Systems and methods for illumination of a sample

Abstract

An illumination device includes a first illumination source arranged to output first illumination light, a second illumination source arranged to output second illumination light, a first collector optic arranged to collect the first illumination light, a second collector optic arranged to collect the second illumination light, a field stop arranged to provide an aperture for light from the first collector optic and second collector optic, and a field optic arranged to image the field stop. The first collector optic is arranged to substantially collimate the first illumination light, and the second collector optic is arranged to form an image of the second illumination source between the second collector optic and the field optic, before or after the field stop so that the image of the second illumination source is defocussed at the field stop.

Description

IOO-I789OIPCSYSTEMS AND METHODS FOR ILLUMINATION OF A SAMPLECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 701,676, filed October 1, 2024, which is assigned to the assignee of the present application and is hereby expressly incorporated by reference in its entirety for all applicable purposes.FIELD

[0002] The disclosure relates to illumination devices, and more particularly to illumination devices for use in an optics module such as a microscope.BACKGROUND

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

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

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

[0006] Some fluorescence microscopes are designed to illuminate, and thereby excite, multiple unique fluorophores in a sample, where each unique fluorophore requires excitation with fluorescence excitation light of a fluorophore-dependent excitation wavelength or spectrum to generate fluorophore-dependent fluorescence emissions. To excite these fluorophores in the sample, fluorescence microscopes may employ two or more different wavelengths (or wavelength spectra) of illumination light (e.g.., the fluorescence microscope has two or more excitation illumination color channels). However, there are challenges in providing uniformity of illumination across a field of view while maintaining high power (e.g., optical flux) to ensure high quality fluorescence images that allow for improved downstream processing (e.g., decodingIOO-I789OIPC high quality blobs) and high sample throughput. For example, in fluorescence microscopes (e.g., epifluorescence microscopes) with a high numerical aperture objective, there are challenges in providing a substantially uniform illumination field across the field of view while maximizing the optical flux delivered to the sample used to excite fluorescently-tagged target molecules within the sample. There are also challenges in providing consistency of illumination when using multiple illumination sources. For example, in fluorescence microscopes, there are challenges in ensuring consistency in optical flux at the sample when using illumination sources having different wavelength spectra.

[0007] Accordingly, there exists a need for a fluorescence microscope with high illumination uniformity and high optical flux in each color channel of multiple excitation illumination color channels (e.g., a microscope having two or more illumination sources).

[0008] Meanwhile, other types of light microscopes having two or more illumination sources may also benefit from high illumination uniformity and high optical flux.SUMMARY

[0009] This summary is provided to introduce in simplified form a selection of concepts that are further described herein. The summary is not intended to identify key or essential features of the invention.

[0010] One or more aspects of an invention are set out in the claims.

[0011] There is provided a device comprising: a first illumination source arranged to output first illumination light; a second illumination source arranged to output second illumination light; a first collector optic arranged to collect the first illumination light; and a second collector optic arranged to collect the second illumination light. The device further comprises a field stop arranged to provide an aperture for light from the first collector optic and second collector optic; and a field optic arranged to image the field stop. The first collector optic is arranged to substantially collimate the first illumination light. The second collector optic is arranged to form an image of the second illumination source between the second collector optic and the field optic, before or after the field stop so that the image of the second illumination source is defocussed at the field stop.

[0012] Optionally, a luminance uniformity of the first illumination source is lower than a luminance uniformity of the second illumination source.

[0013] Optionally, a ratio of a largest dimension of an emitting surface of the first illumination source to a clear aperture of the first collector optic is greater than 0.05, optionally greater than 0.1, optionally greater than 0.15, optionally greater than 0.2.

[0014] Optionally, the second collector optic is arranged such that an irradiance uniformity at the field stop is greater than the luminance uniformity of the second illumination source.IOO-I789OIPC

[0015] The irradiance uniformity is: Emin / Emax, wherein Emin is the minimum irradiance across an entire diagonal or longest dimension of the aperture of the field stop and Emax is the maximum irradiance across the entire diagonal or longest dimension of the aperture of the field stop.

[0016] The luminance uniformity is: Lmin / Lmax, wherein Lmin is the minimum luminance across a central 100%, 95%, 90%, 85%, 80% or 75% of a diagonal or longest dimension of the light-emitting surface of the second illumination source and Lmax is the maximum luminance across the diagonal of the light-emitting surface.

[0017] Optionally, the irradiance uniformity is greater than 0.80, optionally greater than 0.82, optionally greater than 0.84, optionally greater than 0.86, optionally greater than 0.88.

[0018] Optionally, the luminance uniformity is less than or equal to than 0.80, optionally less than 0.78, optionally less than 0.76, optionally less than 0.74, optionally less than 0.72.

[0019] Optionally, a first power efficiency of the first illumination source is within 20% of a second power efficiency of the second illumination source.

[0020] Optionally, the device further comprises an objective disposed to receive the first and second illumination light from the field optic. The first power efficiency is: < E1 / EL1, wherein OEl is the total irradiance optical flux of the first illumination light at the focal plane of the objective, and wherein OLl is the total luminance optical flux of the first illumination light at the light emitting surface of the first illumination source. The second power efficiency is: < E2 / < L2, wherein OE2 is the total irradiance optical flux of the second illumination light at the focal plane of the objective, and wherein OL2 is the total luminance optical flux of the second illumination light at the light emitting surface of the second illumination source.

[0021] Optionally, the second collector optic is arranged to form an image of the second illumination source between the field stop and the field optic.

[0022] Optionally, the second collector optic is arranged to focus the image of the second illumination source at least one sixteenth, at least one eighth or at least one quarter of the focal length of the field optic away from the field stop.

[0023] Optionally, the second collector optic is arranged so that the field stop aperture is fully illuminated by the second illumination light.

[0024] Optionally, the first collector optic is arranged so that the field stop aperture is fully illuminated by the first illumination light.

[0025] Optionally, the device further comprises an objective disposed to receive the first and second illumination light from the field optic, wherein the field stop is positioned at the front focal plane of the field optic, and an optical separation between the field optic and the objective is equal to the sum of the back focal length of the field optic and the front focal length of the objective.IOO-I789OIPC

[0026] Optionally, the distance between the field stop and the first collector optic is equal to the front focal length of the first collector optic.

[0027] Optionally, the distance between the second illumination source and the second collector optic is greater than the back focal length of the second collector optic.

[0028] Optionally, a first optical distance between the first collector optic and the field stop is less than a second optical distance between the second collector optic and the field stop.

[0029] Optionally, the device further comprises a third illumination source arranged to output third illumination light; a third collector optic arranged to collect the third illumination light. The field stop is arranged to provide an aperture for light from the third collector optic. The third collector optic is arranged to substantially collimate the third illumination light, a luminance uniformity of the third illumination source is lower than a luminance uniformity of the second illumination source.

[0030] Optionally, a first optical distance between the first collector optic and the field stop is less than a second optical distance between the second collector optic and the field stop, and wherein a third optical distance between the third collector optic and the field stop is less than the first optical distance and less than the second optical distance.

[0031] Optionally, a difference between the first optical distance and the third optical distance is less than the difference between the first optical distance and the second optical distance.

[0032] Optionally, the device further comprises a fourth illumination source arranged to output fourth illumination light; and a fourth collector optic arranged to collect the fourth illumination light. The field stop is arranged to provide an aperture for light from the fourth collector optic. The fourth collector optic is arranged to form an image of the fourth illumination source between the fourth collector optic and the field optic, before or after the field stop so that the image of the fourth illumination source is defocussed at the field stop. Optionally a luminance uniformity of the fourth illumination source is higher than a luminance uniformity of the first illumination source.

[0033] Optionally, a fourth optical distance between the fourth collector optic and the field stop is greater than a second optical distance between the second collector lens and the field stop.

[0034] Optionally, a difference between the fourth optical distance and the second optical distance is less than the difference between the first optical distance and the second optical distance.

[0035] Optionally, the fourth collector optic is arranged to form an image of the fourth illumination source between the field stop and the field optic.

[0036] Optionally, the device further comprises a first dichroic arranged between the first collector optic and the field stop and a second dichroic arranged between the second collector optic and the field stop. The first dichroic is arranged to reflect the first illumination light and transmit the second illumination light, and the second dichroic is arranged to reflect the second illumination light.IOO-I789OIPC

[0037] Optionally, the first illumination light has a first wavelength and the second illumination light has a second wavelength,

[0038] Optionally, the first wavelength is shorter than the second wavelength.

[0039] Optionally, the second wavelength is shorter than the first wavelength.BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Following drawings are appended to facilitate the understanding of the invention. The drawings show embodiments, which will now be described by way of example only, where:

[0041] 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.

[0042] 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.

[0043] 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.

[0044] FIGS. 4A-4B illustrate cross-sectional views of an optics module in an imaging system.

[0045] FIG. 5 depicts a computing node according to some embodiments disclosed herein.

[0046] FIG. 6A depicts an example of an optics module including a single-source illumination device.

[0047] FIG. 6B depicts an example of an optics module including a dual-source illumination device according to embodiments.

[0048] FIG. 6C depicts a dual-source illumination device according to embodiments.

[0049] FIG. 6D depicts a multi-source illumination device according to embodiments.

[0050] FIG. 6E depicts a multi-source illumination device according to embodiments.

[0051] FIG. 6F depicts a multi-source illumination device according to embodiments.

[0052] FIG. 6G depicts a multi-source illumination device according to embodiments.

[0053] FIG. 6H depicts a multi-source illumination device according to embodiments.

