Optical assemblies and methods for optical assembly alignment

Abstract

Disclosed herein is an assembly for an imaging system. The assembly comprises a base plate comprising a first end, a second end, an upper surface, and a lower surface. The assembly further comprises a first mount removably couplable to the base plate, and an alignment structure coupled to the first mount via one or more first couplings. The assembly further comprises a first optical component contacting the alignment structure to define a first set orientation of the first optical component relative to the first mount, the first optical component being coupled to the first mount in the first set orientation. The assembly further comprises a second optical component coupled to the base plate. When the first mount is removably coupled to the base plate with the first optical component in the first set orientation, the first optical component is aligned relative to the second optical component.

Description

OPTICAL ASSEMBLIES AND METHODS FOR OPTICAL ASSEMBLY ALIGNMENT CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and benefit of U.S. Provisional Application No.63 / 740,540, filed December 31, 2024, which is incorporated by reference herein in its entirety, and is hereby expressly made a part of this specification.TECHNICAL FIELD

[0002] The present disclosure is directed to image analysis techniques for samples, e.g., biological samples. More specifically, the present disclosure describes an assembly for an imaging system.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] Imaging systems may be used as part of these techniques. However, there are challenges in assembling and calibrating imaging systems. Imaging systems typically include a number of optical components that must be correctly aligned with each other to enable optimal functionality of the system. It may be particularly difficult to ensure these components remain aligned with one another, for example if they must be removable from the imaging system in order to enable cleaning, repair, or replacement due to failure. In prior systems, it has been necessary to employ complex and time-consuming re-calibration processes each time a component is removed from the assembly, or replacing the entire imaging system with a brand new imaging system (which is costly).

[0005] Accordingly, there exists a need for an improved assembly for an imaging apparatus for use in automated and high-throughput imaging systems.10XG / 1851PC 1SUMMARY

[0006] 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 an imaging system, an assembly for an imaging system, a method of assembling an assembly for an imaging system, an assembly for an imaging assembly assembled via the method of assembly, a base plate for an optical assembly.BRIEF DESCRIPTION OF THE DRAWINGS

[0007] 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.

[0008] 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. The objective lens focal point is positioned to acquire an image at every Z-slice in a Z-stack. An XZ image of signal distribution (bottom) demonstrates a non-uniform distribution of detected signal within the imaging volume.

[0009] 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.

[0010] FIGS. 4A-4B illustrate cross-sectional views of an optics module in an imaging system, according to some embodiments.

[0011] FIG. 5 depicts a schematic of an assembly according to the present disclosure.

[0012] FIG. 6 depicts a schematic of an assembly according to the present disclosure.

[0013] FIG. 7 depicts part of the assembly depicted in Fig. 6.

[0014] FIG.8 depicts part of the assembly depicted in Fig. 6.

[0015] FIG. 9 depicts part of the assembly depicted in Fig. 6.

[0016] FIG. 10 depicts an assembly according to the present disclosure. The assembly can be described as another implementation of the assembly depicted in Fig. 5.

[0017] FIGS. 11A-11C depict a mount and alignment structure suitable for use with any of the assemblies disclosed herein, and according to the first mount depicted as coupled to the10XG / 1851PC 2assembly in Fig. 10. FIGS. 11D-11F depict another mount and alignment structure suitable for use with any of the assemblies disclosed herein.

[0018] FIG. 12 depicts part of the assembly depicted in Fig. 10.

[0019] FIG. 13 depicts part of the assembly depicted in Fig. 10.

[0020] FIG. 14 depicts part of the assembly depicted in Fig. 10.

[0021] FIGS. 15A-15C depicts a base plate suitable for use with any of the assemblies disclosed herein, and in line with the base plate depicted in Fig. 10.

[0022] FIGS. 16A-16D depicts a base plate assembly including a base plate and a shear plate that are suitable for use with any of the assemblies disclosed herein. FIG. 16E depicts the shear plate coupled to the bottom of the base plate shown in FIGS. 16A-16D.

[0023] FIG. 17 depicts a method of assembly according to the present disclosure.

[0024] 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.

[0025] 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

[0026] 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.

[0027] In brief, the present disclosure relates to an assembly for an imaging system. The assembly comprises a first optical component. The first optical component is removeable from a base plate of the assembly. A benefit of providing an assembly with a removable first optical component is that the component can be removed to enable cleaning, repair, or replacement. An example of a suitable first optical component is an objective lens, along with an accompanying mount, collar, and / or housing.

[0028] To facilitate either the re-coupling of the first optical component to the assembly (e.g., after cleaning) or the coupling of a new first optical component to the assembly (e.g., after10XG / 1851PC 3replacement), a first mount and an alignment structure is provided. The alignment structure is coupled to the mount in a set orientation, which may be described as a preferred or calibrated orientation. Placing the first optical component in contact with the alignment structure brings the first optical component into the same set orientation with respect to the mount. When the first mount is coupled to the base plate with the first optical component in this set orientation, the first optical component is aligned relative to the second optical component.

[0029] Once the assembly has been calibrated, the entire sub-assembly, i.e., both the first mount and the adjustment structure, plus the first optical component, can be removed from the base assembly by un-mounting, i.e. de-coupling, the first mount from the base assembly. After the first optical component has been cleaned and / or repaired, the entire sub-assembly can be remounted, i.e. re-coupled, to the base plate. The orientation of the alignment structure remains locked in a calibrated state with respect to the first mount throughout this process. Because of this locked alignment orientation, when the sub-assembly containing the first optical component is re-mounted to the base plate, the first optical component retains its previous, calibrated orientation relative to the base plate, and is thus immediately in alignment with the second optical component, without the need for time-consuming or complex re-calibration measures.

[0030] In the present disclosure, base plates are sometimes described in the context of use in an imaging or optical system such as a microscope, and more particularly in the context of use in a fluorescence optical system (e.g., an epifluorescence optical system). However, the base plates described herein can be used in any imaging system for which optical alignment between at least two optical components is desirable, and in which at least one of those components is removable and / or replaceable.

[0031] 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.10XG / 1851PC 4

[0032] 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). 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 the 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.

[0033] 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.)

[0034] FIG.3 shows an example workflow of analysis of a biological sample 110 (e.g., cell ortissue 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 labeling 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.

[0035] 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 conditions10XG / 1851PC 5conducive 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.

[0036] 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.

[0037] 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 140.10XG / 1851PC 6

[0038] 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).

[0039] In various embodiments, the ancillary module 170 can be a cooling system of the opto-fluidic 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.

[0040] 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 not10XG / 1851PC 7limited to a camera, an illumination module (e.g., light source such as LEDs), 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.

[0041] 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.

[0042] 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, ora combination thereof. In 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.

[0043] In various embodiments, the opto-fluidic instrument 120 may analyze the sample 110 and may generate the output 190that 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.

[0044] 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 light10XG / 1851PC 8source 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.

[0045] 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.

[0046] 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 adapter configured 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.

[0047] 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., a10XG / 1851PC 9tissue sample, placed in contact with, e.g., a substrate (e.g., a surface of the flow cell or microfluidic device).

[0048] 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.

[0049] 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 some instances, only a portion of the sample carrier device (e.g., an optically transparent "window") can be optically transparent.

[0050] 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, thermal10XG / 1851PC 10bonding, 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).

[0051] FIG. 4A illustrates a cross-sectional view of an optics module 200 in an 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. 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 fora 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. 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 comparative imaging 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).

[0052] 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 probes10XG / 1851PC 11configured 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.

[0053] 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 selected 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 structures10XG / 1851PC 12when 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.

[0054] 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.

[0055] Fig. 5 depicts a schematic of an assembly 500 for an imaging system. In Fig. 5, the assembly 500 is fully assembled (with respect to a subset of optical components in an emission optical channel). In overview, the assembly 500 has a base plate 530, to which several components are coupled. The assembly 500 has a plurality of optical components, each of which is coupled to the base plate 530, either directly or indirectly, including a first optical component 521, a second optical component 522, and an additional optical component 523. As will be explained later, the additional optical component 523 is positioned intermediate the first optical component 521 and the second optical component 522 and may therefore be referred to as an intermediate optical component 523 herein. The assembly 500 may comprise a plurality of other components, including other optical components such as the third optical component 524 depicted in Fig. 5. In some implementations, the second optical component 522 defines the optical axis to which all other optical components on the base plate 530 are aligned.

[0056] In the implementations described in detail herein, the assembly 500 is for an imaging system configured for fluorescence microscopy (e.g., epifluorescence microscopy). The imaging system may be a fluorescence microscope. In these implementations, the first optical component 521 comprises an objective lens, the second optical component 522 comprises a tube lens, the10XG / 1851PC 13intermediate optical component 523 comprises a light director (e.g., folding mirror or dichroic filter), and the third optical component 524 comprises a sensor or sensor array, which may take the form of an image sensor (e.g., CMOS sensor). The first optical component 521 and second optical component 522 are positioned such that, in use of the imaging system, one or more rays of light emitted from a sample 545 travel from the first optical component 521 to the second optical component 522. While in these implementations the optical components take these particular forms, the skilled person will understand that the presently disclosed apparatus and its associated optical components may take other forms and the concepts described herein may be applicable to other types of microscopy, for example optical microscopes such as those configured for brightfield, dark field, phase contrast, confocal, light sheet, and / or super resolution microscopy.

[0057] The base plate 530 may be referred to herein as an optical bench. The base plate takes the form of a plate having a first end 531 and a second end 532. The first end 531 and second end 532 are separated along a length axis of the base plate 530. The length axis of the base plate 530 is a central axis that runs parallel with the x-axis as indicated in Fig. 5. The first end 531 is separated from the second end 532 by a length of the base plate 530. The base plate further comprises an upper surface 533 and a lower surface 534. The upper surface 533 and lower surface 534 are separated by a thickness of the base plate 530. The upper surface 533 and lower surface 534 are separated along a thickness axis of the plate 530. The thickness axis is a central axis that runs parallel to the z-axis as indicated in Fig. 5. The base plate 530 further comprises a first face and a second face (not marked in figure 5) which are separated along a width axis of the plate 530, which is similarly a central axis. The length, width and thickness axes meet at a centerpoint of the plate 530 (not marked in Fig. 5).

[0058] The assembly 500 has a first mount 511. The first mount 511 can be utilized to mount and support the first optical component 521, and define its position in relation to the base plate 530 and the other optical components of the assembly 500. The first mount 511 may be described as a first mounting structure or a first supporting structure herein. The first mount 511 is removably couplable to the base plate 530. For example, as will be described in greater detail elsewhere herein, the first mount 511 may be removably coupled to the base plate 530 via the use of one10XG / 1851PC 14or more fasteners (e.g., helical fasteners such as screws or bolts), and corresponding apertures (e.g., helical threaded apertures) and / or unthreaded slots located in the first mount 511 and an upper surface of the base plate 530. In Fig. 5, the first mount 511 is depicted as being coupled to the base plate 530. In use of the assembly, the first mount 511 will be coupled to the base plate 530 and therefore, in general for each of the implementations disclosed herein, the first mount 511 will be described in relation to other features of the assembly 500 as though it is coupled to the base plate 530. The first mount 511 has an overhang 513, which may be described as an overhanging portion herein. The first mount 511 has an inner surface and an outer surface (not marked in Fig. 5), as well as a first (upper) end and a second (lower) end. The overhang 513 is positioned at the first end of the first mount 511. When the first mount 511 is coupled to the base plate 530, the first mount 511 abuts the base plate 530. In particular, the overhang 513 is positioned over at least a portion of the upper surface 533 of the base plate 530 so as to overlap at least the portion of the upper surface 533, and at least a part of the inner surface of the first mount 511 abuts the second end 532 of the base plate 530. In particular, the alignment structure 512 comprises a complementary surface that is in contact with the inner surface of the first mount 511.

[0059] The assembly 500 also has an alignment structure 512, which may also be described as an adjustment structure herein. As will be explained, in an unlocked state or configuration, the alignment structure 512 can be adjusted, for example during a calibration process. In a locked state or configuration, the alignment structure 512 defines an orientation of the first optical component 521. The alignment structure 512 is coupled to the first mount 511. The alignment structure 512 is positioned on the inner surface of the first mount 511. The alignment structure 512 is coupled to the first mount 511 via one or more first couplings (not shown in Fig. 5). The alignment structure 512 is coupled to the first mount 511 in a particular orientation, which may be set during a calibration process (which may occur during the manufacture or assembly of the assembly 500). As can be seen in Fig. 5, the first optical component 521 contacts the alignment structure 512 (e.g., indirectly via an objective collar). The first optical component 521 contacts the alignment structure 512 to define a first set orientation of the first optical component 521 relative to the first mount 511. By virtue of the contact between the first optical component 52110XG / 1851PC 15and the adjustment structure 512, the orientation of the adjustment structure 512 defines the orientation of the first optical component 521 with respect to the first mount 511. The alignment structure 512 may take the form of a ledge which extends from the inner surface of the first mount 511. The ledge has an upper surface, which may be described as an alignment surface herein. The first optical component 521 rests against, or on, the alignment structure 512, and in particular, may rest against or on the alignment surface. Both the alignment structure 512 and first optical component 521 are positioned under the lower surface 532.

