherein the first optical filter and the second optical filter are mounted using an epoxy.

21. An assembly according to claim 19 or 20, wherein each of the first optical filter and the second optical filter is a dichroic filter.10XG / 1823PC 87100-182300WC)22. An assembly according to any of claims 19-21, wherein the first filter and the second filter each has a width between: about 50mm and about 100mm; or about 70mm and about 80mm.

23. An assembly according to any of claims 19-22, wherein the first filter and the second filter each has a length of between: about 50mm and about 100mm; or about 60mm and about 70mm.

24. An assembly according to any of claims 19-23, wherein the first filter and the second filter each has a thickness of between about 4mm to about 8mm; or about 6mm.

25. An assembly according to any of claims 19-24, wherein the first optical filter has a first filter spectrum, wherein the second optical filter has a second filter spectrum, and wherein the first filter spectrum is different from the second filter spectrum.

26. An assembly according to any of claims 19-25, wherein the first optical filter has a first surface abutting the frame, wherein the second optical filters has a second surface abutting the frame, wherein each of the first surface and the second surface is held in a plane perpendicular to the rotation axis of the first shaft.

27. An assembly according to claim 26, wherein each of the first surface and the second surface abuts the frame in a direction toward a distal end of the first shaft.

28. An assembly according to any of claims 19-27, wherein each of the first and second optical filters has a first dimension and a second dimension in a plane perpendicular to the rotation axis of the first shaft, wherein the first dimension is greater than second dimension.

29. An assembly according to claim 28, wherein the first dimension is greater than the second dimension by: between 5% and 30%, between 10% and 20%, or approximately 15%.

30. An assembly according to any of claim 19-29, wherein each of the first and second optical filters has a polygonal shape.

31. An assembly according to claim 30, wherein the polygonal shape is a parallelogonal shape.

32. An assembly according to any of claims 19-29, wherein each of the first optical filter and second optical filters has a rectangular or rounded-rectangular shape.10XG / 1823PC 88100-182300WC)33. An assembly according to any of claims 19-32, wherein a first comer of the first optical filter is positioned adjacent the rotation axis of the first shaft, and wherein a first corner of the second optical filter is positioned adjacent the rotation axis of the first shaft.

34. A system, comprising: an assembly according to any preceding claim; and an optical bench comprising the at least one reference surface, wherein the clamping member is attached to the optical bench thereby securing the alignment member against the optical bench and aligning the first shaft.

35. A system according to claim 34, wherein the optical bench comprises fastening locations for fastening the clamping member thereto.

36. A system according to claim 35, wherein the fastening locations comprise threaded holes for receiving bolts or screws.

37. A system according to any of claims 34-36, wherein the optical bench comprises a top planar surface and a primary optical axis.

38. A system according to claim 37, wherein the at least one reference surface comprises: a first reference surface, wherein the first reference surface lies in a plane perpendicular to the top planar surface, wherein the alignment surface of the alignment member contacts the first reference surface to define the rotation axis of the first shaft at a predetermined angle with respect to the primary optical axis of the optical bench; and / or a second reference surface, wherein the second reference surface is parallel to the top planar surface, wherein the alignment surface of the alignment member contacts the second reference member to align the frame perpendicular to the top planar surface.

39. A system according to claim 38, wherein the predetermined angle is 30 degrees.

40. A system according to claim 37 or 38, wherein the first reference surface comprises a wall extending perpendicularly from the top planar surface.

41. A system according to any of claims 38-40, wherein the optical bench defines a recess, wherein the recess comprises a first recess portion for receiving the alignment member, wherein first recess portion comprises the at least one reference surface.10XG / 1823PC 89100-182300WC)42. A system according to claim 41, wherein the first reference surface comprises a side wall of the recess.

43. A system according to claim 41 or 42, wherein the second reference surface is a base of the recess.

44. A system according to any of claims 41-43, wherein the recess comprises a second recess portion for receiving the motor.

45. A system according to any of claims 37-44, wherein the optical bench comprises a connection location for connecting a tube lens to the optical bench such that the tube lens is oriented along the primary optical axis.

46. A system according to any of claims 34-45, wherein optical bench comprises a slot for receiving the frame to permit rotation of the frame around the rotation axis of the first shaft.

47. A system according to any of claims 34-46, wherein the system comprises: a plurality of light sources; a first optical filter mounted in the first portion of the assembly; a second optical filter mounted in the second portion of the assembly; an objective; and a tube lens oriented along the primary optical axis; wherein: the plurality of light sources, the objective and the tube lens are attached to the optical bench; the plurality of light sources is configured to generate illumination light and direct the illumination light along an illumination pathway to the first optical filter or the second optical filter; each of the first optical filter and the second optical filter, when positioned in the illumination pathway, is configured to direct at least a portion of the illumination light to the objective; the objective is positioned to direct the illumination light to a sample and receive emission light from the sample and direct the emission light to the first dichroic filter or the second optical filter when positioned in the illumination pathway; each of the first optical filter and the second optical filter, when positioned in the illumination pathway, is configured to direct at least a portion of the emission light to the tube lens.10XG / 1823PC 90100-182300WC)48. A system according to claim 47, wherein assembly is configured to switch positions of the first optical filter and the second optical filter.