[0054] FIG. 61 depicts a multi-source illumination device according to embodiments.

[0055] FIG. 6 J depicts a multi-source illumination device according to embodiments.

[0056] FIG. 6K depicts a multi-source illumination device according to embodiments.IOO-I789OIPC

[0057] FIG. 6L depicts a multi-source illumination device according to embodiments.

[0058] FIG. 7 depicts a Kohler illumination scheme implemented in an illumination device for an optics module.

[0059] FIG. 8 depicts a critical illumination scheme implemented in an illumination device for an optics module.

[0060] FIG. 9 depicts a semi-critical illumination scheme implemented in an illumination device for an optics module.

[0061] 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.

[0062] 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

[0063] 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.

[0064] In brief, the present disclosure relates to an illumination device for use in an optics modules (e.g., in a microscope). The illumination device includes at least two illumination (or light) sources arranged (e.g. , linearly) with each illumination source having a different illumination scheme. In particular, a first illumination source has a substantially Kohler illumination scheme (emphasizing uniformity at the expense of optical flux) and a second illumination source has a semi-critical illumination scheme (emphasizing optical flux at the expense of uniformity). Each illumination scheme is defined by the configuration (e.g., distance) of the collector lens relative to the illumination source, illumination source type (e.g., point or extended source), collector lens parameters (e.g., type, focal length), additional optical components (e.g., filters and / or diffusers), and the configuration (e.g., distance) of the collector lens relative to a field stop. In some embodiments, a field lens is positioned at a distance away from the field stop equal to the focal length of the field lens, such that the field lens relays an image formed at the field stop. In the Kohler illumination scheme, the collector lens is arranged to provide substantially collimated illumination (i.e., substantially parallel rays due to focus at infinity or very far from the field stop) at the field stop. In some embodiments, while ideal Kohler illumination generally requires a point source of light, an extended source of light can still be used to provide substantially Kohler illumination. Kohler illumination provides high uniformity of illumination of the field stop, but at the expense of power (e.g., optical flux). The collector lens in the semi- critical illumination scheme is arranged to form an image of the illumination source between the collector lens and the field lens (other than at the field stop), so as to form a defocussed image of the illumination source at the field stop that optimizes for both uniformity and power (e.g., optical flux). In someIOO-I789OIPC embodiments, the optimization is based on a predetermined set of optical requirements of a system, for example, the selected fluorescent dyes, image sensor parameters (e.g., sensor type, exposure time, frame rate, resolution, etc.), required throughput of an instrument with the optical system. A semi-critical illumination scheme maintains acceptable uniformity of illumination of the field stop (based on requirements of the instrument as a whole) while also ensuring that the optical flux is higher than in the Kohler illumination scheme (e.g., allowing for higher performance of selected fluorescent dyes and / or a greater selection of usable fluorescent dyes). In an implementation, the semi-critical illumination scheme is adopted for illumination sources having higher luminance uniformity when an image of the illumination source is focused at the field stop compared with a illumination source employed in the Kohler illumination scheme.

[0065] In some microscopy applications, the microscope objective images the field stop at the sample plane. Therefore, illumination uniformity at the field stop translates to illumination uniformity at the sample plane. Contemporary microscopes typically employ a Kohler illumination scheme to ensure that the sample under the microscope is uniformly illuminated. Kohler illumination is a technique used for illuminating samples in both transmitted and reflected light (trans- and epi-illuminated) optical microscopy. This technique creates uniform illumination of the sample and prevents the image of the illumination source (such as a halogen lamp filament) from appearing in the final image. Kohler illumination is the primary technique employed in contemporary scientific light microscopy. While Kohler illumination has high uniformity (e.g., is perfectly defocused), Kohler illumination has lower light intensity (i.e., irradiance or optical flux) at the sample. In applications such as fluorescence microscopy, this can result in longer exposure times to create the same contrast or definition in the image and therefore image throughput is lowered.

[0066] Before the introduction of Kohler illumination, critical illumination was the main method used for illuminating samples. In contrast to semi-critical illumination, ideal critical illumination forms an image of the illumination source at the field stop and thus maximizes optical flux from the illumination source; however, the features (e.g., edges, wires, connections, filament or diode structure, etc.) of the illumination source may cause high non-uniformity (e.g., high variability in power) across the FOV. In some microscopes, an in-focus image of the illumination source (i.e., using a illumination source arranged in ideal critical illumination) can be seen in the final image because the image formed at the field stop is relayed to the sample plane. As a result, critical illumination leads to inconsistent illumination of the sample; brighter areas of the illumination source image cause those specific regions of the sample to be illuminated more intensely. This uneven lighting is problematic as it can create artifacts like glare and shadows in the image, or can result in uneven detectability of fluorophores (e.g., some fluorophores receive higher irradiance and fluoresce brightly while other fluorophores receive lower irradiance and fluoresce weakly).

[0067] In applications such as fluorescence microscopy, uniform illumination of the FOV is important for ensuring that the fluorophores within the FOV are equally energized and therefore equally represented in the captured image. Furthermore, if two or more fluorophore types are imaged using respective illumination sources having substantially different uniformity from one another (so as to have a substantive effect onIOO-I789OIPC image quality), the relative concentrations of the fluorophore types across the sample may not be accurately represented in the compound image, thereby reducing image quality.

[0068] To diffuse the filament image in critical illumination, various techniques can be employed, such as lowering the power of the illumination source or using an opal glass bulb or diffuser between the bulb and the sample. While these methods can help reduce uneven illumination to some degree, they also diminish the overall intensity of the light and affect the range of wavelengths that reach the sample.

[0069] To address the above problems and related problems, described herein are devices, assemblies, systems and methods for providing uniform illumination of the sample while maintaining high illumination irradiance, and / or irradiance consistency using illumination sources having differing characteristics (e.g., emission spectra, luminance uniformity, and / or luminance optical flux). High irradiance (or illumination) consistency at the sample means that that illumination using different illumination sources does not result in a large difference in optical flux at the sample (i.e. across the sample plane, or across the FOV of the objective).

[0070] In particular, the uniform illumination, high irradiance and high irradiance consistency is obtained by applying different illumination schemes to different illumination sources depending on the relative uniformity of the illumination sources. In various embodiments, the illumination sources are arranged in a linear configuration such that light from each illumination source travels along the same optical path towards the field stop. In particular, a semi-critical illumination scheme is employed for more uniform sources, whereas for less uniform sources, Kohler illumination is employed. In various embodiments, the more- uniform sources for which semi-critical illumination scheme is employed are relatively less intense (have relatively less irradiance). In various embodiments, the less-uniform sources for which Kohler illumination scheme is employed are relatively more intense (have relatively more irradiance). In various embodiments, the two different illumination schemes are used in the same illumination device. In various embodiments, the same illumination device includes a first plurality of illumination sources arranged in a substantially Kohler illumination scheme and a second plurality of illumination sources arranged in a semi-critical illumination scheme. In various embodiments, the first plurality of illumination sources and the second plurality of illumination sources are arranged linearly such that light from each illumination source follows a same optical path. The configurations of illumination sources described herein can be useful for any type of microscopy in which illumination using different illumination sources (e.g., two or more color channels) is employed. It can be particularly advantageous in fluorescence microscopy where images of different fluorophore types are obtained using different illumination sources. In various embodiments, disparity between the accuracy of fluorophore representation between fluorophores across the image is reduced, minimised or even eliminated.

[0071] Furthermore, the inventors have recognised that using a Kohler illumination scheme for all illumination sources does not allow for an optimal balance between maintaining image uniformity and maintaining high image throughput across all illumination sources (e.g., color channels). By selecting aIOO-I789OIPC semi-critical illumination scheme for more uniform sources (which may be of lower brightness or intensity), intensity levels at the sample are maintained while more acceptable levels of uniformity of illumination can be obtained compared with critical illumination. This increases image throughput compared with devices employing Kohler illumination for all illumination sources.

[0072] In the present disclosure, the term ‘semi-critical’, when used in the context of illumination, refers to an illumination scheme in which the illumination source is imaged by the collector lens at (or the light emanating from the illumination source is focussed by the collector lens to) a position between the collector lens and the field lens, either after or before the field stop. That is, the sharp, in-focus image of the illumination source is not formed (or the light from the collector lens is not focussed) at the field stop itself or downstream of the field lens. The result of semi-critical illumination is that a partially defocussed image of the illumination source appears at the field stop.

[0073] In contrast, Kohler illumination is a scheme in which the illumination light is focussed in the far field or at infinity, or in other words in which the collector lens produces a substantially collimated beam between the collector lens and the field lens. The result of Kohler illumination is that the field stop is more uniformly illuminated than in semi-critical (or critical) illumination. For a point source illumination source, Kohler illumination results in a uniform illumination of the field stop. When real (extended, non-point) sources, such as an LED die, are used in Kohler illumination, the size (diameter or width) of the illumination source results in overlapping collimated beams emerging at different angles from the collector lens. The field stop is placed where these beams overlap, which is usually at the point of minimum width of the overall beam after the collimator lens and which coincides with the back focal plane of the collector lens. This ensures maximal uniformity of illumination of the field stop in Kohler illumination employing real sources. These and other aspects of the arrangement of the illumination source and collector lens in embodiments are directed to addressing the problem of Kohler illumination scheme applied to real sources.

[0074] In the present disclosure, (illumination) devices are sometimes described in the context of use in a microscope, and more particularly in the context of use in a fluorescence microscope. However, the devices (e.g. illumination devices) described herein can be used in any imaging, analysis or observation instrument that illuminates a sample with more than one type of illumination source (e.g. two or more illumination sources having different wavelength spectra).