[0060] The alignment structure 512 is positioned at the second, lower end of the first mount 511. In preferred implementations, the alignment structure is positioned about 0.1 mm to about 10mm from the second end of the first mount 511, and even more preferably the alignment structure 512 is positioned about 0.5 mm from the second end of the first mount 511. As can be appreciated from Fig. 5, when the first mount 511 is coupled to the base plate 530, the first mount 511 extends from the upper surface 533 of the base plate 530 to below the lower surface 534. In a preferred implementation, the first mount 511 extends below the lower surface 534 by about 20 mm to about 150 mm. The overhang 513 has a length of about 25 mm to about 75 mm. The alignment structure 512 is coupled to the first mount 511 by one or more first couplings. The first couplings are each lockable, and can be utilized to lock the orientation of the alignment structure 512 with respect to the first mount 511.

[0061] When the assembly is in an assembled, calibrated state, the alignment structure 512 is locked in an orientation with respect to the first mount 511 such that, when the first optical component 521 is positioned in contact with the alignment structure 512, the optical component is properly aligned with respect to the second optical component 522. In other words, in a calibrated state, the alignment structure is locked in an orientation such that, when the first optical component 522 contacts the alignment structure 512, the first optical component 521 is oriented in the first set orientation. In the locked state, the alignment structure 512 is rigidly coupled to the first mount 511. In the locked state, the orientation of the alignment structure is fixed with respect to the first munt 511. The locked state may be referred to as a fixed, or secured, state or configuration herein.10XG / 1851PC 16

[0062] In an unlocked state, the alignment structure 512 is rotatable with respect to the first mount 511. The alignment structure 512 is constrained, via the one or more first couplings, to rotate within a first plane. This enables the orientation of the alignment structure 512, and thereby the orientation of the alignment surface, to be adjusted relative to the first mount 511, for example during a calibration process which may occur during manufacture. The alignment structure 512 is rotatable in a plane parallel with the inner surface of the first mount 511. In some implementations, this first plane of rotation is defined by the inner surface of the first mount 511. When the first mount 511 is coupled to the base plate 530, as depicted in Fig. 5, the first plane is generally perpendicular to the upper surface 533 of the base plate, and / or is perpendicular to an upper plane defined by the upper surface 533 of the base plate 530. The first plane of rotation is parallel with a plane which comprises both the thickness and width axes of the base plate 530. The first plane is therefore perpendicular to the x-axis, and parallel wot a plane formed by the z- and y- axes. As will be appreciated from Fig. 5, this plane of rotation is also parallel with the second end 532 of the base plate 530.

[0063] The first optical component 521 is coupled to the first mount 511, for example by virtue of helical threaded fasteners and suitably arranged and configured slots (not shown in Fig. 5).Optionally, the first optical component 521 is removably coupled to the first mount 511. The first optical component 521 is positioned in contact with the alignment structure 512 to define a first set orientation of the first optical component 521, and the first optical component 521 is coupled to the first mount 511 in the first set orientation.

[0064] Together, the first mount 511, the alignment structure 512, and the first optical component 521 form a sub-assembly 510. The sub-assembly 510 is removably couplable to the base plate 530 via the first mount 511. When the sub-assembly 510 is removed from the base plate 530, the alignment structure 512 retains its orientation with respect to the first mount 511. The optical component 510 remains in contact with the alignment structure 512, and by virtue of this contact and its coupling to the first mount 511, the first optical component remains in the first set orientation with respect to the first mount 511.

[0065] The first optical component 521 defines a first optical axis 561. The first optical axis 521 is substantially, or entirely, parallel with the z-direction. The first optical component 52110XG / 1851PC 17comprises an objective lens (which may be referred to herein simply as an objective) and an objective lens mount (e.g., an objective collar). The objective lens mount serves as the interface between the objective lens and its surrounding structure, e.g., the first mount 511 and the alignment structure 521. The objective is secured to the objective mount, and the objective lens mount is configured to hold and support the objective lens. The first optical component 521 may also have an objective z-stage. The objective z-stage comprises an actuator that enables precise vertical movement, along the Z-axis, of the objective lens. This actuator may be, for example, a voice coil actuator. The objective mount is secured to the objective z-stage. Depending on the implementation, it may be either the objective lens mount or the objective z-stage which contacts the alignment structure 512.

[0066] The second optical component 522 is coupled to the base plate 530 and defines a second optical axis 562 to which all other optical components on the base plate 530 are aligned ( / .e., the second optical axis defines a master datum optical axis to which all other components are aligned during calibration). The second optical component comprises a tube lens and a tube lens collar configured to hold and support the tube lens.

[0067] The assembly 500 also has an intermediate optical component 523, which is coupled to the base plate 530 via a second mount (not shown in Fig. 5). The second mount 530 is coupled to the base plate 530. The intermediate optical component 523 may comprise both a light directing component such as a mirror, and in particular a fold mirror, in addition to a mirror mount. In some implementations, the intermediate optical component 523 includes an optically clear material (e.g., glass) having a first coating on a first face (the face pointing away from the base plate 530) and a second coating on the second face (the face reflecting light along the optical axis). For example, the first coating and / or the second coating may include an antireflective coating. Additionally, or alternatively, the first coating and / or the second coating may include a dichroic coating (e.g., a thin film dielectric coating configured to selectively transmit certain wavelengths of light while reflecting or attenuating other wavelengths). In a specific example, the first coating may be an anti-reflective coating and the second coating may be a dichroic coating. The mirror is coupled to the mirror mount, and the mirror mount is configured to hold and support the mirror. The mirror mount is rotationally coupled to the second mount such that10XG / 1851PC 18both the mirror and the mirror mount are rotatable about a rotation axis which is substantially parallel with the y axis. This rotation axis is parallel with the width axis of the base plate 530, and is perpendicular to both the first and the second optical axis 561, 562. The intermediate optical component 523 is constrained, via a rotational coupling, to rotate within a second plane. The second plane is parallel to a plane defined by the z- and x- axes, and to a plane which includes the length and thickness axes. The second plane of rotation, in which the intermediate optical component 523 is constrained to rotate, is perpendicular to the first plane of rotation, in which the first optical component 521 is constrained to rotate.

[0068] The assembly further comprises one or more couplings, which couple the second mount to the intermediate optical component 523. These couplings may be referred to as second couplings herein. For example, the one or more second couplings may couple the second mount to the mirror mount. The one or more second couplings have a dual function: they couple the intermediate optical component 523 to the second mount, and enable a degree of rotation to be adjusted. The one or more second couplings may therefore also be referred to as an adjustment structure, or second adjustment structure, herein. The adjustment structure may take the form of a screw, as will be explained elsewhere herein. The adjustment structure is configured to enable adjustment of a degree of rotation of the intermediate optical component 523, and also to enable the intermediate optical component 523 to be locked in a set orientation with respect to the second mount. This set orientation may be referred to as a second set orientation herein. When the assembly 500 is properly calibrated, the intermediate optical component 523 is fixed in the second set orientation via the one or more second couplings. In this orientation, the intermediate optical component 523 directs light passing through the first optical component 521 to the second optical component 522. The one or more second couplings are lockable to prevent misalignment of the intermediate optical component 523 relative to one or more of the base plate 530, the first optical component 521, and the second optical component 522.

[0069] The assembly 500 further has a third optical component 524. The third optical component 524 takes the form of a sensor or sensor array, and is configured to sense and / or generate images. The third optical component 524 may be a camera, for example. The third optical component 524 may also comprise a sensor alignment plate. The assembly 500 also has a third10XG / 1851PC 19mount (not depicted in fig. 5) which is fixed to the base plate 530 toward the first end 531 of the base plate 530. The third mount may be referred to as a camera mount herein. The image sensor is coupled to the sensor alignment plate, which in turn is coupled to the third mount. Each of the sensor alignment plate and third mount comprises an aperture through which light can pass to the lens of the sensor.

[0070] The assembly 500 further has a sample module 540 which may take the form of the sample module 160 described above. The sample module may take the form of a sample stage configured to move a sample 545 in the x-y directions, in a manner similar to that described above with respect to Figs. 1-3. The assembly 500 has a sample stage mount 542 which connects to the base plate 530. The sample stage mount 542 provides a surface on which the sample stage or sample module 540 is positioned. The sample stage mount 542 is coupled at or toward the first end 531 of the base plate 530. The sample stage mount 542 may be coupled to an underside of the base plate 530, e.g., to a lower surface 534 of the base plate 530. The sample stage mount 542 is adjustable with respect to the base plate 530, so that the distance of the sample stage or module 540 from the base plate 530 may be adjusted as part of calibration processes. The orientation, and in particular the angle, of the surface on which the sample stage 540 is positioned is also adjustable. This is accomplished via suitable couplings between the sample stage mount 542 and base plate 530, which will be explained in greater detail with respect to assembly 600.

[0071] The assembly 500 is broadly similar in function and application to the optics module 200 described above in relation to Fig. 2 and excessive repetition will be avoided herein for brevity. The assembly 500 depicted in Fig. 5 shows the components associated with collection of light from the sample ( / .e., the emission channel), and does not show the component which are associated with excitation of the sample ( / .e., the excitation channel), nor does it show any features such as a controller or processor configured to perform any subsequent processing of the signals received at the sensor 524. However, in an implementation of the assembly 500 as part of an imaging system configured for fluorescence microscopy then the skilled person will understand that the imaging system will comprise several additional features. For example, the imaging system may additionally comprise one or more LEDs, one or more excitation dichroics,10XG / 1851PC 20an excitation fold mirror, a field lens, one or more emission filters, additional dichroic filters, one or more beamsplitters, other lenses, etc.

[0072] At a high level, the first and second optical components 521, 522 are positioned such that, in use of the imaging system, one or more rays of light emitted from the sample 545 travel through the first optical component 521 to the second optical component 522. In more detail, during use of the imaging system, the objective lens in the first optical component 521 focuses excitation light onto the sample 545. The excitation light illuminates the sample 545, and light emitted by the sample 545 in response to the excitation light travels along the first optical axis 561. The first optical component 521 collects the emitted light (e.g., from fluorescently tagged oligonucleotides hybridized to target molecules within the sample). The emission light passes through the first optical component 521 and continues to travel along the first optical axis 561 toward the intermediate optical component 523. The intermediate optical component 523 directs the light toward the second optical component 522. In particular, the mirror forming part of the intermediate optical component 523 redirects light travelling along the first optical axis 561 to travel instead along the second optical axis 562, toward the second optical component 522. The tube lens forming part of the second optical component 522 collects light from the objective lens (i.e., substantially parallel rays), as redirected via the fold mirror. The second optical component 522 focuses the light to form an image at an image plane (that is coplanar with the sensor surface of the third optical component 524, e.g., a CMOS sensor), and the focused image is detected by the third optical component 524.

[0073] The assembly 500 can be calibrated in several ways. A particularly advantageous method of calibrating the assembly 500 will be described herein. But, at a high level, each of the first optical component 521, second optical component 522, third optical component 524, and intermediate optical component 523 are positioned and oriented to enable the functionality described above. For example, the second optical component 522 may be coupled to the base plate 530 to define an orientation of the second optical axis 562 with respect to the base plate 530 (to which all other optical components on the base plate 530 are aligned). The orientation of the intermediate optical component 523 is adjusted, via the adjustment structure, until it is oriented in the second set orientation. This may be accomplished, for example, by shining light10XG / 1851PC 21along the second optical axis 562 and rotating the intermediate optical component 523 until the light is directed down to the sample stage 540 at a location that a sample may be positioned. While the sub-assembly 510 is coupled to the base plate 530, the orientation of the alignment structure 512 is then adjusted, while it is in an unlocked state, until the first optical component 521 is aligned with respect to the second optical component 522. When the first optical component 521 is properly aligned, this defines the first set orientation. The first optical component 521 is then coupled to the first mount 511 in the first set orientation. The position and orientation of the third optical component 524 can then be similarly adjusted until light passing from the objective, via the tube lens, is properly focused at the camera. Once the assembly 500 has been calibrated in this manner, the orientation of the alignment structure (and hence the first optical component 521) is locked. The orientation of the intermediate optical component is also fixed in place, in the second set orientation. The position and orientation of the third optical component is also fixed.

[0074] After calibration of the assembly 500, a user may need to clean, repair, or replace the first optical component 521, for example due to wear or contamination. In prior arrangements, such an operation would require the assembly to undergo extensive recalibration processes once the cleaned, repaired or new component has been fitted back into the assembly. In contrast, the current assembly 500 enables the cleaning, repair or replacement of the component without requiring such extensive recalibration. With the presently disclosed assembly 500, the subassembly 510 can be easily removed by uncoupling the first mount 511 from the base plate 530. When the mount 511 is removed, both the first optical component 521 and, optionally, the alignment structure 512 and / or z-stage is removed with the first optical component 521. Importantly, the relative orientations and alignments of each component of the sub-assembly remain fixed. In particular, the first optical component 521 remains in the first set orientation with respect to the first mount 511.

[0075] Once the sub-assembly 510 has been removed, the objective lens or other component part of the first optical component 521 can be cleaned or replaced in a straightforward manner, and the sub-assembly 510 can be re-coupled to the base plate 530. As the first mount 511 is recoupled to the base plate 530, the first optical component 521 is brought back to its former10XG / 1851PC 22position and orientation relative to the base plate 530 and relative to the second optical component 522. Accordingly, no complex or time-consuming re-calibration steps are required. As will be described in greater detail elsewhere herein, the base plate 530 and the first mount 511 may comprise corresponding engagement features to ensure that the first mount 511 is accurately re-coupled to the base plate 530 in the same, preferred position each time.