49. A system according to claim 47 or 48, wherein the first optical filter has a first reflection band, a first transmission band, a second reflection band, a second transmission band, a third reflection band, and a third transmission band.

50. A system according to claim 49, wherein: the first reflection band of the first optical filter is between about 300nm and about 425nm; the first transmission band of the first optical filter is between about 425nm and about 500nm; the second reflection band of the first optical filter is between about 51 Onm and about 550nm; the second transmission band of the first optical filter is between about 550nm and about 600nm; the third reflection band of the first optical filter is between about 600nm and about 650nm; and / or the third transmission band of the first optical filter is between about 650nm and about 750nm.

51. A system according to any of claims 47-50, wherein the second optical filter has a first reflection band, a first transmission band, a second reflection band, and a second transmission band.

52. A system according to claim 51, wherein: the first reflection band of the second optical filter is between about 425nm and about 500nm; the first transmission band of the second optical filter is between about 500nm and about 550nm; the second reflection band of the second optical filter is between about 550nm and about 600nm; and the second transmission band of the second optical filter is between about 600nm and about 675nm.

53. A method for mounting an assembly according to any of claims 1-33 onto an optical bench, the method comprising:10XG / 1823PC 91securing the assembly to the optical bench to thereby contact the alignment surface of the alignment member against the at least one reference surface.

54. A method of using an assembly according to any of claims 19-33, wherein the alignment surface of the alignment member contacts the reference surface throughout the method, wherein the method comprises: providing a torque from a motor; rotating the first shaft using the torque thereby rotating the frame.

55. A method according to claim 54, the method comprising: controlling the motor to rotate the frame and move the first optical filter from a first position in an illumination pathway and to move the second optical filter to the first position in the illumination pathway.

56. A method of using a system according to any of claims 47-52, the method comprising: rotating the frame such that the first optical filter is positioned in the illumination pathway to receive first illumination light; energizing a first light source of the plurality of light sources to direct first illumination light to the first optical filter, wherein the first illumination light has a first wavelength and the first optical filter has a first reflectance band that includes the first wavelength; rotating the frame such that the second optical filter is positioned in the illumination path to receive second illumination light; and energizing a second light source of the plurality of light sources to direct second illumination light to the second optical filter, wherein the second illumination light has a second wavelength and the second optical filter has a second reflectance band that includes the second wavelength.

57. A method according to claim 56, the method comprising: rotating the frame such that the first optical filter is positioned in an illumination path to receive third illumination light; energizing a third light source of the plurality of light sources to direct third illumination light to the first optical filter, wherein the third illumination light has a third wavelength and the first optical filter has a third reflectance band that includes the third wavelength; rotating the frame such that the second optical filter is positioned in the illumination path to receive fourth illumination light; and energizing a fourth light source of the plurality of light sources to direct fourth illumination light to the second optical filter, wherein the fourth illumination light has a second10XG / 1823PC 92100-182300WC) wavelength and the second optical filter has a second reflectance band that includes the fourth wavelength.

58. A method according to claim 57, wherein the second wavelength is less than the first wavelength, the third wavelength is less than the second wavelength, and the fourth wavelength is less than the third wavelength.

59. A method according to claim 57, wherein the second wavelength is greater than the first wavelength, the third wavelength is greater than the second wavelength, and the fourth wavelength is greater than the third wavelength.

60. A method according to any of claims 56-59, wherein, when the first optical filter is positioned in the illumination pathway, the first optical filter is positioned to receive third and / or fifth illumination light, wherein the method comprises, before rotating the frame such that the second optical filter is positioned in the illumination path: energizing a third light source of the plurality of light sources to direct third illumination light to the first optical filter, wherein the third illumination light has a third wavelength and the first optical filter has a third reflectance band that includes the third wavelength; and / or energizing a fifth light source of the plurality of light sources to direct fifth illumination light to the first optical filter, wherein the fifth illumination light has a fifth wavelength and the first optical filter has a fifth reflectance band that includes the fifth wavelength.

61. A method according to any of claims 56-60, wherein, when the second optical filter is positioned in the illumination pathway, the second optical filter is positioned to receive fourth illumination light, wherein the method comprises, after rotating the frame such that the second optical filter is positioned in the illumination path: energizing a fourth light source of the plurality of light sources to direct fourth illumination light to the second optical filter, wherein the fourth illumination light has a fourth wavelength and the second optical filter has a fourth reflectance band that includes the fourth wavelength.10XG / 1823PC 93

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