[0075] 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.

[0076] 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 moleculesIOO-I789OIPC 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.

[0077] 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.)

[0078] FIG. 3 shows an example workflow of analysis of a biological sample 110 (e.g., cell or tissue sample) using an opto-fluidic instrument 120, according to various embodiments. In various embodiments, the sample 110 can be a biological sample (e.g., a tissue) that includes molecules such as DNA, RNA, proteins, antibodies, etc. For example, the sample 110 can be a sectioned tissue that is treated to access the RNA thereof for labelling with circularizable DNA probes. Ligation of the probes may generate a circular DNA probe which can be enzymatically amplified and bound with fluorescent oligonucleotides, which can create bright signal that is convenient to image and has a high signal-to-noise ratio.

[0079] In various embodiments, the sample 110 may be placed in the opto-fluidic instrument 120 for analysis and detection of the molecules in the sample 110. In various embodiments, the opto-fluidic instrument 120 can be a system configured to facilitate the experimental conditions conducive for the detection of the target molecules. For example, the opto-fluidic instrument 120 can include a fluidics module 140, an optics module 150, a sample module 160, and an ancillary module 170, and these modules may be operated by a system controller 130 to create the experimental conditions for the probing of the molecules in the sample 110 by selected probes (e.g., circularizable DNA probes), as well as to facilitate the imaging of the probed sample (e.g., by an imaging system of the optics module 150). In various embodiments, the various modules of the opto-fluidic instrument 120 may be separate components in communication with each other, or at least some of them may be integrated together.

[0080] 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 isIOO-I789OIPC 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.

[0081] 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.

[0082] 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).

[0083] 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.IOO-I789OIPC

[0084] As discussed above, the opto-fluidic instrument 120 may include an optics module 150 which include the various optical components of the opto-fluidic instrument 120, such as but not limited to a camera, an illumination module (e.g. any of the illumination devices described in the present disclosure including two or more illumination sources), 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.

[0085] 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.

[0086] In various embodiments, the system controller 130 may be configured to control the operations of the opto-fluidic instrument 120 (e.g., and the operations of one or more modules thereof). In some instances, the system controller 130 may take various forms, including a processor, a single computer (or computer system), or multiple computers in communication with each other. In various embodiments, the system controller 130 may be communicatively coupled with data storage, set of input devices, display system, or a combination thereof. 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.

[0087] In various embodiments, the opto-fluidic instrument 120 may analyze the sample 110 and may generate the output 190 that includes indications of the presence of the target molecules in the sample 110. For instance, with respect to the example embodiment discussed above where the opto-fluidic instrument 120 employs a hybridization technique for detecting molecules, the opto-fluidic instrument 120 may cause the sample 110 to undergo successive rounds of fluorescent probe hybridization (using two or more sets of fluorescent probes, where each set of fluorescent probes is excited by a different color channel) and be imaged to detect target molecules in the probed sample 110. In such cases, the output 190 may include optical signatures (e.g., a codeword) specific to each gene, which allow the identification of the target molecules.

[0088] 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 illumination source configured to illuminate the sample carrier device. In some instances, the assembly includes a heat exchanger (e.g., a fluid block having a cooling fluid flowing therethrough). In some instances, an assembly for transilluminating can include sample carrier device (e.g., a sample substrate), an optically transparent substrate, a illumination source configured to illuminate the optically transparent substrate, a light scattering layer configured to scatter light from the illumination source, and / or a thermal control module configured to control the temperature of the sample carrier device and / or optically transparent substrate.IOO-I789OIPC

[0089] 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.

[0090] 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.

[0091] In some instances, the one or more sample carrier devices can be designed for performing a variety of chemical analysis, biochemical analysis, nucleic acid analysis, cell analysis, or tissue analysis applications. In some instances, for example, the sample carrier device (e.g., flow cells and microfluidic devices) may comprise a sample, e.g., a tissue sample. In some instances, the sample carrier device (e.g., flow cells and microfluidic devices) may comprise a sample, e.g., a tissue sample, placed in contact with, e.g., a substrate (e.g., a surface of the flow cell or microfluidic device).

[0092] 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.

[0093] 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.IOO-I789OIPCAlternatively, in some instances, only a portion of the sample carrier device (e.g., an optically transparent “window”) can be optically transparent.

[0094] The sample carrier devices for the disclosed systems (e.g. , substrates configured to receive a sample, microscope slides, substrates comprising one or more etched microfluidic channels, flow cells or microfluidic devices comprising one or more microfluidic channels or sample chambers, etc.) can be fabricated using any of a variety of techniques known to those of skill in the art, where the choice of fabrication technique is often dependent on the choice of material used, and vice versa. Examples of suitable sample carrier device fabrication techniques include, but are not limited to, extrusion, drawing, precision computer numerical control (CNC) machining and boring, laser photoablation, photolithography in combination with wet chemical etching, deep reactive ion etching (DRIE), micro-molding, embossing, 3D- printing, thermal bonding, adhesive bonding, anodic bonding, and the like (see, e.g., Gale, et al. (2018), “A Review of Current Methods in Microfluidic Device Fabrication and Future Commercialization Prospects”, Inventions 3, 60, 1 - 25, which is hereby incorporated by reference in its entirety).

[0095] FIG.4A illustrates a cross-sectional view of an optics module 200 in a comparative imaging system. One or more illumination sources 210, e.g. , one or more light emitting diodes (LEDs), provides light through one or more optical components and an objective lens 220 to thereby illuminate a sample 230 in a sample holder 250. The one or more illumination sources 210 may be two or more illumination sources forming part of any of the illumination devices described in the present disclosure including those described with reference to FIGS. 6B, 6C and 6D. In various embodiments, the optical components include a collimator 211. In various embodiments, the optical components include a field stop 212. In various embodiments, the optical components include one or more excitation filters 213. In various embodiments, the one or more excitation filters 213 are configured to filter light from the illumination source(s) 210 for a predetermined range of wavelengths (e.g., each filter has one or more blocking band(s) and / or transmission band(s) that may be different or may overlap at least in part) and each excitation filter 213 is aligned with appropriate illumination sources (e.g., blue LEDs, green LEDs, yellow LEDs, red LEDs, ultraviolet LEDs, etc.). In various embodiments, the optical components include a condenser 214. In various embodiments, the optical components include a beam splitter 215. 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).

[0096] 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 opticsIOO-I789OIPC and / or the sensor array. In various embodiments, the sample 230 is probed with fluorescent probes configured to bind to a target (e.g., DNA or RNA) that, when illuminated with a particular wavelength (or range of wavelengths) of light, emit light signals that can be detected by the sensor array 260. In various embodiments, the sample 230 is repeatedly probed with two or more (e.g., two, three, four, five, six, etc.) different sets of probes. In various embodiments, each set of probes corresponds to a specific color (e.g., blue, green, yellow, or red) such that, when illuminated by that color, probes bound to a target emit light signals. In some embodiments, the sensor array 260 is aligned with the optical axis 251 of the objective lens 220 (i.e., the optical axis of the camera is coincident with and parallel to the optical axis of the objective lens 220). In various embodiments, the sensor array 260 is positioned perpendicularly to the objective lens 220 (i.e., the optical axis of the camera is perpendicular to and intersects the optical axis of the objective lens 220). In various embodiments, a tube lens 261 is mounted in the optical path to focus light on the sensor array 260 thereby allowing for image formation with infinity-corrected objectives. Descriptions of optical modules and illumination assemblies for use in opto-fluidic instruments can be found in U.S. patent application publication no. 2024-0171833 and U.S. patent application publication no. 2024-0167956 each of which is incorporated by reference in its entirety.

[0097] 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-thy mine -rich regions in DNA. In various embodiments, illumination of the sample causes autofluorescence of the sample. In various embodiments, autofluorescence is the natural emission of light by biological structures when they have absorbed light, and may be used to distinguish the light originating from artificially added fluorescent markers. In various embodiments, fluorescence of the sample through fluorescent probes, autofluorescence, and / or a fluorescent stain can be used with the methods described herein to determine one or more focus metrics of a tissue sample.

[0098] 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 illumination 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.IOO-I789OIPC

[0099] Referring now to FIG. 5, a schematic of an example of a computing node is shown. Computing node 10 is only one example of a suitable computing node and is not intended to suggest any limitation as to the scope of use or functionality of embodiments described herein. Regardless, computing node 10 is capable of being implemented and / or performing any of the functionality set forth hereinabove.

[0100] In computing node 10 there is a computer system / server 12, which is operational with numerous other general purpose or special purpose computing system environments or configurations. Examples of well-known computing systems, environments, and / or configurations that may be suitable for use with computer system / server 12 include, but are not limited to, personal computer systems, server computer systems, thin clients, thick clients, handheld or laptop devices, multiprocessor systems, microprocessorbased systems, set top boxes, programmable consumer electronics, network PCs, minicomputer systems, mainframe computer systems, and distributed cloud computing environments that include any of the above systems or devices, and the like.

[0101] Computer system / server 12 may be described in the general context of computer system-executable instructions, such as program modules, being executed by a computer system. Generally, program modules may include routines, programs, objects, components, logic, data structures, and so on that perform particular tasks or implement particular abstract data types. Computer system / server 12 may be practiced in distributed cloud computing environments where tasks are performed by remote processing devices that are linked through a communications network. In a distributed cloud computing environment, program modules may be located in both local and remote computer system storage media including memory storage devices.