[0076] The presently disclosed assembly 500 is also beneficial in scenarios in which the first optical component 521 must be removed from the first mount 511, e.g., so that the first optical component 521 or one of its component parts can be cleaned, repaired or replaced (without replacing the entire assembly). In this scenario, the first optical component 521 can be uncoupled from the first mount 521. For example, the first optical component 521 may be unscrewed from the first mount 521. During this process, the alignment structure 512 remains locked in place, and the relative orientation of the alignment structure 512 and the first mount 511 remains constant. Once the first optical component 521 has been cleaned, repaired or replaced, it can be re-positioned in contact with the alignment structure. This contact brings the first optical component 521 back into the first set orientation with respect to the first mount 511, and the first optical component 521 can be re-coupled to the mount 511 to re-fix the first optical component in the first set orientation. As described above, re-coupling the sub-assembly 510 to the base plate 530 via the first mount 511 brings the first optical component 521 back into alignment with respect to the other features of the assembly 500 and, in particular, with respect to the second optical component 522. In this way, the objective lens is swappable while maintaining the first set orientation of the objective lens relative to the first mount 511. Accordingly, the presently disclosed assembly 500 enables quicker, more efficient cleaning, repair, and / or replacement of optical components without the need for extensive, timeconsuming re-calibration.

[0077] Fig. 6 depicts an assembly 600 for an imaging system in accordance with the present disclosure. Figs. 7-9 depict different views and components of the same assembly 600 depicted in Fig. 6. The assembly 600 is a specific implementation of the assembly described with respect to the schematic assembly 500 depicted in Fig. 5. The components have like forms and functions to those described above with respect to Fig.5 unless where noted otherwise, and repetition will10XG / 1851PC 23be kept to a minimum to ensure brevity and improve readability. Like reference numerals are used, where appropriate and possible, to denote like parts.

[0078] As with the assembly 500 depicted in Fig. 5, the assembly 600 has a first optical component 621, a second optical component 622, an intermediate optical component 623, and a third optical component 624.

[0079] The first optical component 621 is coupled to a first mount 611. The first mount 611 comprises an overhang 613. The overhang 613 comprises a plurality of screw holes 614a, 614b which enable the first mount 611 to be coupled to, and uncoupled from, the base plate 630. The screw holes 614a, 614b are configured to align with corresponding screw holes formed in the upper surface 633 of the base plate 630, and both sets of screw holes can correspond with screws to enable the first mount 611 to be screwed to the base plate 630. In this manner, the first mount 611 is removably couplable to the base plate 630.

[0080] The first optical component 621 is positioned in, and / or aligned with, an aperture in the base plate 630. The aperture may be formed by the structure of the base plate 630, and the first mount 611. In this way, light can pass from the first optical component 621, to the second optical component 622, via the intermediate optical component 623.

[0081] The second optical component 623 is rotationally coupled to a second mount 651. The second mount 651 is rigidly coupled to the base plate 630, for example via screws or other suitable fasteners. In addition, Fig. 6 depicts the sensor alignment plate 653 coupled to a third mount 652, and the image sensor 624 coupled to the sensor alignment plate 653.

[0082] The assembly 600 depicted in Fig. 6 has a particular form of alignment, or adjustment, structure 612. As described in relation to the assembly 500 of Fig. 5, the alignment structure is configured, and constrained, to rotate in a first plane of rotation. The first plane of rotation is parallel with the second end 632 of the base plate 630, and perpendicular to a second plane of rotation in which the intermediate optical component 623 is constrained to rotate. However, the alignment structure 612 is further constrained to rotate about a first rotational axis 661 with respect to the first mount 611. The first rotational axis 661 is perpendicular to the second rotational axis 662. The first rotational axis 661 is substantially parallel with the x axis, and also parallel with the length axis of the base plate 630.10XG / 1851PC 24

[0083] As with assembly 500, the assembly 600 has a sample stage 640. The sample stage 640 is supported by the sample stage mount 642. The sample stage mount 642 is adjustably coupled to the base plate at two points on the underside 634 of the base plate 630. These two points are separated along the y-axis, i.e., in a direction parallel with the second optical axis 662. A first of these couplings 643 is depicted in Fig. 6, with the other being positioned on the opposite side of the base plate 630. The couplings are positioned on left and right sides of the base plate 630, towards the front (i.e., toward the second end 632). The couplings 643 take the form of threaded adjusters which enable a degree of vertical adjustment. This movement may be accommodated by one or more pivotable couplings such as swivel washers which are used to couple the sample stage mount 642 to the base plate 630 toward the first end 631 of the base plate. As the skilled person is aware, a swivel washer is a specialized type of washer designed to allow some degree of movement or adjustability between two connected components. The one or more swivel washers, or other pivotable couplings, enable the angle that the sample stage 640 makes with the base plate 630 to be adjusted, since vertical adjustment of the couplings 643 causes a slight pivoting motion about the pivotable couplings. Therefore, the sample stage 640 is adjustable with respect to the base plate 630. In some implementations, the sample stage 640 includes two linear stages (e.g., a first linear stage for x-axis motion and a second linear stage perpendicularly coupled to the first linear stage for y-axis motion). In some implementations, the linear stages include one or three phase linear servo motors. In some implementations the linear stages include lead screws driven by stepper motors.

[0084] Fig.7 depicts a simplified schematic of the sub-assembly 610 of assembly 600, as coupled to the base plate 630. The alignment structure 612 is configured to rotate about a first rotation axis 662 by virtue of a rotational coupling 616. The rotational coupling 616 may take the form of a screw, a shaft, and / or a bearing. The alignment structure 612 is rotatable about the first rotational axis 662, which is in turn defined by the rotational coupling 616. In this implementation, the alignment structure 612 has a face which is substantially triangular or wedge-shaped, and a lower surface that extends substantially perpendicular to the triangular face to form a ledge on which the first optical component can rest. The alignment structure 612 is constrained, via the rotational coupling 616, to rotate within the first plane.10XG / 1851PC 25

[0085] The rotational coupling 616 is lockable by virtue of a plurality of through-holes formed in the alignment structure 612. The first mount 611 also has a plurality of elongated slots positioned, sized, and otherwise configured to correspond with the through-holes 617a, 617b. Fixation elements such as screws or bolts can be positioned through the through-holes 617a, 617b, and / or slots. Because the slots are elongated, a certain degree of rotation of the alignment surface 612 with respect to the first mount 611 is enabled. In a calibration phase, the alignment structure 612 is rotated about the first rotational axis 662 until the alignment structure 612 reaches the first set orientation with respect to the first mount 611. At this orientation, as discussed herein, the first optical component s properly positioned in a calibrated position and orientation. The alignment structure 612 can then be locked in the first set orientation by using fastening means such as screws, nuts, and bolts, which co-operate with through-holes 617a, b. In principle, the sub-assembly can be returned to an unlocked state by loosening these fastening means, but after the assembly 600 has been calibrated one, for example as part of a manufacturing process, there is no need to unlock these couplings.

[0086] Fig. 7 also depicts a mirror 655 and mirror mount 656, which together form part of the second optical component 623. The mirror mount 656 extends along at least two sides of the mirror 655 and has a groove into which the mirror sits. The mirror mount 656 is rotationally coupled to the second mount 651 to define the second rotational axis 662.

[0087] Fig.8 depict a simplified schematic of the sub-assembly 610 when it is not coupled to the base plate 630, and rotated to show the first optical component 621 coupled to the inner surface 615 of the first mount 611. The first mount 611 has a ledge 614 that, when the sub-assembly is coupled to the base plate 630, extends under the lower surface of the base plate 630. The ledge has an upper surface which may be referred to as an alignment surface herein. In each implementation described herein, the alignment surface is a precision flat surface. Here, the first optical component 621 has an objective mount configured to hold the objective lens. The objective mount has a flat lower surface. Once the orientation of the alignment structure 611 has been fixed in the first set orientation with respect to the first mount 611, the flat lower surface of the objective mount is positioned in contact with the alignment surface of the ledge 614. The flat lower surface of the alignment structure 611 lies flat against the flat alignment surface of the10XG / 1851PC 26ledge 614. In this way, the orientation of the ledge 614 defines the orientation of the first optical component 621. In the depicted implementation, the first optical component 621 also has a z-stage. In some implementations, the z-stage may contact the alignment surface via its lower flat surface.

[0088] Fig. 9 depicts a similar view of the assembly 600 as Fig. 7, but with the first mount 611 rendered transparent, and with several components on the upper surface 633 of the base plate 630 removed for simplicity of illustration. In Fig.9, it can be appreciated that the objective mount is coupled to the first mount 611 by virtue of a plurality of couplings. The couplings take the form of threaded fasteners, such as screws or the like. The couplings co-operate with apertures 669a-669d formed in the first optical component. In particular, the apertures 669a-669d may be formed in the objective mount. While four apertures 669a-669d are marked with reference numerals in Fig. 9, the skilled person will appreciate that there may be more, or fewer, and the apertures 669a-669d may be positioned differently according to the requirements of the particular implementation. The apertures may be any of slots, holes, or recesses formed on the objective mount. It will be understood that there are corresponding slots, holes, apertures or recesses formed on the inner surface of the mount 611. The apertures formed on the first optical component are large enough so that they do not define only one orientation of the first optical component 621 with respect to the first mount 611. In other words, the apertures 669a-669d are large enough so that they allow fora range of possible orientations of the first optical component 621 with respect to the first mount 611. This range may be described as a degree of 'play' in the coupling. Once the first alignment structure 612 has been oriented in the first set orientation, the first optical component 621 can be secured in place against the inner surface 615 of the first mount 621 using the first couplings 669a-669d, for example using threaded fasteners such as screws or the like.

[0089] Fig. 9 also depicts base orientation structures 691, 692 formed on an upper surface 633 of the base plate 630. The base orientation structures may also be referred to as base engagement or base alignment features herein. As will be detailed elsewhere herein, the base orientation structures 691, 692 are shaped, positioned, and otherwise configured so as to enable components to be positioned and alignment accurately with respect to the base 630. The first10XG / 1851PC 1base orientation structure 691, or structures 691, form a first subset of the base orientation structures. The first mount 611 also comprises one or more first mount engagement or mount alignment structures, which are configured to co-operate and correspond with the base orientation structure 691. The first subset of base orientation structures 691 and one or more first mount alignment structures correspond with one another to define a preferred alignment of the first mount 611 with respect to the base plate 630. For example, in the arrangement depicted in Fig. 9, the first base orientation structure comprises a recess that is exactly shaped and formed so as to receive the overhang 613 of the first mount 611.

[0090] As shown in Fig. 9, the assembly 600 also has third base orientation structure 692. The third base orientation structure is configured to receive the intermediate optical component. This structure will be described in greater detail with respect to Figs. 15A-15C and Figs. 16A-16E.

[0091] Fig. 10 depicts an assembly 1000 for an imaging system in accordance with the present disclosure. Figs. 10-14 depict different views and components of the same assembly 1000 depicted in Fig. 10. The assembly 1000 is a specific implementation of the assembly described with respect to the schematic assembly 500 depicted in Fig.5, and is similar in form and function to assembly 500 as well as assembly 600 depicted in Figs. 6-9. The components have like forms and functions to those described above with respect to Fig. 5 unless noted otherwise, and repetition will be kept to a minimum to ensure brevity and improve readability. Like reference numerals are used, where appropriate and possible, to denote like parts. In particular, the assembly 1000 is depicted in Fig. 10 only with those components which have significantly different aspects and features to describe in comparison with assemblies 500 and 600. In particular, the assembly 1000 depicts a different implementation of the first mount 1011 and alignment structure, and a different implementation of the second mount 1051.

[0092] In a manner similar to that described above in relation to assembly 500 and assembly 600, the assembly 1000 has a first mount 1011 which is removably couplable to the base plate 1030. In Fig. 10, the first mount 1011 is depicted as (removably) coupled to the base plate 1030. The first mount 1011 comprises an overhang 1013. The overhang 1013 overlaps the upper surface 1033 of the base plate 1030. When the first mount 1011 is removably coupled to the base plate 1011, it contacts the upper surface 1033 of the base plate 1030, and extends below the lower10XG / 1851PC 28surface of the base plate 1030. The first mount 1011 is in contact with a region of the second end 1032 of the base plate 1030 while it is coupled to the base plate 1030. The region may be a central region of the second end 1032.

[0093] The overhang 1013 comprises a plurality of screw holes 1014a, 1014b which enable the first mount 1011 to be coupled to, and uncoupled from, the base plate 1030. The screw holes 1014a, 1014b are configured to align with corresponding screw holes formed in the upper surface 1033 of the base plate 1030, and both sets of screw holes 1014a, 1014b are configured to correspond with screws or similar threaded fasteners to enable the first mount 1011 to be screwed to the base plate 1030.