[0102] As shown in FIG. 5, computer system / server 12 in computing node 10 is shown in the form of a general-purpose computing device. The components of computer system / server 12 may include, but are not limited to, one or more processors or processing units 16, a system memory 28, and a bus 18 that couples various system components including system memory 28 to processor 16.

[0103] Bus 18 represents one or more of any of several types of bus structures, including a memory bus or memory controller, a peripheral bus, an accelerated graphics port, and a processor or local bus using any of a variety of bus architectures. By way of example, and not limitation, such architectures include Industry Standard Architecture (ISA) bus, Micro Channel Architecture (MCA) bus, Enhanced ISA (EISA) bus, Video Electronics Standards Association (VESA) local bus, and Peripheral Component Interconnect (PCI) bus.

[0104] Computer system / server 12 typically includes a variety of computer system readable media. Such media may be any available media that is accessible by computer system / server 12, and it includes both volatile and non-volatile media, removable and non-removable media.

[0105] System memory 28 can include computer system readable media in the form of volatile memory, such as random-access memory (RAM) 30 and / or cache memory 32. Computer system / server 12 may further include other removable / non-removable, volatile / non-volatile computer system storage media. By way of example only, storage system 34 can be provided for reading from and writing to a non-removable, nonvolatile magnetic media (not shown and typically called a “hard drive”). Although not shown, a magneticIOO-I789OIPC disk drive for reading from and writing to a removable, non-volatile magnetic disk (e.g., a “floppy disk”), and an optical disk drive for reading from or writing to a removable, non-volatile optical disk such as a CD- ROM, DVD-ROM or other optical media can be provided. In such instances, each can be connected to bus 18 by one or more data media interfaces. As will be further depicted and described below, memory 28 may include at least one program product having a set (e.g., at least one) of program modules that are configured to carry out the functions of embodiments described herein.

[0106] Program / utility 40, having a set (at least one) of program modules 42, may be stored in memory 28 by way of example, and not limitation, as well as an operating system, one or more application programs, other program modules, and program data. Each of the operating system, one or more application programs, other program modules, and program data or some combination thereof, may include an implementation of a networking environment. Program modules 42 generally carry out the functions and / or methodologies of embodiments described herein.

[0107] Computer system / server 12 may also communicate with one or more external devices 14 such as a keyboard, a pointing device, a display 24, etc.', one or more devices that enable a user to interact with computer system / server 12; and / or any devices (e.g., network card, modem, etc.) that enable computer system / server 12 to communicate with one or more other computing devices. Such communication can occur via Input / Output (VO) interfaces 22. Still yet, computer system / server 12 can communicate with one or more networks such as a local area network (LAN), a general wide area network (WAN), and / or a public network (e.g., the Internet) via network adapter 20. As depicted, network adapter 20 communicates with the other components of computer system / server 12 via bus 18. It should be understood that although not shown, other hardware and / or software components could be used in conjunction with computer system / server 12. Examples, include, but are not limited to microcode, device drivers, redundant processing units, external disk drive arrays, RAID systems, tape drives, and data archival storage systems, etc.

[0108] The present disclosure includes systems, methods, and / or computer program products. The computer program product may include a computer readable storage medium (or media) having computer readable program instructions thereon for causing a processor to carry out aspects of the present disclosure.

[0109] The computer readable storage medium can be a tangible device that can retain and store instructions for use by an instruction execution device. The computer readable storage medium may be, for example, but is not limited to, an electronic storage device, a magnetic storage device, an optical storage device, an electromagnetic storage device, a semiconductor storage device, or any suitable combination of the foregoing. A non-exhaustive list of more specific examples of the computer readable storage medium includes the following: a portable computer diskette, a hard disk, a random access memory (RAM), a readonly memory (ROM), an erasable programmable read-only memory (EPROM or Flash memory), a static random access memory (SRAM), a portable compact disc read-only memory (CD-ROM), a digital versatile disk (DVD), a memory stick, a floppy disk, a mechanically encoded device such as punch-cards or raised structures in a groove having instructions recorded thereon, and any suitable combination of the foregoing.IOO-I789OIPCA computer readable storage medium, as used herein, is not to be construed as being transitory signals per se, such as radio waves or other freely propagating electromagnetic waves, electromagnetic waves propagating through a waveguide or other transmission media (e.g., light pulses passing through a fiberoptic cable), or electrical signals transmitted through a wire.

[0110] Computer readable program instructions described herein can be downloaded to respective computing / processing devices from a computer readable storage medium or to an external computer or external storage device via a network, for example, the Internet, a local area network, a wide area network and / or a wireless network. The network may comprise copper transmission cables, optical transmission fibers, wireless transmission, routers, firewalls, switches, gateway computers and / or edge servers. A network adapter card or network interface in each computing / processing device receives computer readable program instructions from the network and forwards the computer readable program instructions for storage in a computer readable storage medium within the respective computing / processing device.

[0111] Computer readable program instructions for carrying out operations of the present disclosure may be assembler instructions, instruction-set-architecture (ISA) instructions, machine instructions, machine dependent instructions, microcode, firmware instructions, state-setting data, or either source code or object code written in any combination of one or more programming languages, including an object oriented programming language such as Smalltalk, C++ or the like, and conventional procedural programming languages, such as the “C” programming language or similar programming languages. The computer readable program instructions may execute entirely on the user's computer, partly on the user's computer, as a stand-alone software package, partly on the user's computer and partly on a remote computer or entirely on the remote computer or server. In the latter scenario, the remote computer may be connected to the user's computer through any type of network, including a local area network (LAN) or a wide area network (WAN), or the connection may be made to an external computer (for example, through the Internet using an Internet Service Provider). In some embodiments, electronic circuitry including, for example, programmable logic circuitry, field-programmable gate arrays (FPGA), or programmable logic arrays (PLA) may execute the computer readable program instructions by utilizing state information of the computer readable program instructions to personalize the electronic circuitry, in order to perform aspects of the present disclosure.

[0112] Aspects of the present disclosure are described herein with reference to flowchart illustrations and / or block diagrams of methods, apparatus (systems), and computer program products according to embodiments of the disclosure. It will be understood that each block of the flowchart illustrations and / or block diagrams, and combinations of blocks in the flowchart illustrations and / or block diagrams, can be implemented by computer readable program instructions.

[0113] These computer readable program instructions may be provided to a processor of a general purpose computer, special purpose computer, or other programmable data processing apparatus to produce a machine, such that the instructions, which execute via the processor of the computer or other programmable data processing apparatus, create means for implementing the functions / acts specified in the flowchart and / orIOO-I789OIPC block diagram block or blocks. These computer readable program instructions may also be stored in a computer readable storage medium that can direct a computer, a programmable data processing apparatus, and / or other devices to function in a particular manner, such that the computer readable storage medium having instructions stored therein comprises an article of manufacture including instructions which implement aspects of the function / act specified in the flowchart and / or block diagram block or blocks.

[0114] The computer readable program instructions may also be loaded onto a computer, other programmable data processing apparatus, or other device to cause a series of operational steps to be performed on the computer, other programmable apparatus or other device to produce a computer implemented process, such that the instructions which execute on the computer, other programmable apparatus, or other device implement the functions / acts specified in the flowchart and / or block diagram block or blocks.Illumination Assemblies

[0115] FIG. 6A illustrates an optics module 600A. The optics module 600A can be as described below and optionally can be based on (and include any or all features of) the optics module and / or the imaging system and / or the computer node described with reference to Figures 1-5. For convenience of explanation, where the optics module can be used as a microscope, the optics module 600A may alternatively be referred to as a microscope (e.g., due to the ability of the optics module to resolve microscopic objects / features). The optics module 600A includes an objective 602, an imaging dichroic 604 (which may be referred to as an illumination dichroic), a tube lens 606, an image sensor 608, and an illumination device 620A. In some examples, the image sensor 608 is (at least a) part of a camera (e.g., a digital camera). In some examples, the image sensor is a CCD or CMOS image sensor. In this setup, the objective 602 is an infinity corrected objective arranged to focus the illumination light at a focal plane (e.g., a plane intersecting a sample) and arranged to collect emission light from a sample 610 (e.g. , emission light from the focal plane of the objective 602 or in the vicinity thereof) and collimate the emission light. In general, when light is collected collected from outside the focal plane, the light will appear defocused at the image sensor (e.g., by an amount proportional to a distance that the light source is from the focal plane). The collimated emission light is transmitted from the objective 602 to the tube lenses 606a, 606b through the infinity space 612 (i.e., the portion of the optical path in which the collimated rays travel). In some embodiments, a z-distance between the objective and sample is adjusted one or more times to thereby image additional focal planes (i.e. , z-slices) within a field of view (FOV) of the objective 602. In some embodiments, a plurality of z-slices of a FOV form a z-stack of images representing an image volume. For each z-slice of the plurality of z-slices of a FOV, there may be in-focus light and out-of-focus light imaged. In some embodiments, post-processing may be performed by a computer node on each z-slice to sharpen the image. For example, deconvolution or deblurring may be performed using an approximated or experimentally obtained point spread function (PSF) of the optical system to thereby sharpen the images. In some embodiments, the deconvolution or deblurring is performed by a computing node before z-slices are presented to a user and / or before one or more analysis steps (e.g., decoding steps) are performed on the z-slices.IOO-I789OIPC

[0116] The imaging dichroic 604 (also known as a dichroic plate, mirror or filter) is positioned to intersect the illumination light path in the infinity space 612. The imaging dichroic 604 has wavelength dependent reflectivity such that it reflects the illumination light from the illumination device 620A to fold the optical path of the illumination light 622 toward the objective 602. The imaging dichroic 604 is also arranged to transmit emission light 615 from the sample to allow it to pass substantially undeviated (e.g., with minimal wavefront error) from the objective 602 to the tube lens 606. In some embodiments, the imaging dichroic 604 includes one or more coatings (e.g., thin-film dielectric coating, anti-reflective coating, etc.) on one or both sides of a substrate (e.g., non-crystalline silica glass).