[0094] The second mount 1051 is coupled to the base plate 1030, for example via screws or the like. As explained above in relation to Fig.5, in addition to the rotational coupling which couples the intermediate optical component 1023 to the second mount 1051, one or more second couplings also couple the intermediate optical component 1023 to the second mount 1051. These second couplings act to lock an orientation of the intermediate optical component 1023 with respect to the second mount 1051, to prevent misalignment of the intermediate optical component 1023 relative to one or more of the base plate 1030, the first optical component 1021, and the second optical component (not depicted in Fig. 10). As will be explained in greater detail with respect to Figs. 13 and 14, in the implementation depicted in Figs. 10-14, the one or more second couplings may take the form of a screw or multiple screws which co-operate with corresponding slots, holes, apertures or recesses formed in both the intermediate optical component 1023 and the second mount 1051.

[0095] Figs. 11A-11C depict a first mount 1011. The first mount 1011 is the mount depicted coupled to the assembly 1000 in Fig. 10. At a high-level, the form and function of the first mount 1011 is similar to that described above with respect to assemblies 500 and 600, though the alignment structure 1012 coupled to the first mount 1011 takes a different form. As shown in Fig.11A and 11C, the alignment structure 1012 is an elongate bar. The elongate bar is coupled to the first mount 1011 via one or more first couplings. The one or more first couplings are rotational couplings, which enable the orientation of the elongate bar to be adjusted relative to the first mount 1011.10XG / 1851PC 29

[0096] The alignment structure 1012 has four elongated surfaces which run from the first end to the second end of the alignment structure 1012. The four elongated surfaces include a complementary surface, such that, when the alignment structure 1012 is positioned on the inner surface 1015 of the first mount, the complementary surface is in contact with the inner surface of the first mount 1011. Another of the four elongated surfaces is the alignment surface 1018. The alignment surface 1018 forms a ledge on which, in use, the first optical component 1021 rests. The alignment surface 1018 is a precision flat surface to ensure that the orientation of the first optical component 1021 can be defined to a high degree of accuracy.

[0097] The elongate bar extends from a first end to a second end, and has a first through-hole 1017a positioned at or toward the first end, and a second through-hole 1017b positioned at or toward the second end. The through-holes 1017a, 1017b enable the elongate bar to be coupled to the first mount 1011 via the first couplings. Each coupling may be any of a screw, a shaft, and / or a bearing. The first mount 1011 has a plurality of slots. The plurality of slots is positioned on the inner surface 1015 of the first mount 1011. The slots are each positioned to align with the through-holes 1017a, 1017b and are each configured to receive a coupling of the one or more first couplings. The slots are elongated in the z-direction, and may be described as vertical slots or vertical elongated slots herein.

[0098] Each coupling passes through one of the through-holes 1017a, 1017b in the alignment structure 1012, and into a corresponding elongated slot in the first mount 1011, thereby coupling the alignment structure 1012 to the first mount 1011. Each elongated slot is position at ortoward the second (lower) end of the first mount 1011, to enable the alignment structure 1012 to be positioned at or toward the second (lower) end of the first mount 1011. The alignment structure 1012 is rotatable, at least to a certain degree (e.g., ± about 5 degrees, ± about 4.5 degrees, ± about 4 degrees, ± about 3.5 degrees, ± about 3 degrees, ± about 2.5 degrees, ± about 2 degrees, ± about 1.5 degrees, ± about 1 degree), about a rotation axis defined by each first coupling. Because the slots in the first mount 1011 are elongated, they allow for movement and adjustment of the alignment structure 1012 in a degree of freedom defined by the elongation of the slots. In an implementation in which the slots are elongated in a direction parallel with a length axis of the first mount 1011, e.g., in a direction from the first end to the second end of the10XG / 1851PC 30first mount 1011, then each coupling allows the alignment structure 1012 to be adjusted, at least to a certain degree, in the same direction. When the first mount 1011 is coupled to the base plate 1030, the slots can be considered to be elongated in a vertical / z-axis, and accordingly each coupling allows the alignment structure 1012 to be adjusted, at least to a certain degree, in the vertical / z-direction.

[0099] As with the assemblies 500, 600 described above, the alignment structure 1012 is rotatable with respect to the first mount. The alignment structure 1012 is constrained, via the one or more first couplings, to rotate within a first plane. In the depicted implementation in which the alignment structure 1012 comprises two through-holes 1017a, 1017b which correspond with two elongated slots on an inner surface 1015 of the first mount 1011, the first plane is parallel with, and defined by, the inner surface 1015 of the first mount 1011. When the first mount 1011 is removably coupled to the base plate 1030, this first plane is perpendicular to an upper plane defined by the upper surface 1033 of the base plate 1030 and / or is perpendicular to the upper surface 1033 itself. When the first mount 1011 is removably coupled to the base plate 1030, at least part of the inner surface 1015 is in planar contact with the second end 1032 of the base plate 1030, and therefore the first plane is parallel not only with the inner surface 1015, but also with the second end 1032 of the base plate 1030.

[0100] The one or more first couplings may each additionally comprise a fixation member. The fixation member may be, for example, a screw, a bolt, a rivet, a shaft, a nut, or any combination of these components. The fixation members are configured to fix, i.e., lock, the orientation of the alignment structure 1012 with respect to the first mount 1011. The one or more first couplings can therefore be used to fix the orientation of the alignment structure 1012 when it has been adjusted to the first set orientation. As described above, the orientation of the alignment structure 1012 defines the orientation of the first optical component 1032 when the first optical component 1021 is in contact with the alignment structure 1012. When the first mount 1011 is removably coupled to the base plate 1030 with the first optical component 1021 in the first set orientation, the first optical component 1021 is aligned relative to the second optical component 1022.10XG / 1851PC 31

[0101] The first mount 1011 also comprises a plurality of apertures 1071a-1071d. The apertures 1071a-1071d may extend all the way through the first mount 1011 ( / .e., are a through-hole), or may take the form of a recess. The apertures 1071a-1071d are shaped, positioned, and otherwise configured to receive a coupling / fastener such as a screw. The apertures 1071a-1071d align with similar apertures 1071a-1071d on the first optical component 1021 to enable the first optical component 1021 to be rigidly coupled, via the use of suitable couplings, to the first mount 1011. The apertures on the first optical component 1021 may take a similar form as the apertures 669a— 669d depicted in Fig. 9. As described in relation to Fig. 9, the apertures in the first optical component are large enough to accommodate a range of possible orientations of the first optical component 1021.

[0102] The function of the alignment structure 1012 will now be briefly described, though it should be understood that the functionality of the first mount 1011 and alignment structure 1012 is broadly similar to that functionality described above with respect to assemblies 500, 600. While the fixation elements of the one or more first couplings are in an unlocked state, and when the orientation of the alignment structure 1012 is therefore not in a fixed state, the orientation of the alignment structure 1012 is adjusted. When the orientation of the alignment structure 1012 relative to the first mount 1011 is in the first set orientation, the orientation of the alignment structure 1012 is fixed by the fixation members. The first optical component 1011 can be oriented in the first fixed orientation by positioning a lower, flat surface of the first optical component 1011 in contact with the precision flat alignment surface 1018. In this way, the first optical component 1021 rests on the ledge defined by the alignment structure 1012. The orientation of the first optical component, when positioned with its flat lower surface in contact with the alignment surface 1018, is defined by the orientation of the alignment structure 1012. The orientation of the first optical axis is also therefore defined by the orientation of the orientation of the alignment structure 1012. The first optical component 1021 is then rigidly coupled to the inner surface 1015 of the first mount 1011 in order to fix the first optical component 1021 in the first set orientation. This coupling may be accomplished via the use of the apertures 1071a-1071d, corresponding apertures on the first optical component 1021, and couplings / fasteners which pass the apertures and lock the first optical component 1021 to the first mount 1011.10XG / 1851PC 32

[0103] Figs. 11D-11F depict another embodiment of a first mount 1111 suitable for use with any of the assemblies disclosed herein (e.g., capable of being coupled to the base plate 630, 1030). Similar to the first mount 1011 depicted in Figs. 11A-C, the first mount 1111 can be coupled to the assembly 1000 in Fig. 10 or the assembly 1600 of Figs. 16A-16D. In addition to the features of the first mount 1011, the first mount 1111 further includes a plurality of holes 1020 for coupling one or more masses 1021a, 1021b. Coupling of one or more masses 1021a, 1021b to the first mount 1051 may beneficially impart properties of a tuned mass damper to the first mount 1111, allowing for improved performance (e.g., faster operation) of a z-stage coupled thereto and configured to move the objective lens along the z-axis. In various embodiments, the plurality of holes 1020 is configured to receive threaded screws (e.g., each hole of the plurality of holes 1020 is tapped). In various embodiments, the plurality of holes 1020 is provided below a center of mass (e.g., at the bottom) of the first mount 1111. In various embodiments, the plurality of holes 1020 are positioned symmetrically about a vertical axis that intersects the center of mass of the first mount 1111. For example, as shown in Fig. 11F, a first mass 1021a is coupled to a first side at the bottom of the first mount 1051 and a second mass 1021b is coupled to a second side at the bottom of the first mount 1051. The first mass 1021a and / or the second mass 1021b may be sized such that the surfaces of the masses are substantially flush with one or more (e.g., all) surfaces of the first mount 1111. Alternatively, the first mass 1021a and / or the second mass 1021b may be sized such that the masses extend beyond one or more surfaces of the first mount 1111 (e.g., the masses have a thickness that is greater than the thickness of the first mount 1111 at the location where the masses couple and / or the masses extend below the bottom surface of the first mount 1111). In various embodiments, a single mass may extend across the bottom of the first mount 1111 from the first side to the second side and be coupled to the plurality of holes 1020.

[0104] Fig. 12 depicts a first optical component 1021 in contact with the alignment structure 1014 and coupled to the first mount 1011 to form a sub-assembly 1010. As described above, for example with respect to figure 8, the sub-assembly is removably couplable to the base plate 1030. Here, the first optical component 1021 has multiple sub-components including an objective lens, an objective lens mount, and a z-stage which itself comprises a voice coil actuator10XG / 1851PC 33configured to precisely translate a carriage in the plus or minus z-directions. As explained above with respect to assemblies 500 and 600, the sub-assembly 1010 is removably couplable to the base plate 1030 to facilitate cleaning, repair, and replacement of the first optical component 1021 or any of its individual sub-components. While the first optical component 1021 remains rigidly coupled to the first mount 1011, it is kept in the first set orientation. Accordingly, when the sub-assembly 1010 is re-introduced to the base plate 1030, the first optical component 1021 is brought back into alignment with the other optical components and, in particular, with the second optical component. When cleaning, repair or replacement requires the first optical component 1021 to be uncoupled from the first mount 1011, or when the first optical component 1021 or one of its sub-components must be replaced, then the cleaned, repaired, or new optical component can be easily brought back into the first set orientation via the first alignment surface 1014. This enables the process to take place without extensive, complex and time-consuming recalibration processes.

[0105] Fig. 13 depicts the sub-assembly 1010 of Fig. 12 and the second mount 1051 coupled to the base plate 1030. Fig. 14 depicts the second mount 1051 and intermediate optical component 1022 from a different angle. The implementation of the second mount 1051 depicted in Figs. 13 and 14 is depicted together with the implementation of the alignment structure 1012 in which the alignment structure 1012 is an elongate bar. However, it should be understood that this implementation of the second mount 1051 may be used with any of the implementations of the sub-assembly described herein, for example the sub-assembly 610 described above with respect to Fig. 6.

[0106] As can be appreciated from the figures, in this implementation, the second mount 1051 extends upward from the upper surface 1033 of the base plate 1030. The second mount 1051 comprises a first portion 1084 that extends upward from the base plate 1030. As described above, the intermediate optical component 1023 is rotationally coupled to the second mount 1051, and in this implementation the intermediate optical component 1023 is rotationally coupled to an inner face of the first portion 1084. The second mount 1051 also has a second portion, which extends inwardly from the first portion 1885. The second portion 1085 extends in10XG / 1851PC 34a direction parallel to the rotation axis of the intermediate optical component to a position at least partly above the intermediate optical component 1023.

[0107] The second portion 1085 of the second mount 1051 has an angled face 1082. The angled face 1082 is formed at substantially 45° with respect to the second optical axis, which is defined by the second optical component. The angled face 1082 is also formed at substantially 45° with respect to the first optical axis, which is defined by the first optical component 1021. Equivalently, the angled face 1082 is formed at substantially 45° with respect to both the x- and the z-axes.

[0108] Here, the intermediate optical component 1023 has sub-components which include both a mirror 1055, in particular a fold mirror, and a mirror mount 1056. While reference is made to a mirror or fold mirror herein, the component may be any light director, for example a suitably configured dichroic filter. The mirror mount 1056 holds and supports the mirror 1055, and contains suitable structure to enable the intermediate optical component 1023 to be rotationally coupled to the second mount 1051. The mirror mount 1056 also contains suitable structure to allow the intermediate optical component 1021 to be coupled to the second mount 1051 via at least one additional point, via at least one second coupling 1081. This at least one second coupling 1081 is configured to enable adjustment of the orientation of the intermediate optical component 1023, and also to lock or fix the degree of orientation. In this way, the one or more second coupling 1081 are each lockable to lock the orientation of the intermediate optical component 1021 with respect to the second mount 1051.