[0117] An emission filter (not shown) may be positioned between the imaging dichroic 604 and the tube lens 606 to ensure that only light with a wavelength of interest (i.e., emission of fluorophore) reaches the sensor and all other wavelengths (e.g., the reflected / scattered illumination light in particular) is blocked. In an alternative embodiment, the emission filter (not shown) may be positioned between the tube lens 606 and the image sensor 608. For example, a filter wheel having a plurality of emission filters may be positioned in the optical path between the tube lens 606 and the image sensor 608.

[0118] In some embodiments, the filter wheel includes a plurality of emission filters. In some embodiments, the plurality of emission filters includes two, three, four, five, or more filters. In some embodiments, the number of filters equals the number of different color channels for the optics module. In some embodiments, the number of filters is greater than the number of different color channels for the optics module. In some embodiments, for example, where multispectral or hyperspectral imaging is implemented (e.g., with spectral unmixing), the number of filters is greater than the number of color channels. In a multispectral imaging example, the emission filters may include 3 to 15 filters. In a hyperspectral imaging example, the number of emission filters may include up to 100 filters or up to 1000 filters.

[0119] In some embodiments, the color channels include one or more of near ultraviolet (nUV), blue, green, yellow, and red. In some embodiments, an emission filter of the plurality of emission filters corresponding to the nUV channel has a transmission band of about 400 nm to about 500 nm. In some embodiments, an emission filter of the plurality of emission filters corresponding to the nUV channel has a transmission band of about 415 nm to about 495 nm. In some embodiments, an emission filter of the plurality of emission filters corresponding to the nUV channel has a transmission band of about 420 nm to about 490 nm. In some embodiments, an emission filter of the plurality of emission filters corresponding to the nUV channel has a transmission band of about 425 nm to about 485 nm. In some embodiments, the lower bound of the transmission band is selected to be a wavelength in the range of about 400 nm to about 435 nm. In some embodiments, the upper bound of the transmission band is selected to be a wavelength in the range of about 470 nm to about 500 nm.

[0120] In some embodiments, an emission filter of the plurality of emission filters corresponding to the blue channel has a transmission band of about 490 nm to about 560 nm. In some embodiments, an emission filter of the plurality of emission filters corresponding to the blue channel has a transmission band of aboutIOO-I789OIPC495 nm to about 550 nm. In some embodiments, an emission filter of the plurality of emission filters corresponding to the blue channel has a transmission band of about 500 nm to about 545 nm. In some embodiments, an emission filter of the plurality of emission filters corresponding to the blue channel has a transmission band of about 505 nm to about 540 nm. In some embodiments, the lower bound of the transmission band is selected to be a wavelength in the range of about 485 nm to about 515 nm. In some embodiments, the upper bound of the transmission band is selected to be a wavelength in the range of about 530 nm to about 560 nm.

[0121] In some embodiments, an emission filter of the plurality of emission filters corresponding to the green channel has a transmission band of about 545 nm to about 595 nm. In some embodiments, an emission filter of the plurality of emission filters corresponding to the green channel has a transmission band of about 550 nm to about 590 nm. In some embodiments, an emission filter of the plurality of emission filters corresponding to the green channel has a transmission band of about 555 nm to about 585 nm. In some embodiments, an emission filter of the plurality of emission filters corresponding to the green channel has a transmission band of about 560 nm to about 580 nm. In some embodiments, the lower bound of the transmission band is selected to be a wavelength in the range of about 540 nm to about 570 nm. In some embodiments, the upper bound of the transmission band is selected to be a wavelength in the range of about 570 nm to about 600 nm.

[0122] In some embodiments, an emission filter of the plurality of emission filters corresponding to the yellow channel has a transmission band of about 600 nm to about 650 nm. In some embodiments, an emission filter of the plurality of emission filters corresponding to the yellow channel has a transmission band of about 605 nm to about 645 nm. In some embodiments, an emission filter of the plurality of emission filters corresponding to the yellow channel has a transmission band of about 610 nm to about 640 nm. In some embodiments, an emission filter of the plurality of emission filters corresponding to the yellow channel has a transmission band of about 615 nm to about 635 nm. In some embodiments, the lower bound of the transmission band is selected to be a wavelength in the range of about 600 nm to about 630 nm. In some embodiments, the upper bound of the transmission band is selected to be a wavelength in the range of about 620 nm to about 650 nm.

[0123] In some embodiments, an emission filter of the plurality of emission filters corresponding to the red channel has a transmission band of about 650 nm to about 755 nm. In some embodiments, an emission filter of the plurality of emission filters corresponding to the red channel has a transmission band of about 655 nm to about 750 nm. In some embodiments, an emission filter of the plurality of emission filters corresponding to the red channel has a transmission band of about 660 nm to about 745 nm. In some embodiments, an emission filter of the plurality of emission filters corresponding to the red channel has a transmission band of about 665 nm to about 740 nm. In some embodiments, the lower bound of the transmission band is selected to be a wavelength in the range of about 645 nm to about 675 nm. In some embodiments, the upper bound of the transmission band is selected to be a wavelength in the range of about 730 nm to about 760 nm.IOO-I789OIPC

[0124] Because the optics module 600A of FIG. 6A includes the illumination device 620A, the optics module 600A finds use in illuminating a sample 610 having one or more target analytes, where each target analyte is tagged with a fluorophore selected from a plurality of fluorophores. Each fluorophore has a specific excitation wavelength band and a specific emission wavelength band that correspond to one or more color channels of excitation illumination light, e.g., red, yellow, green, blue, near ultraviolet. In some embodiments, each fluorophore is sequentially excited by illumination light from the illumination device 620A. The excitation light used to excite for each fluorophore includes at least a portion of the respective fluorophore -dependent excitation wavelengths for exciting each fluorophore in the sample. In response to the excitation light, the fluorophores emit emission light that is collected (or captured) through the objective 602, which then directs the collected emission light 615 through the infinity space 612, the imaging dichroic 604 and to the tube lens 606, which focuses the emission light on the image sensor 608. In some embodiments, the emission light 615 passes through an emission filter as described above. For example, the emission light 615 may pass through a filter wheel positioned in front of the image sensor 608. An image of the emission light 615 is then captured by the image sensor 608.

[0125] In an alternative configuration, one or more of the image sensors may be replaced by an eyepiece allowing a user to view the sample directly, through the optics module. In some embodiments, the image sensors 608, 608a, 608b include any suitable image sensors for detecting the emission light, such as at least one photodiode array, at least one CCD sensor or camera, and / or at least one CMOS sensor or camera. The emission light is focused (z.e., rays are converged) by the tube lens 606 onto the image sensors.

[0126] The illumination device 620 A comprises an illumination unit 621 including an illumination source 623, a collector lens 625, an excitation filter (not shown), a field stop 627, and a field lens 629. The illumination device 620A is arranged to provide excitation illumination light and introduce said excitation illumination light into the infinity space 612 between the objective 602 and the tube lens 606 for onwards reflection to the objective 602 via the imaging dichroic 604, and thereby illuminate the sample (e.g., at or near the focal plane of the objective 602).

[0127] The illumination unit 621 houses the illumination source 623 and may include other components which support the operation of the illumination source, such as thermal management components and driving circuitry. The illumination source 623 comprises, for example, an LED die, lamp, laser, or other light emitting structure and is arranged to emit illumination light 622 at a suitable wavelength for the application of the optics module. The wavelength is, in the example of a fluorescence microscope, an excitation wavelength of a microscopic fluorophore molecule in the sample 610.

[0128] The collector lens 625 is arranged in the path of the illumination light 622 such that it collects a substantial proportion of the illumination light. The collector lens 625 is arranged with its optical axis parallel to or collinear with a normal of the centre (or central area) of the illumination source 623. The collector lens 625 is of positive optical power.IOO-I789OIPC

[0129] In various embodiments, an excitation filter (not shown) is arranged in the path of the illumination light 622 such that the excitation filter transmits only a defined band of wavelengths of illumination source 623. For example, a unique excitation filter may be positioned between the respective collector lens 625 and the field stop 627 for each illumination unit 621 in the illumination device 620A. In various embodiments, the transmission bands of the excitation filter and the emission filter do not overlap to prevent reflected / scattered excitation light from reaching the sensor 608.

[0130] In some embodiments, an excitation filter corresponding to the nUV channel has a transmission band of about 340 nm to about 420 nm. In some embodiments, an excitation filter corresponding to the nUV channel has a transmission band of about 345 nm to about 415 nm. In some embodiments, an excitation filter corresponding to the nUV channel has a transmission band of about 350 nm to about 410 nm. In some embodiments, an excitation filter corresponding to the nUV channel has a transmission band of about 355 nm to about 405 nm. In some embodiments, the lower bound of the transmission band is selected to be a wavelength in the range of about 330 nm to about 370 nm. In some embodiments, the upper bound of the transmission band is selected to be a wavelength in the range of about 390 nm to about 430 nm.

[0131] In some embodiments, an excitation filter corresponding to the blue channel has a transmission band of about 410 nm to about 495 nm. In some embodiments, an excitation filter corresponding to the blue channel has a transmission band of about 415 nm to about 490 nm. In some embodiments, an excitation filter corresponding to the blue channel has a transmission band of about 420 nm to about 485 nm. In some embodiments, an excitation filter corresponding to the blue channel has a transmission band of about 425 nm to about 480 nm. In some embodiments, the lower bound of the transmission band is selected to be a wavelength in the range of about 400 nm to about 440 nm. In some embodiments, the upper bound of the transmission band is selected to be a wavelength in the range of about 465 nm to about 500 nm.

[0132] In some embodiments, an excitation filter corresponding to the green channel has a transmission band of about 500 nm to about 550 nm. In some embodiments, an excitation filter corresponding to the green channel has a transmission band of about 505 nm to about 545 nm. In some embodiments, an excitation filter corresponding to the green channel has a transmission band of about 510 nm to about 540 nm. In some embodiments, an excitation filter corresponding to the green channel has a transmission band of about 515 nm to about 535 nm. In some embodiments, the lower bound of the transmission band is selected to be a wavelength in the range of about 490 nm to about 530 nm. In some embodiments, the upper bound of the transmission band is selected to be a wavelength in the range of about 520 nm to about 560 nm.

[0133] In some embodiments, an excitation filter corresponding to the yellow channel has a transmission band of about 550 nm to about 600 nm. In some embodiments, an excitation filter corresponding to the yellow channel has a transmission band of about 555 nm to about 595 nm. In some embodiments, an excitation filter corresponding to the yellow channel has a transmission band of about 560 nm to about 590 nm. In some embodiments, an excitation filter corresponding to the yellow channel has a transmission band of about 565 nm to about 585 nm. In some embodiments, the lower bound of the transmission band isIOO-I789OIPC selected to be a wavelength in the range of about 540 nm to about 580 nm. In some embodiments, the upper bound of the transmission band is selected to be a wavelength in the range of about 570 nm to about 610 nm.

[0134] In some embodiments, an excitation filter corresponding to the red channel has a transmission band of about 600 nm to about 645 nm. In some embodiments, an excitation filter corresponding to the red channel has a transmission band of about 605 nm to about 640 nm. In some embodiments, an excitation filter corresponding to the red channel has a transmission band of about 610 nm to about 635 nm. In some embodiments, an excitation filter corresponding to the red channel has a transmission band of about 615 nm to about 630 nm. In some embodiments, the lower bound of the transmission band is selected to be a wavelength in the range of about 590 nm to about 630 nm. In some embodiments, the upper bound of the transmission band is selected to be a wavelength in the range of about 620 nm to about 660 nm.

[0135] The field stop 627 is arranged on the opposite side of the collector lens 625 to the illumination source 623 and comprises an aperture arranged to provide an aperture stop (defining the size and area of the illuminated field, thus, acting as a physical limit to the field of view of the image sensor) for the illumination light 622 collimated or focussed by the collector lens 625. In some embodiments, the illumination device 620A includes a field stop 627 for each illumination color channel (z.e., for each illumination unit 621). In some embodiments, as shown below in FIGS. 6J-6K, the illumination device 620 A includes a single field stop through which all excitation illumination color channels direct respective excitation light.

[0136] The field lens 629 is arranged on the opposite side of the field stop 627 from the collector lens 625 and is arranged to form an image of the field stop 627 between the field lens 629 and the objective 602 (z.e., in the infinity space, or between the imaging dichroic 604 and the field lens 629). In some embodiments, the field lens 629 is arranged external to the illumination device 620A and in the optical path of the excitation illumination light.

[0137] FIG. 6B shows an example of the path of illumination (e.g., excitation) light and the path of sample luminance (e.g., emission light from the sample) in an optics module (which may also be described as a microscope) 600B which includes a dual source illumination device. The optics module 600B takes the structure and function of the optics module 600A described with reference to FIG. 6A, except where stated otherwise. The components having the same or similar reference numerals take the structure and function all components (or their variants) described with reference to FIG. 6A, except where stated otherwise.

[0138] As in the optics module 600A of FIG. 6A, the optics module 600B of FIG. 6B includes an objective (not shown), imaging dichroic 604, tube lens (not shown), and an illumination device 620B arranged relative to each other in the same way as described with reference to FIG. 6A. The optics module 600B of FIG. 6B is also arranged to illuminate and image a sample 610 as described with reference to FIG. 6A. The optics module 600B of FIG. 6B also includes an emission dichroic 611 (which may alternatively be referred to as an imaging dichroic plate), and includes a first image sensor 608a and a second image sensor 608b in place of the image sensor 608 of FIG. 6A. The optics module 600B of FIG. 6B includes a dual-source illumination device 620B in place of the single-source illumination device 620A described with reference to FIG. 6A.IOO-I789OIPC

[0139] The dual source illumination device 620B includes a first illumination unit 621a, a first collector lens 625a, field stop 627 and field lens 629, which take the structure and function as described with reference to the same components in FIG. 6A (or any of the variants described) and are arranged in the same configuration. The dual-source illumination device 620B additionally includes a second illumination unit 621b, a second collector lens 625b, and a first and second excitation filters 626a, 626b associated with the collector lens 625 and second collector lens 625b, respectively, as well as a wavelength-dependent reflector plate 628b.

[0140] The second illumination unit 621b is substantially identical in structure and function as the first illumination unit 621a except that it houses a second illumination source 623b different from the first illumination source 623a. The second illumination source 623b has a different luminance uniformity than that of the first illumination source 623a. The second illumination source 623b also has a different wavelength spectrum (for example including a different peak wavelength) than that of the first illumination source 623a. However, the arrangement in FIG. 6B is not limited thereto and, depending on application, the second illumination source 623b may have another difference in light emitting characteristics to the first illumination source in addition to a difference in uniformity (e.g. brightness or polarisation). In general terms, the first and second illumination sources 623a, 623b have a difference in luminance uniformity as well as a difference in at least one other light emitting characteristic so that the illumination sources illuminate the sample in different ways.

[0141] The second collector lens 625b is arranged in the path of second illumination light 622b emitted by the second illumination source 623b such that the second collector lens collects a substantial proportion of the second illumination light. The second collector lens 625b is arranged with its optical axis parallel to or collinear with a normal of the centre (or central area) of the second illumination source. The second collector lens 625b is of positive optical power.

[0142] In implementations requiring different wavelengths of illumination light at the sample, a first bandpass filter 626a is optionally included between the first collector lens 625a and the wavelengthdependent reflector plate 628b and a second band pass filter 626b is optionally included between the second collector lens 625b and the wavelength-dependent reflector plate 628b. The first and second bandpass filter are arranged to pass a portion of the spectrum of the first and second illumination light, respectively. The pass band of the first bandpass filter is different from the passband of the second bandpass filter so that the wavelength spectrum of the first illumination light 622a reaching the sample is different from that of the second illumination light 622b. In other implementations in which a difference in polarisation at the sample is important, the bandpass filters may be replaced by polarisation filters such that the first illumination light 622a reaching the sample has a different polarisation than that of the second illumination light 622b.

[0143] The wavelength-dependent reflector plate 628b is positioned in the optical path of the first illumination light 622a between the first collector lens 625a and the field stop 627 as well as in the optical path of the second illumination light 622b on the other side of the second collector lens 625b from the secondIOO-I789OIPC illumination unit 621b. The wavelength-dependent reflector plate 628b is, for example, a dichroic plate arranged at 45 degrees to the optical axis of the first and second collector lenses 625a, 625b. The wavelengthdependent reflector plate 628b is arranged to transmit the first illumination light 622a and to reflect the second illumination light 622b through 90 degrees such that the first illumination light 622a and second illumination light 622b emerge from the wavelength-dependent reflector plate 628b overlapping with their optical axes substantially colinear with each other.

[0144] The field stop 627 is placed in the path of the overlapping beams of first illumination light 622a and second illumination light 622b. The first and second illumination light follow a path to the sample via the imaging dichroic 604 and the objective as described with reference to FIG. 6A. Emission light 615a, 615b emanating from the sample as a result of the illumination light 622a, 622b then follows the same path as described with reference to FIG. 6A through the objective, imaging dichroic 604 and tube lens to a respective image sensor 608a, 608b. In the example of fluorescence microscopy, first emission light 615a and second emission light 615b is emitted from the sample due to excitation of fluorophores in the sample by the first illumination light 622a and second illumination light 622b, respectively. However, other applications may result in light emanating from the sample as a result of irradiation by illumination light via mechanisms other than fluorescence, for example via simple reflection from the surface.

[0145] A difference between the optics module 600A of FIG. 6A and the optics module 600B of FIG. 6B is the arrangement of image sensors. As in the optics module 600A of FIG. 6A, the optics module 600B of FIG. 6B includes an image sensor arranged downstream of the tube lens to capture the emission light from the fluorophores. However, the setup of FIG. 6B includes a first image sensor 608a arranged to capture the first emission light 615a and a second image sensor 608b arranged to capture the second emission light 615b. The first image sensor 608a is arranged downstream of an imaging dichroic plate 611 which is positioned in the optical path of the first emission light 615a between the tube lens and the first image sensor 608a. The second image sensor 608b is arranged downstream of the imaging dichroic plate 611 which is positioned in the optical path of the second emission light 615b between the tube lens and the second image sensor 608b. The imaging dichroic plate 611 is positioned at, e.g., 45 degrees to the optical axis of the tube lens and is arranged to reflect the first emission light 615a through, e.g., 90 degrees toward the first image sensor 608a and to transmit the second emission light 615b to allow it to pass to the second image sensor 608b.