[0109] In this implementation, the at least one second coupling 1081 is screw that connects, i.e., couples, the second mount 1051 to the intermediate optical component 1023. In particular, the screw connects the second mount 1051 to the intermediate optical component 1023 via a slot or aperture formed in the angled face 1082 of the second mount 1051, and via a corresponding slot in the mirror mount 1056. Tightening or loosening the screw brings the intermediate optical component 1023 closer, and further away, from the second mount 1051 respectively. When the screw is not being adjusted, the intermediate optical component 1023 is held in place and its orientation with respect to the second mount 1051 is fixed.

[0110] When the assembly 1000 is in a calibrated state, the first optical axis associated with the first optical component 1021 meets the second optical axis associated with the second optical10XG / 1851PC 35component at the intermediate optical component 1023. The second coupling 1081 is used to lock the mirror 1055 in an orientation such that light passing from the sample and through the first optical component 1021 is directed to the second optical component. When the second coupling is 'locked', e.g., when the screw is not being turned, the second coupling 1081 prevents misalignment of the intermediate optical component 1023 relative to one or more of the base plate 1030, the first optical component 1021, and / or the second optical component.

[0111] Figs. 15A-15C depict a base plate 1500. The base plate 1500 is suitable for use with any of the assemblies described or depicted herein. The base plate 1500 has one or more orientation structures. The orientation structures are generally positioned, shaped, and otherwise configured to enable components to be positioned in the correct orientation with respect to the base 1500 to ensure proper alignment of the various components. The orientation structures are configured to engage with the various components, for example via corresponding orientation structure(s) on those components. The orientation structures on both the base 1500 and the various components may be described as engagement structures, or alignment structures, herein. The orientation structures may take the form or recesses, slots and / or apertures, which are configured to receive and position the optical components. The orientation structures are shaped, positioned, and otherwise configured to define a particular configuration of the optical components such that the components may be coupled to the base plate 1500 in a particular configuration with respect to one another. The base plate has a plurality of base orientation structures, which can be separated into subsets of base orientation structures according to which component they are configured to orient.

[0112] As explained with respect to the base plates for the assemblies described above, the base plate 1500 has a first end and a second end, as well as an upper surface 1590 and a lower surface. The base plate 1500 has a cut-out 1501 in its second end, to accommodate the first optical component. The cut-out 150 enables light to pass from the fold mirror via the first optical component and to the sample, and vice versa.

[0113] The base plate 1500 has first base orientation structures 1510 a, 1510b, which may be described as a first subset of the plurality of base orientation structures. The first base orientation structures 1510a, 1510b take the form of recesses positioned either side of the cut-out 1501. The10XG / 1851PC 36recesses 1510a, 1510b may be substantially symmetrical about the second optical axis. The first base orientation structures 1510a, 1510b enable the first mount 511 to be properly oriented with respect to the base plate 1500 when it is coupled to the base plate 1500. The first base orientation structures 1510a, 1510b are shaped to interact and engage with corresponding orientation structure on the first mount, in particular, a lower surface of the overhang of the first mount. The overhang may also have extensions or protrusions which are shaped to co-operate with the first base orientation structures 1510a, 1510b. In other words, the first base orientation structures 1510a, 1510b take the form of recesses formed in the upper surface 1590 of the base plate 1500. The first mount has first mount orientation structure(s) in the form of an overhang configured to overlap the upper surface 1590 of the base plate 1500, and fit within the recesses of the first subset of base orientation structures 1010a, 1010b.

[0114] The first base orientation structures 1510a, 1510b each have a floor, which may be described as a lower surface of the recess. The floor is positioned at a first depth below the upper surface 1590 of the base plate 1500. The first base orientation structures 1510a, 1510b each also have at least one mounting pad 1511 raised above the floor. The mounting pads 1511 are at a second depth below the upper surface 1590, where the second depth is less than the first depth. The first base orientation structures 1510a, 1510b are shaped and configured to receive the first mount, which when positioned in the first base orientation structures 1510a, 1510b will rest on the mounting pads 1511. In this way, the mounting pads 1511 define a preferred height for the first mount with respect to the base plate. Accordingly, the mounting pads 1511 also define a preferred height for the first optical component.

[0115] Each mounting pad 1511 also has a height, measured from the floor or the first base orientation structures 1510a, 1510b. In the depicted implementation, the mounting pads 1511 have a height of about 0.1mm to about 1mm. Preferably, the mounting pads 1511 have a height of about 0.5mm. The mounting pads 1511 also have a surface area. The surface area of the mounts 1511 is less than the surface area of the floor of the first base orientation structure 1510a, and may be significantly less. Each mounting pad 1511 in each recess may have an area, for example, of about 50 mm2to about 3000 mm2.10XG / 1851PC 37

[0116] The base plate 530 also has second engagement features which enable the second optical component 522 to be mounted to the base plate 530. The base plate 530 also has third engagement features which enable the third (i.e., intermediate) optical component 523 to be mounted to the base plate 530, and fourth engagement features which enable the fourth optical component 524 to be mounted to the base plate 530. The base plate 531 has a top surface 531 and a lower surface 532.

[0117] The plurality of base orientation structures may comprise several recesses, each with one or more mounting pads. For example, the base has a base orientation structure 1570 which takes the form of a large recess in the base plate. As with the first base orientation structures 1510a, 1510b described above, the orientation structure 1570 has at least one mounting pad. Here, the recess has a plurality of mounting pads 1571, 1572, 1573, and in particular three mounting pads. Each of the mounting pads 1571, 1572, and 1573 have the same height above the floor. The mounting pads 1571, 1572, 1573 define a preferred height for an optical component. The mounting pads 1571, 1572, 1573 may have precision flat surfaces on their upper surfaces so that a component seated on the mounting pads 1571, 1572, 1573 can be positioned at a particular height, and can be aligned with respect to other components of the assembly, with a high accuracy. The base orientation structure 1570 has a depth measured from the upper surface 1590 to the floor, and each mounting pad 1571, 1572, 1573 has a height measured from the floor. The depth of the recess is greater than the height of the mounting pads.

[0118] An advantage of using mounting pads relates to ease and accuracy of manufacture. The mounting pads in a particular recess have a combined upper surface area that is less than the surface area floor of the recess, and may even have a combined surface area that is significantly less than the floor of the recess. This means that the total area which must be a precision flat surface is reduced, facilitating milling of the base plate. Milling the recesses, particularly larger recesses such as base orientation structure 1570, with a milling machine or other cutting tool can cause the blade (and workpiece) to heat up. A high RPM is required to mill recesses in a base plate. The present inventors have recognized that thermal fluctuations in the cutting tool associated with prolonged use of the cutting tool at high RPM can result in fluctuations and warping in the material. These fluctuations may be slight, but are significant when it is desirable10XG / 1851PC 38to enable an optical component to be seated with high accuracy in a recess. Because the surface area of each mounting pad is small, the use of the cutting tool needn't be prolonged, and the cutting tool is less likely to heat up to a degree that could cause warps or fluctuations in the material. In addition, the floor of each recess can be cut to a reduced level of accuracy, improving efficiency and reducing the manufacturing time associated with the base plate 1500.

[0119] In some implementations, each mounting pad is positioned in contact with a wall of its recess. This can be seen in the base orientation structure 1570, where each of the mounting pads 1571, 1572, and 1573 are in contact with a wall of the base orientation structure 1570. This enables a further efficiency of manufacture: the recess can be cut to a reduced level of accuracy in a shape defined by the surface area of the recess floor. After, the milling machine can be set with a z-axis depth suitable for cutting the mounting pads, the cutting tool can be placed in the recess, and each mounting pad can be cut one after each other without adjusting the z-axis setting. Since each adjustment of the cutting tool introduces a level of uncertainty within a small tolerance, this method of manufacture helps ensure the mounting pads in the recess are all at the same height.

[0120] The base plate 1500 also has a second subset of base orientation structures 1520. The second subset of base orientation structure 1520, or second base orientation structures 1520, are best seen in Fig. 15B. The second base orientation structures 1520 are configured to receive the second optical component, and thereby define a preferred alignment for the second optical component with respect to the base plate 1500.

[0121] The second subset of base orientation structures 1520 take the form of at least two flat surfaces: a first flat surface 1521 and a second flat surface 1522. When the apparatus is in an assembled state, the first flat surface 1521 and the second flat surface 1522 are positioned either side of the second optical axis. As can be appreciated from Fig. 15B, the first and second flat surfaces 1521, 1522 are angled with respect to one another. The first and second flat surfaces 1521, 1522 are angled with respect to one another such that the depth of the flat surfaces, measured with respect to an upper surface 1590 of the base plate 1500, increases toward a central point. The first flat surface 1521 and the second flat surface 1522 increase in height in directions away from the second optical axis. In other words, the first flat surface 1521 and the10XG / 1851PC 39second flat surface 1522 slope downwardly toward each other. The first flat surface 1521 and the second flat surface 1522 may meet at the central point, however in the implementation depicted in Figs. 15A-15C the first flat surface 1521 and the second flat surface 1522 do not meet at the central point. In other words, the first flat surface 1521 and the second flat surface 1522 may be continuous, i.e., may meet at a central point, or may be discontinuous. Regardless, the first flat surface 1521 and the second flat surface 1522 may be described as a V-shaped groove or simply a v-groove since, when viewed along the x-axis, the first flat surface 1521 and the second flat surface 1522 generally form a V-shape. When the second optical component is seated in the V-groove, the first flat surface 1521 and the second flat surface 1522 are substantially symmetrical about the second optical axis defined by the sconed optical component 522.

[0122] In use, the first flat surface 1521 and the second flat surface 1522 guide the second optical component to a lowest point, ensuring the second optical component is centered. The second base orientation structure 1520 prevents movement of the second optical component in directions perpendicular to the second optical axis.

[0123] The second optical component 522 has complementary component-orientation structure which is configured to co-operate with the second orientation structure. This componentorientation structure may take the form of an outer surface of the tube lens housing, for example. Optionally, the second optical component 522 may have an outer flange which extends from an outer radial surface of the second optical component. In particular, the flange extends from an outer radial surface of the tube lens collar. This optional flange is depicted extending from the second optical component 622. As depicted in Fig. 15B, the first flat surface 1521 may have a groove cut into it, in which case the first flat surface 1521 is formed by two parallel first flat surfaces 1521a and 1521b. Similarly, the second flat surface 1522 may have a groove cut into it, in which case the second flat surface 1522 is formed by two parallel second flat surfaces 1522a and 1522b. The grooves, or slots, in the flat surfaces 1521, 1522 are shaped, positioned and otherwise configured to receive the flange. The first flat surfaces 1521a, 1521b, engage with the flange on a first side of the second optical component, and the second flat surfaces 1522a, 1522b, engage with the flange on a second, opposing side of the second optical component 522. This10XG / 1851PC 40helps to ensure the second optical component 522 is accurately oriented with respect to the base plate 1590 and other optical components of the assembly.

[0124] The second subset of base orientation structures may also have an axis stop 1525 formed in the base plate 1500. The axis stop 1525 may take any of several forms. The axis stop 1525 is positioned substantially perpendicular to the second optical axis, or equivalently to the x-axis of the plate. As depicted in Fig. 15B, the axis stop 1525 may take the form of a backward-facing step or wall configured to abut the second optical component. This prevents movement of the second optical component in at least one direction parallel to the second optical axis. While the axis stop 1525 depicted in Fig. 15B is a step or wall, the axis stop may be any of a lip, ledge, or protrusion.

[0125] The base assembly 1500 also has third orientation structure 1530 configured to receive, and orient, the second mount. As explained elsewhere herein, the second mount holds the intermediate optical component. The third base orientation structure 1530 is formed between the first base orientation structure 1510a and the second base orientation structure 1520. In other words, the third base orientation structure 1530 is formed intermediate the first base orientation structure 1510a and the second base orientation structure 1520. The base 1500 also has fourth base orientation structure 1540. The fourth base orientation structure 1540 is configured to receive a third mount. As with the other base-orientation structures, the fourth base orientation structure 1540 is configured to receive and engage with a component to enable it to be correctly aligned and oriented with the base plate 1500. The one or more fourth base orientation structure(s) 1540 is formed at the first end of the base plate 1500, opposing the first base orientation structures 1510a, 1510b. Each of the third and fourth base-orientation structures may take the form of a recess with one or more mounting plates, in a manner similar to that described above with respect to other base orientation structures.

[0126] The base plate 1500 also has fifth base orientation structure 1550 configured to receive a dichroic flipper. The fifth base orientation structure 1550 enables, or allows, the dichroic flipper to be aligned relative to the optical axis.

[0127] The dichroic flipper may have multiple optical filters, and an assembly for mounting the flippers onto the base plate. The plurality of optical filters may be mounted onto a frame that is rotated about a rotational axis defined by a first shaft. The first shaft is aligned directly to an10XG / 1851PC 41optical bench or other component thereby also aligning the optical filters relative to a predetermined optical axis. The fifth base orientation structure 1550 has at least one reference surface. The at least one reference surface has a first reference surface that is substantially horizonal and a second reference surface that is substantially vertical. 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 base plate includes 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 the optical axis defined by the 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 ignored by 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,10XG / 1851PC 42a 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.

[0128] The fourth base orientation structure 1550 may define a slot for receiving a frame to permit rotation of the frame around the rotation axis of the dichroic flipper assembly. The slot is elongated, and forms an angle of substantially 45° with respect to the optical axis defined by the tube lens. Having this slot for the frame allows the axis of rotation of the first shaft and frame to be below the upper surface 1590, which increases the stability of the assembly. In various implementations, the slot does not extend through the entire thickness of the base plate (e.g., the frame rotates within a recess in the base plate 1500). Alternatively, the slot may extend through the entire thickness of the base plate, as shown in FIG. 15A, if the depth needed for the slot is greater than the depth of the optical bench.

[0129] The fourth base orientation structure 1550 may also define a recess, with a first recess portion configured to receive an alignment member of the dichroic flipper assembly, the shaft of the dichroic flipper assembly, and (e.g.) a clamping member of the assembly. The recess also has a second recess portion for receiving a motor. Positioning the assembly in the recess of the fourth base orientation structure lowers the center of mass of the assembly with respect to the base plate, which reduces vibrations as the frame rotates back and forth. The first recess portion has attachment points for receiving screws used to attach the clamping member to the base plate. In various implementations, first recess portion has a square or rectangular cross-sectional shape, e.g., having a recess base which is flat and parallel to the upper surface 1590 of the base plate 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 exerts a force on one or more alignment members (e.g., alignment member) to cause the alignment member(s) to press against the reference surface (or10XG / 1851PC 43reference surfaces) of the first recess portion to stably fix the alignment member(s) and securely align the first shaft with the reference surface(s).

[0130] The base plate 1550 also has seventh base orientation structure. The seventh base orientation takes the form of a recess 1570 configured to receive an excitation illumination assembly. The seventh base orientation structure allows the excitation illumination assembly to be aligned relative to the optical axis, and in particular to the optical axis defined by the tube lens

[0131] The base plate 1550 also has sixth base orientation structure 1570 configured to receive a field lens. The seventh base orientation structure 1570 enables, and allows, the field lens to be aligned relative to the optical axis.

[0132] The base plate of any of claims 110 to 1120, further comprising an eighth base orientation structure configured to receive an excitation fold mirror, wherein the eighth base orientation structure allows the excitation fold mirror to be aligned relative to the optical axis.

[0133] Each of the components, once positioned in the correct orientation via the corresponding base- orientation structure, may be additionally secured in position. This can be achieved via threaded fasteners such as bolts, screws, or the like. Alternative means of securement include epoxy or other adhesives.

[0134] Figs. 16A-16D depicts a base plate assembly 1600 including a base plate 1630 and a shear plate 1680 that are suitable for use with any of the assemblies disclosed herein. The base plate 1630 of Figs. 16A-16D is substantially similar to the base plate 1530 of Figs. 15A-15C, but the second subset of base orientation structures 1520 for receiving the second optical component 1526 (e.g., a tube lens) has been replaced with a collar 1523 having a substantially flat back surface 1524 configured to engage a flange 1528 of the second optical component 1526 (and to define the second optical axis). The collar 1523 may be secured to the base plate 1630 via fixation members (e.g., threaded screws). To secure the second optical component 1526 to the base plate 1630, the second optical component 1526 is positioned within the collar 1523 such that the flange 1528 of the second optical component 1526 engages the substantially flat back surface 1524 of the collar 1523 and the second optical component is secured to the collar 1523 via one or more fixation members (e.g., threaded screws) through one or more through holes in the flange 1528. The collar 1523 (with second optical component 1526 secured thereto) is then10XG / 1851PC 44coupled to the base plate 1630 (e.g., with fixation members) thereby preventing all motion (e.g., translation and rotation) of the second optical component 1526 relative to the base plate 1630and defining the second optical axis 562 to which other optical components (e.g., an objective lens, camera, autofocus module, imaging dichroics, emission filters, etc.) may be aligned. Fig. 16D illustrates the second optical component 1526 (e.g., a tube lens) secured within the collar 1523 and coupled to the base plate 1630thereby defining the second optical axis 562 to which all other optical components may be aligned.

[0135] Fig. 16E depicts the shear plate 1680 coupled to the bottom of the base plate 1630shown in Figs. 16A-16D. The shear plate may be coupled to the bottom of the base plate 1630to provide additional stiffness to the base plate 1630and / or improve damping of vibrations (e.g., higher frequency vibrations). The shear plate 1680 may be about 1mm thick to about 20mm thick. Preferably, the shear plate 1680 is about 6mm to about 12mm thick. As shown in Fig. 16E, the shear plate includes a plurality of through holes 1682 configured to receive a fixation member. As shown in Fig. 16E, the plurality of through holes 1682 are spaced about the perimeter of the shear plate 1680. In various embodiments, the fixation member(s) include a bolt, rivet, screw (e.g., shoulder screw), or other suitable threaded member. The fixation member(s) couple the shear plate 1680 to the bottom of the base plate 1630. In various embodiments, an intermediate layer (not shown) is positioned between the shear plate 1680 and the base plate 1630. The intermediate layer may have vibration dampening properties across one or more ranges of vibrational frequencies. For example, the intermediate layer includes a polymer foam (e.g., acrylic, polystyrene, polyurethane, polypropylene, polyethylene, polyvinyl chloride, ethylene vinyl acetate, etc.) or a rubber (e.g., chloroprene, Ethylene Propylene Diene Monomer, silicone, etc.). In various embodiments, the intermediate layer includes an adhesive (e.g., an acrylic adhesive) on one or both sides. In various embodiments, the intermediate layer is about 100 microns to about 1000 microns thick. In various embodiments, the intermediate layer is a double-sided transfer tape with very high bonding adhesive on each side.

[0136] Fig. 17 depicts a method 1700 of assembling an assembly for an imaging system. The method 1700 is suitable for assembling any of the assemblies described herein, including assembly 500, assembly 600, and assembly 1000. The method of assembly 1700 may also be10XG / 1851PC 45described as a calibration method or method of calibration, because the end-result of the method 1700 is a correctly assembled and calibrated assembly.

[0137] The method 1700 is suitable for use to assemble any assembly referred to herein. In particular, the resulting assembly has a base plate with an upper (or 'top') surface, a lower (or 'bottom') surface, and a thickness therebetween. The base plate extends from a first end to a second end. The base plate has a plurality of base orientation structures formed in the upper surface, the base orientation structures comprising first, second, third and fourth base orientation structure. The assembly has a first optical component including an objective lens and defining a first optical axis; and a second optical component including a tube lens and defining a second optical axis. The second optical component is coupled to the second base orientation structure, resulting in the second optical component being coupled to the base plate in its proper orientation. The assembly has a first mount having an inner surface and an outer surface. The first mount extends from a first (upper) end to a second (lower) end. The first end of the first mount is coupled to the first base orientation structure. The first base orientation structure is formed at the second end of the base plate. The first mount has an alignment structure configured to rotate in a first plane and align the objective lens. The resulting assembly also has an intermediate optical component including a fold mirror configured to rotate in a second plane. The second optical component, including the objective lens, is coupled to the inner surface of the first mount. The assembly has a second mount coupled to the third base orientation structure of the plurality of base orientation structures. The third base orientation structure is formed between the firs base orientation structure and the second base orientation structure. The fold mirror is rotatably coupled to the second mount. The assembly also has a third mount coupled to the fourth base orientation structure of the plurality of base orientation structures. The fourth base orientation structure is formed at the first end of the base plate. The assembly also has an image sensor coupled to the third mount. When such an assembly is assembled via the method 1700, the result is a properly calibrated and aligned assembly for an imaging system.

[0138] At block 1710, the second optical component is coupled to the second base orientation structure, to define an orientation of the second optical axis. For example, with reference to Figs.6-16E, the second optical component 622 is positioned on the flat surfaces 1521, 1522.10XG / 1851PC 46Optionally, a flange of the second optical component 622 is positioned in a groove in surfaces 1521, 1522. The second optical component 622 is positioned against the axis stop 1525 such that the axis stop 1525 abuts the second optical component 622.

[0139] At block 1720, the second mount is secured to the third base orientation structure. For example, with reference to Figs. 6-16E, the second mount 1051 is positioned in a recess 1530, to rest on one or more mounting pads in the recess. These features ensure the second mount 1051 is positioned in a preferred orientation. The second mount may be secured in place in the recess 1530 via the use of screws or other threaded fasteners in the manner described above.

[0140] At block 1730, the fold mirror is aligned based on the second optical axis by rotating the fold mirror within its plane of rotation. Once the fold mirror is properly aligned, it is locked in the aligned position. This position may be referred to as a second set orientation. For example, with reference to Figs. 6-16E, the intermediate optical component 1023 is rotated with respect to the second mount 1051. The orientation of the fold mirror 1055 is adjusted until the fold mirror 1055 is positioned in the aligned position. The aligned position can be found, for example, by directing light through the second optical component, toward the intermediate optical component, and checking that the fold mirror properly directs the light down toward the proper location on the sample stage.

[0141] At block 1740, the first mount is secured to the first base alignment structure. This occurs after locking the fold mirror in the aligned position. For example, with reference to Figs. 6-16E, the overhang 1013 of first mount 1011 is positioned in the first base orientation structures 1510a, 1510b. The lower surface of the overhand 1013 is seated on the mounting pads, e.g., mounting pad 1511. The first mount 1011 is removably secured to the base plate via threaded fasteners in the manner described above.

[0142] At block 1750, the alignment structure is aligned based on the second optical axis by rotating the alignment structure within the first plane of rotation, which is perpendicular to a second plane of rotation in which the fold mirror is constrained to rotate. The alignment structure is locked in an aligned position, which may be referred to as the first set orientation herein. For example, with reference to Figs. 6-16E, the elongate bar 1012 is adjusted within the elongated slots of the first mount 1011 until the elongate bar 1012 is oriented in the first set orientation.10XG / 1851PC 47When the first optical component is in this first set orientation, it is aligned relative to the second optical component. Light passing along the first optical axis, from the sample, travels via the objective lens to the fold mirror and is directed along the second optical axis toward the tube lens. The elongate bar 1012 is locked in the aligned position by virtue of one or more first couplings which interact with through-holes 1017a, 1017b, in the manner described above.

[0143] At block 1760, after the alignment structure is locked, the first optical component is secured to the first mount such that the objective lens is aligned with the second optical component via the alignment structure. For example, with reference to Figs. 6-16E, after the elongate bar 1012 is locked in the first set (aligned) orientation, the first optical component 1021 is secured to the first mount 1011. This may be accomplished via threaded fasteners configured to co-operate with the plurality of apertures 1071a-1071d in the first mount 1011 in the manner described above.

[0144] At block 1770, the third mount is secured to the fourth base orientation structure. For example, with reference to Figs. 6-16E, the third mount 652 is positioned in fourth base orientation structure 1540. The fourth base orientation structure 1540 ensures that the third mount is oriented correctly, and the third mount is then secured to the fourth base orientation structure 1540, and thereby to the base plate, by virtue of threaded fasteners or the like.

[0145] At block 1780, the image sensor is aligned with the second optical axis and the image sensor is secured to the third mount. For example, with reference to Figs.6-16E, the image sensor 624 may be first coupled to a sensor alignment place 653. The sensor alignment plate 653 enables adjustment of the position of the image sensor 653 with respect to the third mount 652. The sensor alignment plate has over-sized through-holes which, when the image sensor 624 is coupled to the third mount 652 via the sensor alignment plate 653, enable the image sensor 624 to be adjusted in each of the x and y directions, as well as a degree of tip and tilt with respect to the third mount 652. Once the image sensor 524, 624 is aligned with the second optical axis 562, the sensor alignment plate 653 is attached with threaded fasteners such as screws and epoxied to the third mount 652.

[0146] In the resulting assembly, each of the optical components is aligned with one another, and the apparatus is configured for operation. Light travelling from the sample may pass through10XG / 1851PC 48the objective to the fold mirror, and be directed toward the tube lens. The tube lens acts to focus the light such that an image is formed at the image sensor. Upon inspection of Fig. 17 it will be appreciated that, by positioning the second optical component first at block 1710, the optical axis associated with the second optical axis is determined as a first step in the process. In this way, the second optical axis acts as a 'master datum'. The second optical component therefore acts as primary reference that serves as the foundation for defining the orientation of each other component. This ensures consistency and precision during calibration of the apparatus. The resulting apparatus can be assembled and calibrated much more quickly and effectively in comparison with prior methods of assembly.General terminology:

[0147] 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.

[0148] 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.

[0149] 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.

[0150] 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.10XG / 1851PC 49

[0151] 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, ranges 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.

[0152] 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.

[0153] 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.10XG / 1851PC 50

[0154] 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:

[0155] 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).

[0156] The phrase "barcode diversity" refers to the total number of unique barcode sequences that may be represented by a given set of barcodes.

[0157] 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 probe that targets the analyte. In some instances, a particular barcode can be unique relative to other barcodes.

[0158] 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.).10XG / 1851PC 51

[0159] 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.

[0160] A "digital barcode" (or "digital barcode sequence") is a representation of a corresponding physical barcode (ortarget 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 some 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.10XG / 1851PC 52

[0161] 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 x 106, at least 5 x 106, at least 6 x 105, at least 7 x 106, at least 8 x 106, at least 9 x 106, at least 107, 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.

[0162] 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 these instances, a decoded barcode (i.e., a series of letters or code words) may serve as a proxy for the target analyte.