[0146] The arrangement of FIG. 6B is provided to show how two illumination sources can be used in a device according to the present disclosure. However, the present disclosure is not limited thereto and devices having more than two illumination sources are also envisaged. Furthermore, more than two image sensors are also envisaged, for example one per illumination source, or even just a single image sensor arranged to detect all emission light (e.g., both the first and second emission light 615a, 615b). Further still, the arrangement of the illumination sources and collector lenses relative to each other can be different from that illustrated in FIG. 6B. In other implementations, no image sensor is provided and an eyepiece for viewing the sample is provided instead.IOO-I789OIPC

[0147] For example, in another implementation, instead of being positioned as shown in FIG. 6B, the first illumination source 623a and first collector lens 625a are positioned in the same orientation as, and to one side of, the second illumination source 623b and second collector lens 625b, respectively. In this case, a reflector (e.g. , a second wavelength-dependent reflector plate 628a) is provided in the same orientation, and to the left (with respect to FIG. 6B) of the wavelength-dependent reflector plate 628b and in the path of the first illumination light (and the optical axis of the first collector lens). The path of first illumination light 622a is thereby turned through 90 degrees by the reflector so that it follows the same path to the field stop as the second illumination light 622a shown in FIG. 6B. The second illumination light 622b passes through the second wavelength-dependent reflector plate 628a so that it emerges therefrom parallel to the path of the first illumination light 622a towards the field stop 627. In this arrangement, the illumination sources are placed side by side in a row.

[0148] In a modified version of this arrangement shown in the dual-source illumination device 620C of FIG. 6C, the first and second illumination sources 623a, 623b may be angled obliquely to the optical axis of the field stop 627 (the optical axis being perpendicular to the plane of the aperture of the field stop). The optical axes of first and second collector lenses 625a, 625b are also angled obliquely to the optical axis of the field stop. In this setup, the first wavelength-dependent reflector 628a and second wavelength-dependent reflector 628b are angled to align the optical axes of the reflected beams of first illumination light and second illumination light respectively with the optical axis of the field stop upon reflection therefrom. In this arrangement, the first and second illumination light 622a, 622b is turned through an angle of less than 90 degrees by the first wavelength-dependent reflector 628a and second wavelength-dependent reflector 628b, respectively. In some embodiments, wavelength-dependent reflectors may have varying performance based on the angle at which illumination light is incident on the reflective surface (e.g., performance at varying angles may depend on the coating). As shown in FIG. 6C, the angle between the excitation illumination beam for each illumination source 623a, 623b and the optical axis of the field stop 627 is, e.g., 30 degrees. This arrangement of illuminators, collector lenses, combiner and reflector relative to the optical axis of the field stop allows a larger diameter collector lens to be used while minimising the footprint of the illumination device. A larger diameter collector lens allows an illumination scheme which is closer to true Kohler illumination to be implemented.

[0149] In various embodiments, as shown in FIG. 6C, the first illumination source 621a and the second illumination source 621b are arranged (e.g. linearly) such that the illumination source with the longest wavelength (i.e., first illumination source 623a) is closest to the field stop 627 and illumination sources with progressively shorter wavelengths (e.g. , second illumination source 623b) are further from the field stop 627. In this embodiment, where progressively shorter wavelength colors of illumination source are positioned further from the field stop 627, the first wavelength-dependent reflector 628a and second wavelengthdependent reflector 628b allow for shorter wavelengths to pass and reflect longer wavelengths (e.g., are short-pass dichroic filters that transmit wavelengths below a threshold and reflect wavelengths above the threshold). In various embodiments, this arrangement is reversed where the first illumination source and theIOO-I789OIPC second illumination source are arranged linearly such that the illumination source with the shortest wavelength is closest to the field stop 627 and illumination sources with progressively longer wavelengths are further from the field stop 627. In this embodiment, where progressively longer wavelength colors of illumination source are positioned further from the field stop 627, the first wavelength-dependent reflector and second wavelength-dependent reflector allow for longer wavelengths to pass and reflect shorter wavelengths (e.g., are long-pass dichroic filters that transmit wavelengths above a threshold and reflect wavelengths below the threshold).

[0150] The present disclosure is not limited to dual-source illumination devices. Illumination devices having more than two illumination sources, for example three, four, five, six, seven or more illumination sources, are also envisaged. In various embodiments, the power efficiency and uniformity of each illumination source within the illumination device is considered when choosing an appropriate illumination scheme for the illumination source. As in the dual-source illumination device, in a multi-source illumination device, a first illumination source is configured in a Kohler illumination scheme and a second illumination source is configured in a semi-critical illumination scheme. The multi-source illumination device further includes, for example, at least one additional illumination source configured in a Kohler illumination scheme and / or at least one additional illumination source configured in a semi-critical illumination scheme.

[0151] FIG. 6D shows an example of a multi-source illumination device 620D. The multi-source illumination device 620D shown in FIG. 6D includes all of the components of the dual-source illumination device 620C described with reference to FIG. 6C and their configuration and position relative to each other. In the dual-source illumination device of FIG. 6C and / or in the multi-source illumination device of FIG. 6D, the illumination sources are arranged so that the first illumination source 623a is configured in a Kohler illumination scheme and the second illumination source 623b is configured in a semi-critical illumination scheme. In various embodiments, as shown in FIG. 6D, the first illumination source 623a is positioned closer to the field stop 627 than the second illumination source 623b. In one example, a first optical distance between the first collector lens 625a and the field stop 627 is less than a second optical distance between the second collector lens 625b and the field stop. In another example, a first optical distance between the first illumination source 623a and the field stop 627 is less than a second optical distance between the second illumination source 623b and the field stop 627. As shown in FIGS. 6C and 6D, the multi-source illumination devices 620C, 620D are arranged in a linear combiner configuration where the optical path of each illumination source shares the same exit path via the field stop 627 (and an output aperture to the extent the illumination device 620C, 620D is packaged into a modular housing).

[0152] The multi-source illumination device 620D further includes a first heat sink 624a that is thermally coupled to the first illumination source 623a, fourth illumination source 623d, and fifth illumination source 623e to thereby draw away and diffuse heat generated therefrom. The illumination device 620D further includes a second heat sink 624b separate from the first heat sink 624a that is thermally coupled to the second illumination source 623b and the third illumination source 623c to thereby draw away and diffuse heat generated therefrom. In some embodiments, the heat sinks 624a, 624b are made from a thermally conductiveIOO-I789OIPC material, such as a metal (e.g., aluminum, copper, silver, etc.), or other suitable thermally conductive material. In some embodiments, the heat sinks 624a, 624b include fins (e.g., skived fins) extending therefrom to improve convective heat transfer to the surrounding air. In some embodiments, the heat sinks 624a, 624b include one or more fans configured to force air across the heatsink (e.g., through the fins). In some embodiments, the heat sinks 624a, 624b may include a vapor chamber. In some embodiments, the vapor chamber includes one or more heat pipes extending vertically from an upper surface of the vapor chamber and through a radiator (e.g., a plurality of thin plates configured to dissipate heat from the one or more heat pipes to ambient air, which may be forced air via the one or more fans).

[0153] The multi-source illumination device 620D further includes a third illumination source 623c arranged to output third illumination light and a third collector lens 625c is arranged to collect the third illumination light. The field stop 627 is arranged to provide an aperture for light from the third collector lens 625c reflected via a third wavelength-dependent reflector 628c. The third collector lens 625c is arranged to substantially collimate the third illumination light (as per the Kohler illumination scheme). Optionally a luminance uniformity of the third illumination source 623c is lower than a luminance uniformity of the second illumination source 623b. The term ‘luminance uniformity’ when used in the present disclosure may be described as (spatial) emission uniformity. The third illumination source 623c is placed closer to the field stop 627 than the first illumination source 623a. That is, a third optical distance between the third collector lens 625c and the field stop is less than the aforementioned first optical distance. In various embodiments, as shown in FIG. 6D, the third illumination source 623c has a shorter wavelength (or spectra) than the first illumination source 623a and, thus, is positioned closer to the field stop 627 (e.g., a third optical distance between the third illumination source 623c and the field stop 627 is less than the first optical distance between the first illumination source 623a and the field stop 627).

[0154] The multi-source illumination device 620D further includes a fourth illumination source 623d arranged to output fourth illumination light and a fourth collector lens 625d is arranged to collect the fourth illumination light. The field stop 627 is arranged to provide an aperture for light from the fourth collector lens 625d reflected via a fourth wavelength-dependent reflector 628d. The fourth collector lens 625d is arranged to form an image of the fourth illumination source 623d between the fourth collector lens 625d and the field optic 629, before or after the field stop 627, so that the image of the fourth illumination source 623d is defocussed at the field stop 627 (as per the semi-critical illumination scheme). Optionally, a luminance uniformity of the fourth illumination source 623d is greater than a luminance uniformity of the first illumination source 623a. The fourth illumination source 623d is placed further from the field stop 627 than the second illumination source 623b. That is, a fourth optical distance between the fourth collector lens 625d and the field stop 627 is greater than the aforementioned second optical distance.