[0163] 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.10XG / 1851PC 53Probe terminology:

[0164] The term "probe" may refer eitherto 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.

[0165] 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.

[0166] 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.

[0167] 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 another molecule 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 another10XG / 1851PC 54molecule, 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:

[0168] 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)).

[0169] 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 18:112.10XG / 1851PC 55Samples:

[0170] 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-cellulartissue 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.

[0171] 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.10XG / 1851PC 56

[0172] 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.

[0173] 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.

[0174] 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.

[0175] 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.

[0176] 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.10XG / 1851PC 57Endogenous analytes:

[0177] 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.

[0178] 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.

[0179] 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.10XG / 1851PC 58

[0180] 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 noncoding 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 RNA (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 doublestranded 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).

[0181] 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.

[0182] 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.

[0183] 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 least10XG / 1851PC 59about 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.

[0184] 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 is identical 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:

[0185] 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 reporter10XG / 1851PC 60oligonucleotide 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.

[0186] 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.

[0187] 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, 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, 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.

[0188] 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 binding10XG / 1851PC 61molecule, a receptor ligand, a small molecule, a bi-specific antibody, a bi-specific 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.

[0189] 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 / orcomponent had not been stained or stained. Forexample, 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 be localized 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.

[0190] 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 by10XG / 1851PC 62extracting 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 referto 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.

[0191] 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, antibodies, 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.10XG / 1851PC 63

[0192] 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.

[0193] 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-lntersciences (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.

[0194] 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 of analyte 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). In10XG / 1851PC 64some 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).

[0195] 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.

[0196] 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.

[0197] 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 been10XG / 1851PC 65developed and are readily available. See, e.g., U.S. Pat. No. 6,265,552, which 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).

[0198] 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.

[0199] 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 the10XG / 1851PC 66biological 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), or 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:

[0200] 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.

[0201] 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 a10XG / 1851PC 67fluorophore. The probes are sequentially hybridized to gene sequences (e.g., DNA) or gene transcript sequences (e.g., mRNA) sequences, and visualized as diffraction-limited 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 ll(4):360-361).

[0202] 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.10XG / 1851PC 68

[0203] 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.).

[0204] 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 a padlock 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.Products of endogenous analytes and / or labelling agents:

[0205] 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 an10XG / 1851PC 69amplification 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.

[0206] 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 decoding schemes 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:

[0207] 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 specific10XG / 1851PC 70probe 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.

[0208] 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.

[0209] 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. 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 situ10XG / 1851PC 71hybridization (PUSH) probe set. See, e.g., U.S. Pat. Pub. 2020 / 0224243 which is hereby incorporated by reference in its entirety.

[0210] 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.

[0211] 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-i-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 a DNA-splinted DNA ligase activity. In some instances, the ligase is a ligase that has an RNA-splinted DNA ligase activity.

[0212] 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 fora 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., 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, the10XG / 1851PC 72gap 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.

[0213] 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.

[0214] 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.

[0215] 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 nucleic acid molecules of interest (see, e.g., U.S. Patent No. 7,264,929, the entire contents of which are incorporated10XG / 1851PC 73herein 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 singlestranded 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:

[0216] 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.

[0217] 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, 3'10XG / 1851PC 74termini). 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.

[0218] 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 single-stranded 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 Sac 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.

[0219] In some instances, the amplification is performed at a temperature between or between about 20^C and about BO^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, or49°C.

[0220] 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 non-isothermal10XG / 1851PC 75amplification. 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 ( / .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. Sci. 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 :IO95- 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 (4)29) polymerase, Klenow fragment, Bacillus stearothermophilus 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.

[0221] 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- hydroxysuccinimide 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, ora 7-Deaza-7-Propargylamino-dATP moiety modification.

[0222] 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 the10XG / 1851PC 76probes 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 RCA template 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 (i.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.

[0223] 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 thereof10XG / 1851PC 77(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:

[0224] 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 probe hybridization, 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).

[0225] 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 reduce10XG / 1851PC 78the number of target sequences required for detection (e.g., the length of the RCA product can be reduced).

[0226] 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.

[0227] The detectable reactive molecules may comprise tyramide, such as used in tyramide signal amplification (TSA) or multiplexed catalyzed reporter deposition (CARD)-FISH. In some 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.

[0228] 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;10XG / 1851PC 79Niu 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 not exposed), 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 a10XG / 1851PC 80nicked 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.

[0229] 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.

[0230] 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 some instances, 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,10XG / 1851PC 81the 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.

[0231] 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 exam pie, 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.

[0232] 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.10XG / 1851PC 82In 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:

[0233] 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.

[0234] 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.

[0235] 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 for targeting 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.10XG / 1851PC 83

[0236] 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).

[0237] 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, they can also be designed to feature error detection and correction mechanisms, see, e.g., U.S. Pat.10XG / 1851PC 84Pub. 20190055594 and WO2019199579A1, which are hereby incorporated by reference in their entirety.Sequential hybridization:

[0238] 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 temporally-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.

[0239] 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,10XG / 1851PC 85the 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 instances, the method comprises detecting a plurality of analytes in a sample.

[0240] 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.

[0241] In any of the implementations disclosed herein, the detection of signalscan 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.10XG / 1851PC 86

[0242] 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 of microscope 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.

[0243] 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.

[0244] 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 new10XG / 1851PC 87nucleic 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).

[0245] 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 probe or 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.

[0246] 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 other10XG / 1851PC 88components (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.

[0247] 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 L-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 displacement probe 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.

[0248] 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.10XG / 1851PC 89Decoding:

[0249] 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.Anchor probes:

[0250] 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 contacted10XG / 1851PC 90with 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.

[0251] 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.

[0252] 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 some instances, 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).10XG / 1851PC 91

[0253] 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.

[0254] 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.

[0255] 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.10XG / 1851PC 92

[0256] 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 is insoluble 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.

[0257] 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.

[0258] 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 reading10XG / 1851PC 93and 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 drawings are 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 / 1851PC 94

Claims

1. CLAIMS:

1. An assembly for an imaging system, the assembly comprising:a base plate comprising a first end, a second end, an upper surface, and a lower surface; a first mount removably couplable to the base plate;an alignment structure coupled to the first mount via one or more first couplings;a first optical component contacting the alignment structure to define a first set orientation of the first optical component relative to the first mount, the first optical component being coupled to the first mount in the first set orientation; anda second optical component coupled to the base plate;wherein, when the first mount is removably coupled to the base plate with the first optical component in the first set orientation, the first optical component is aligned relative to the second optical component.

2. The assembly of claim 1, wherein the first mount is removably coupled to the base plate.

3. The assembly of any preceding claim, wherein the imaging system is configured for fluorescence microscopy, and the first and second optical components are positioned such that, in use of the imaging system, one or more rays of light emitted from a sample travel from the first optical component to the second optical component.

4. The assembly of any preceding claim, wherein the one or more first couplings are configured to enable an orientation of the alignment structure to be adjusted relative to the first mount.

5. The assembly of claim 4, wherein the alignment structure is rotatable with respect to the first mount.

6. The assembly of claim 4 or claim 5, wherein the alignment structure is constrained, via the one or more first couplings, to rotate within a first plane.10XG / 1851PC 957. The assembly of claim 6, wherein, when the first mount is removably coupled to the base plate, the first plane is perpendicular to an upper plane defined by the upper surface of the base plate.

8. The assembly of claim 6 or claim 7, wherein, when the first mount is removably coupled to the base plate, the first plane is parallel to the second end of the base plate.

9. The assembly of any of claims 6 to 8, wherein the first plane is defined by an inner surface of the first mount.

10. The assembly of claim 9, wherein the alignment structure comprises a complementary surface that is in contact with the inner surface of the first mount.

11. The assembly of claim 9 or claim 10, wherein the adjustment structure is positioned on the inner surface of the first mount.

12. The assembly of any preceding claim, wherein the first mount has a first end and a second end, and the alignment structure is positioned at or toward the second end of the first mount.

13. The assembly of claim 12, wherein the alignment structure is positioned about 0.1 mm to about 10mm from the second end of the first mount.

14. The assembly of claim 12 or claim 13, wherein the alignment structure is positioned about 0.5 mm from the second end of the first mount.

15. The assembly of any preceding claim, wherein the one or more first couplings are each lockable to lock the orientation of the alignment structure with respect to the mount.10XG / 1851PC 9616. The assembly of claim 15, wherein the alignment structure is locked, via the one or more first couplings, in an orientation such that, when the first optical component contacts the alignment structure, the first optical component is positioned in the first set orientation.

17. The assembly of any preceding claim wherein, when the first mount is removably coupled to the base plate, the first mount extends below the lower surface of the base plate.

18. The assembly of claim 17, wherein, when the first mount is removably coupled to the base plate, the first mount extends from the upper surface of the base plate to below the lower surface.

19. The assembly of claim 17 or claim 18, wherein the first mount extends below the lower surface by about 20 mm to about 150 mm.

20. The assembly of any preceding claim, wherein the first mount comprises an overhang that overlaps the upper surface of the base plate.

21. The assembly of claim 20, wherein the overhang has a length of about 25 mm to about 75 mm.

22. The assembly of any preceding claim, wherein the alignment structure comprises an elongate bar extending from a first end to a second end.

23. The assembly of claim 22, the alignment structure comprising a first through-hole at the first end and a second through-hole at the second end.

24. The assembly of any preceding claim, wherein the one or more first couplings comprise one or more rotational couplings.10XG / 1851PC 9725. The assembly of claim 24, wherein each of the one or more rotational couplings comprises a screw, a shaft, and / or a bearing.

26. The assembly of any preceding claim, wherein the first mount comprises a plurality of slots configured to receive the one or more first couplings.

27. The assembly of claim 26, wherein the plurality of slots comprise elongated slots.

28. The assembly of claim 27, wherein the alignment structure comprises an elongate bar extending from a first end to a second end, and wherein the plurality of slots comprises a first slot configured to align with the first end of the alignment structure and a second slot configured to align with the second end of the alignment structure.

29. The assembly of any preceding claim, wherein the one or more first couplings each comprises one or more fixation members.

30. The assembly claim 29, wherein the one or more fixation members comprises a screw, a bolt, a rivet, a shaft, and / or a nut.

31. The assembly of any preceding claim, wherein the first optical component comprises an objective lens, optionally wherein the objective lens is swappable while maintaining the first set orientation of the first optical component relative to the first mount.

32. The assembly of claim 31, wherein the first optical component further comprises an objective lens mount, optionally wherein the objective lens mount contacts the alignment structure.10XG / 1851PC 9833. The assembly of claim 32, wherein the objective lens mount comprises a z-stage, optionally wherein the objective lens mount is secured to the z-stage, and further optionally wherein the objective z-stage contacts the alignment structure.

34. The assembly of claim 33, wherein the z-stage comprises a voice coil actuator.

35. The assembly of any preceding claim, wherein the first mount has a height of about 50 mm to about 200 mm.

36. The assembly of any preceding claim, wherein the base plate comprises a thickness between the upper and lower surfaces, and the thickness is about 10 mm to about 75 mm.

37. The assembly of any preceding claim, wherein the second optical component comprises a tube lens.

38. The assembly of claim 37, wherein the second optical component further comprises a tube lens collar.

39. The assembly of any preceding claim, wherein the alignment structure comprises a ledge comprising an alignment surface on which the first optical component rests.

40. The assembly of any preceding claim, further comprising a second mount coupled to the base plate, and an intermediate optical component rotationally coupled to the second mount.

41. The assembly of claim 40, wherein:the alignment structure is constrained to rotate within a first plane; andthe intermediate optical component is constrained to rotate within a second plane that is perpendicular to the first plane.10XG / 1851PC 9942. The assembly of claim 40 or claim 41, wherein the first optical component is associated with a first optical axis and the second optical component is associated with a second optical axis, wherein the first and second optical axes meet at the intermediate optical component.

43. The assembly of any of claims 40 to 42, wherein the intermediate optical component is configured to direct light passing through the first optical component to the second optical component.

44. The assembly of any of claims 40 to 43, wherein the intermediate optical component is further coupled to the second mount via one or more second couplings.

45. The assembly of claim 44, wherein the one or more second couplings are configured to lock the orientation of the intermediate optical component with respect to the second mount.

46. The assembly of claim 45, wherein the intermediate optical component is locked, via the one or more second couplings, in an orientation such that light passing through the first optical component is directed to the second optical component.

47. The assembly of any of claims 44 to 46, wherein the one or more second couplings are configured to prevent misalignment of the intermediate optical component relative to one or more of the base plate, the first optical component, and the second optical component.

48. The assembly of any of claims 44 to 47 wherein the at least one second coupling comprises a screw.

49. The assembly of claim 48, wherein the second mount comprises an angled face which is angled with respect to the second optical axis, and both the intermediate optical component and the angled face comprises a slot configured to receive the screw.10XG / 1851PC 10050. The assembly of any preceding claim, wherein the base plate comprises a plurality of base orientation structures.

51. The assembly of claim 50, wherein the plurality of base orientation structures comprises a first subset of base orientation structures, and the first mount comprises one or more first mount orientation structures, wherein the first subset of base orientation structures and the one or more first mount orientation structures correspond with one another to define a preferred orientation of the first mount with respect to the base plate.

52. The assembly of claim 51, wherein the first subset of base orientation structures comprises recesses formed in the upper surface of the base plate, and the one or more first mount orientation structures comprise an overhang configured to overlap the upper surface of the base plate and fit within the recesses of the first subset of base orientation structures.

53. The assembly of claim 52, wherein each of the recesses comprises a floor and at least one mounting pad raised above the floor to define a preferred height of the first mount with respect to the base plate.

54. The assembly of any of claims 50 to 53, wherein the plurality of base orientation structures comprises a second subset of base orientation structures, and the second optical component comprises one or more second component orientation structures, wherein the second subset of base orientation structures and the one or more second component orientation structures correspond with one another to define a preferred orientation of the second optical component with respect to the base plate.

55. The assembly of claim 54, wherein the second subset of base orientation structures comprises a first flat surface and a second flat surface.10XG / 1851PC 10156. The assembly of claim 55, wherein the first and second flat surfaces are positioned either side of the second optical axis.

57. The assembly of claim 55 or claim 56, wherein the first and second flat surfaces are angled with respect to one another, such that the height of the first flat surface increases in a first direction and the height of the second surface increases in a second, opposite direction, where both the first and second direction are perpendicular to the second optical axis.

58. The assembly of any of claims 54 to 57, wherein the first and second angled surfaces form a V-shape.

59. The assembly of any of claims 54 to 58, wherein the first flat surface and the second flat surface are continuous.

60. The assembly of any of claims 54 to 58, wherein the first flat surface and the second flat surface are discontinuous.

61. The assembly of any of claims 54 to 60, wherein the second subset of base orientation structures further comprises an axis stop formed in the base plate.

62. The assembly of claim 61, wherein the axis stop is substantially perpendicular to the second optical axis.

63. The assembly of claim 61 or claim 62, wherein the axis stop comprises a backward-facing step configured to abut the second optical component.

64. The assembly of any of claims 50 to 63, wherein at least a subset of the plurality of base orientation structures comprises a recess formed in the upper surface of the base plate.10XG / 1851PC 10265. The assembly of claim 64, wherein each recess comprises at least one mounting pad.

66. The assembly of claim 65, wherein the at least one mounting pad in each recess has a height of about 0.1mm to about 1mm.

67. The assembly of claim 66, wherein the at least one mounting pad in each recess has a height of about 0.5mm.

68. The assembly of any of claims 65 to 67, wherein the at least one mounting pad in each recess has an area of about 50 mm2to about 3000 mm2.

69. An assembly for an imaging system, the assembly comprising:a base plate comprising a first end, a second end, an upper surface, and a lower surface, and at least one base orientation structure;wherein the at least one base orientation structure comprises a recess formed in the upper surface of the base plate, the recess being shaped and configured to receive one or more optical components, and comprising a floor and at least one mounting pad raised above the floor;the assembly further comprising at least one optical component positioned in the recess, resting on the one or more mounting pads;wherein the one or more mounting pads define a preferred height and / or alignment of the at least one optical component with respect to the base plate.

70. The assembly of claim 69, wherein the recess comprises a first depth measured from the upper surface to the floor, and the at least one mounting pad comprises a first height above the floor, wherein the first depth is greater than the first height.

71. The assembly of any of claim 69 or claim 70, wherein the at least one mounting pad is a plurality of mounting pads, such that the recess comprises more than one mounting pad raised above its floor.10XG / 1851PC 10372. The assembly of any of claims 69 to 71, wherein the one or more mounting pads each have an upper surface.

73. The assembly of claim 72, wherein the upper surface of each of the one or more mounting pads is flat.

74. The assembly of claim 72 or claim 73, wherein a surface area of the floor in each recess is greater than a combined surface area of the upper surfaces of the one or more mounting pads.

75. The assembly of any of claims 69 to 74, wherein the assembly is the assembly of any of claims 1 to 68.

76. A method of assembling an assembly for an imaging system, the assembly comprising:a base plate comprising an upper surface, a lower surface, and a thickness therebetween, the base plate extending from a first end to a second end, the base plate having a plurality of base orientation structures formed in the top surface, the base orientation structures comprising first, second, third and fourth base orientation structure;a first optical component comprising an objective lens and defining a first optical axis; a second optical component comprising a tube lens and define a second optical axis, wherein the second optical component is coupled to the second base orientation structure;a first mount having an inner surface and an outer surface, wherein the first mount extends from a first end to a second end, wherein the first end of the first mount is coupled to the first base orientation structure, the first base orientation structure being formed at the second end of the base plate, and the first mount comprising an alignment structure configured to rotate in a first plane and align the objective lens;an intermediate optical component comprising a fold mirror configured to rotate in a second plane, wherein the objective lens is coupled to the inner surface of the first mount;a second mount coupled to the third base orientation structure of the plurality of base10XG / 1851PC 104orientation structures, the third base orientation structure formed between the first base orientation structure and the second base orientation structure;a fold mirror rotatably coupled to the second mount;a third mount coupled to a fourth base orientation structure of the plurality of base orientation structures, the fourth base orientation structure formed at the first end of the base plate; andan image sensor coupled to the third mount;wherein the method comprises:coupling the second optical component to the second base orientation structure to define an orientation of the second optical axis;securing the second mount to the third base orientation structure; aligning the fold mirror based on the second optical axis by rotating the fold mirror within the second plane and locking the fold mirror in an aligned position;securing the first mount to the first base orientation structure;after locking the fold mirror in the aligned position, aligning the alignment structure based on the second optical axis by rotating the alignment structure within the first plane, the first plane being perpendicular to the second plane, and locking the alignment structure in an aligned position;after locking the alignment structure, securing the first optical component to the first mount such that the objective lens is aligned with the second optical component via the alignment structure;securing the third mount to the fourth base orientation structure; and aligning the image sensor with the second optical axis and securing the image sensor to the third mount.

77. The method of claim 76, wherein the assembly is the assembly of any of claims 1 to 68.

78. The method of claim 76 or 77, wherein the objective lens is constrained to rotate within the first plane.10XG / 1851PC 10579. The method of any of claims 76 to 78, wherein the fold mirror is constrained to rotate within the second plane.

80. The method of any of claims 76 to 78, wherein the second base orientation structure comprise a first flat surface and a second flat surface.

81. The method of claim 80, wherein the first flat surface and second flat surface form a V-shape.

82. The method of claim 80 or claim 81, the method further comprising positioning the second optical component on the first and second flat surfaces.

83. The method of any of claims 76 to 82, wherein the second base orientation structure comprises an axis stop formed in the base plate.

84. The method of claim 83, wherein the axis stop is substantially perpendicular to the second optical axis.

85. The method of claim 83 or claim 84, wherein the axis stop comprises a backward-facing step configured to abut the second optical component.

86. The method of any of claims 83 to 85, wherein the method further comprises positioning the second optical component to abut the axis stop.

87. The method of any of claims 76 to 86, the method further comprising positioning the alignment structure on the inner surface of the first mount.

88. The method of claim 87, further comprising positioning the alignment structure at the second end of the first mount.10XG / 1851PC 10689. The method of claim 88, the method comprising positioning the alignment structure about 0.1 mm to about 10mm from the second end of the first mount.

90. The method of claim 89, the method comprising positioning the alignment structure about 0.5 mm from the second end of the first mount.

91. The method of any of claims 76 to 90, wherein the alignment structure comprises an elongate bar having a first end and a second end, the elongate bar comprising a first through-hole at the first end and a second through-hole at the second end.

92. The method of claim 91, wherein locking the alignment structure in the aligned position comprises positioning a first coupling through the first through-hole to couple the first end of the elongate bar to the first mount, and positioning another first coupling through the second through-hole to couple the second end of the elongate bar to the first mount.

93. The method of any of claims 76 to 92, wherein each of the plurality of base orientation structures comprises a recess, and the method comprises forming the recesses in the base plate.

94. The method of claim 93, wherein each recess comprises at least one mounting pad, and the method comprises forming the mounting pads after forming the recesses.

95. The method of claim 94, wherein forming the recesses comprises using a milling machine set at a first z-axis height, and forming the mounting pads comprises using the milling machine at a second, different z-axis height.

96. The method of claim 95, wherein the one or more mounting pads comprises a plurality of mounting pads such that each recess comprises more than one mounting pad and, within a particular recess, each of the plurality of mounting pads are formed without adjusting the milling machine from the second, different z-axis height.10XG / 1851PC 10797. The method of any of claims 76 to 96, wherein the first optical component further comprises an objective z-stage.

98. The method of any of claims 76 to 96, wherein the first optical component further comprising an objective mount secured to the objective z-stage.

99. The method of claim 98, further comprising positioning the objective z-stage in contact with the alignment structure.

100. The method of any of claims 76 to 99, the first optical component further comprising an objective mount, and the method comprising securing the objective mount to the first mount.

101. The method of claim 100, further comprising positioning the objective mount in contact with the alignment structure.

102. The method of any of claims 76 to 101, the intermediate optical component further comprising a mirror mount, wherein the fold mirror is coupled to the mirror mount and the mirror mount is rotatably coupled to the second mount.

103. The method of claim 102, the assembly further comprising an adjustment structure coupling the second mount to the mirror mount.

104. The method of claim 103, wherein the adjustment structure comprises a screw.

105. The method of claim 104, further comprising adjusting a degree of rotation of the fold mirror via rotation of the screw.10XG / 1851PC 108106. The method of any of claims 76 to 105, the assembly further comprising a sensor alignment plate coupled to the third mount, wherein the image sensor is coupled to the sensor alignment plate.

107. The method of claim 106, further comprising permanently affixing the sensor alignment plate to the third mount after the image sensor is aligned to the optical axis.

108. The method of any of claims 76 to 107, further comprising a stage positioned under the first mount, wherein the method further includes aligning the stage based on the optical axis after locking the fold mirror.

109. A assembly for an imaging system assembled via the method of any of claims 76 to 108.

110. A base plate for an optical assembly, the base plate comprising a plurality of base orientation structures, the plurality of base orientations structures comprising:a first base orientation structure configured to receive a tube lens defining an optical axis; a second base orientation structure configured to receive a first mount for coupling an objective lens;a third base orientation structure configured to receive a second mount for coupling a fold mirror;a fourth base orientation structure configured to receive a third mount for coupling an image sensor;wherein, when the tube lens is coupled to the first base orientation structure, the second base orientation structure allows the objective lens to be aligned relative to the optical axis, the third base orientation structure allows the fold mirror to be aligned relative to the optical axis, and the fourth base orientation structure allows the image sensor to be aligned relative to the optical axis.10XG / 1851PC 109111. The base plate of claim 110, further comprising a fifth base orientation structure configured to receive a dichroic flipper, wherein the fifth base orientation structure allows the dichroic flipper to be aligned relative to the optical axis.

112. The base plate of claim 110 or claim 111, wherein the fifth base orientation structure comprises at least one reference surface.

113. The base plate of claim 112, wherein the at least one reference surface comprises a first reference surface that is substantially horizonal and a second reference surface that is substantially vertical.

114. The base plate of any of claim 111 to claim 113, wherein the fifth base orientation structure comprises a slot through the base plate.

115. The base plate of any of claims 110 to 114, further comprising a seventh base orientation structure configured to receive an excitation illumination assembly, wherein the seventh base orientation structure allows the excitation illumination assembly to be aligned relative to the optical axis.

116. The base plate of any of claims 110 to 115, further comprising a sixth base orientation structure configured to receive a field lens, wherein the sixth base orientation structure allows the field lens to be aligned relative to the optical axis.

117. The base plate of any of claims 110 to 116, wherein the second base orientation structure further comprises a V-groove.

118. The base plate of claim 117, wherein the V-groove comprises a first flat surface and a second flat surface.10XG / 1851PC 110119. The base plate of claim 117 or claim 118, wherein the first flat surface and the second flat surface are continuous.

120. The base plate of claim 117 or claim 118, wherein the first flat surface and the second flat surface are discontinuous.

121. The base plate of any of claims 110 to 120, further comprising an eighth base orientation structure configured to receive an excitation fold mirror, wherein the eighth base orientation structure allows the excitation fold mirror to be aligned relative to the optical axis.

122. The base plate of any of claims 110 to 120, wherein each of the plurality of base orientation structures comprises a recess.

123. The base plate of claim 122, wherein each recess comprises at least one mounting pad.

124. The base plate of claim 123, wherein the at least one mounting pad in each recess has a height of about 0.1mm to about 1mm.

125. The base plate of claim 124, wherein the at least one mounting pad in each recess has a height of about 0.5mm.

126. The base plate of claim 125, wherein the at least one mounting pad in each recess has an area of about 50 mm2to about 3000 mm2.

127. The base plate of any of claims 110 to 126, wherein the second base orientation structure further comprises an axis stop formed in the base plate.

128. The base plate of claim 127, wherein the axis stop is substantially perpendicular to the optical axis.10XG / 1851PC 111129. The base plate of claim 127 or claim 128, wherein the axis stop comprises a backwardfacing step configured to abut the tube lens.

130. The base plate of any of claims 110 to 129, further comprising a tube lens secured against the second base orientation structure.

131. The base plate of any of claims 110 to 130, further comprising a first mount positioned on the first base orientation structure of the plurality of base orientation structures, the first mount configured to receive an objective lens.10XG / 1851PC 112

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