[0155] The multi-source illumination device 620D further includes a fifth illumination source 623e arranged to output fifth illumination light and a fifth collector lens 625e is arranged to collect the fifth illumination light. The field stop 627 is arranged to provide an aperture for light from the fifth collector lens 625e reflected via a fifth wavelength-dependent reflector 628e. The fifth collector lens 625e is arranged toIOO-I789OIPC form an image of the fifth illumination source 623e between the fifth collector lens 625e and the field optic 629, before or after the field stop 627 so that the image of the fifth illumination source is defocussed at the field stop 627 (as per the semi-critical illumination scheme). Optionally, a luminance uniformity of the fifth illumination source 623e is higher than a luminance uniformity of the first illumination source. The fifth illumination source 623e is placed further from the field stop 627 than the fourth illumination source 623d. That is, a fifth optical distance between the fifth collector lens 625e and the field stop 627 is greater than the aforementioned fourth optical distance.

[0156] In various embodiments, the collector lenses 625a, 625b, 625c, 625d, 625e are the same type and / or form of collector lens. For example, all collector lenses 625a, 625b, 625c, 625d, 625e may be aspheric lenses. In various embodiments, at least one of the collector lenses 625a, 625b, 625c, 625d, 625e is a different collector lens. For example, one or more collector lenses 625a, 625b, 625c, 625d, 625e may be a plano-convex lens. In various embodiments, an additional filter, lens, or diffuser is positioned between the collector lens and the wavelength-dependent reflectors. For example, a plano-concave lens may be positioned after the collector lens 625a, 625b, 625c, 625d, 625e and before the respective wavelengthdependent reflector (e.g. , positioned between the collector lens and an excitation filter). In another example, a diffuser may be positioned after the collector lens 625a, 625b, 625c, 625d, 625e and before the respective wavelength-dependent reflector (e.g., positioned between the collector lens and an excitation filter).

[0157] Where the term ‘between’ is used to describe the relative position of components, it may be understood to mean ‘between’ along the optical path, rather than strictly spatially between.

[0158] The third wavelength-dependent reflector 628c is transmissive to the first, second, fourth and fifth illumination light 622a, 622b, 622d, 622e and reflective to the third illumination light 622c. The first wavelength-dependent reflector 628a is transmissive to the second, fourth and fifth illumination light 622b, 622d, 622e and reflective to the first illumination light 622a. The second wavelength-dependent reflector 628b is transmissive to the fourth and fifth illumination light 622d, 622e and reflective to the second illumination light 622d. The fourth wavelength-dependent reflector 628d is transmissive to the fifth illumination light 622e and reflective to the fourth illumination light 622d. The fifth wavelength-dependent reflector 628e is reflective to the fifth illumination light 622e. In a convenient arrangement, the first to fifth illumination sources are positioned in order of increasing or decreasing wavelength and the wavelengthdependent reflectors 628a-628e act as either high or low pass filters to provide the reflectance and transmission behaviours described above.

[0159] In the embodiment shown in FIG. 6D, the illumination sources are arranged such that the shortest wavelength illumination source (i.e., the third illumination source 623c) is closest to the field stop 627 with progressively longer wavelength colors of illumination sources (i.e., first illumination source 623a, second illumination source 623b, fourth illumination source 623d, and fifth illumination source 623e) positioned further from the field stop 627. As such, the arrangement of wavelength-dependent reflectors 628a, 628b, 628c, 628d, 628e allow for longer wavelengths to pass and reflect shorter wavelengths (e.g., are long-passIOO-I789OIPC dichroic filters that transmit wavelengths above a threshold and reflect wavelengths below the threshold). In various embodiments, the arrangement of illumination sources shown in FIG. 6D can be reversed (not shown) such that the illumination source having the longest wavelength (i.e., the fifth illumination source 623e) is the closest to the field stop 627 with progressively shorter wavelength colors of illumination sources (i.e., fourth illumination source 623d, second illumination source 623b, first illumination source 623a, and third illumination source 623c) positioned further from the field stop 627. In this embodiment, the arrangement of wavelength-dependent reflectors 628a, 628b, 628c, 628d, 628e allow for shorter wavelengths to pass and reflect longer wavelengths (e.g., are short-pass dichroic filters that transmit wavelengths below a threshold and reflect wavelengths above the threshold).

[0160] In some embodiments, the illumination sources in a multi-source illumination device are physically grouped into a first group for those applied in a Kohler illumination scheme and a second group for those applied in a semi-critical illumination scheme. For example, the first and third illumination sources 621a, 621c and their respective collector lenses 625a, 625c and wavelength-dependent reflectors 628a, 628c belong to a first illumination group 660A; and the second, fourth and fifth illumination sources 621b, 621d, 621e and their respective collector lenses 625b, 625d, 625e and wavelength-dependent reflectors 628b, 628d, 628e belong to a second illumination group 660B.

[0161] The spacing between the centres of adjacent illumination sources in the same illumination group may be different than the spacing between the centres of adjacent illumination sources in different illumination groups. Due to the layout of the illumination sources within the illumination device, the spacing between the centres of illumination sources is equivalent to the difference in optical path length between one illumination source and the field stop on the one hand, and the adjacent illumination source and the field stop on the other.

[0162] For example, in FIG. 6D, the centres of the first illumination source 623a and second illumination source 623b are separated by a distance Sab, the centres of the first illumination source 623a and third illumination source 623c are separated by a distance Sca, and the centres of the second illumination source 623b and fourth illumination source 623d are separated by a distance Sbd- Scais less than Sab. Sbdis less than Sab.

[0163] The positioning of the illumination sources applied in the Kohler illumination scheme closer to the field stop than those applied in the semi-critical scheme permits the use of shorter focal length collector lenses for those illumination sources in the Kohler illumination scheme and / or allows the field stop to be placed closer to the focal plane of those collector lenses. This enables these illumination sources to be implemented in a scheme which is closer to true Kohler illumination (in which the front focal plane of the collector lens matches the position of the field stop).

[0164] A degree of freedom in the design of the collector lens for semi-critical illumination is the magnification of the collector lens which is given by the formula M = di Zdo, wherein 1 / f = l / do+ 1 / di. As illustrated in FIG. 9, dois the distance of the object (in this case the illumination source) from the collectorIOO-I789OIPC lens plane 933, and di is the distance of the image (in this case the image plane 934i) from the collector lens principal plane 933. And f is the focal length of the collector lens. Adjusting the magnification as well as the image plane 943i position can allow an appropriate balance to be struck to allow the field stop aperture to be fully filled, while at the same time improving non-uniformity of the field stop illumination and maintaining high power efficiency. In some systems, such as in microscopes, size of field stop is dictated by the magnification of the objective. The field stop size determines magnification of collector lens, which is determined by image and object distance from collector lens. Therefore, for semi critical illumination, the effect of moving the collector lens further away from the field stop is to allow the field stop to be fully illuminated while also meeting the power efficiency and uniformity requirements.

[0165] FIG. 6E depicts an example of a multi-source illumination device 620E. The multi-source illumination device 620E is similar to the multi-source illumination device 620D in FIG. 6D and includes a first illumination source 623a having a first collector lens 625a, first excitation filter 626a, and a first wavelength-dependent reflector 628a; a second illumination source 623b having a second collector lens 625b, second excitation filter 626b, and a second wavelength-dependent reflector 628b; a third illumination source 623c having a third collector lens 625c, third excitation filter 626c, and a third wavelength-dependent reflector 628c; a fourth illumination source 623d having a fourth collector lens 625d, fourth excitation filter 626d, and a fourth wavelength-dependent reflector 628d; and a fifth illumination source 623e having a fifth collector lens 625e, fifth excitation filter 626e, and a fifth wavelength-dependent reflector 628e. As shown in FIG. 6E, the multi-source illumination devices 620E is arranged in a linear combiner configuration where the optical path of each illumination source shares the same exit path via the field stop 627 (and an output aperture to the extent the illumination device 620E is packaged into a modular housing). In some embodiments, wavelength-dependent reflectors may have varying performance based on the angle at which illumination light is incident on the reflective surface (e.g., performance at varying angles may depend on the coating). As shown in FIG. 6E, the angle between the excitation illumination beam for each illumination source 623a-623e and the optical axis of the field stop 627 is, e.g., 30 degrees (i.e., the angle, a, of wavelength-dependent reflectors is 60 degrees relative to the optical axis of the field stop 627).

[0166] The multi-source illumination device 620E further includes a monolithic heat sink 624 that is thermally coupled to all illumination sources 623a-623e to thereby draw away and diffuse heat generated therefrom. In some embodiments, the heat sink 624 is made from a thermally conductive material, such as a metal (e.g., aluminum, copper, silver, etc.), or other suitable thermally conductive material. In some embodiments, the heat sink 624 includes fins (e.g., skived fins) extending therefrom to improve convective heat transfer to the surrounding air. In some embodiments, the heat sink 624 includes one or more fans configured to force air across the heatsink (e.g., through the fins). In some embodiments, one or more fans are positioned external to the multi-source illumination device 620E and configured to force air over the heat sink 624. In some embodiments, the heat sink 624 may include a vapor chamber. In some embodiments, the vapor chamber includes one or more heat pipes extending vertically from an upper surface of the vaporIOO-I789OIPC chamber and through a radiator (e.g. , a plurality of thin plates configured to dissipate heat from the one or more heat pipes to ambient air, which may be forced air via the one or more fans).

[0167] FIG. 6F depicts an example of a multi-source illumination device 620F. The multi-source illumination device 620F is similar to the multi-source illumination device 620D in FIG. 6D and includes a first illumination source 623a having a first collector lens 625a, first excitation filter 626a, and a first wavelength-dependent reflector 628a; a second illumination source 623b having a second collector lens 625b, second excitation filter 626b, and a second wavelength-dependent reflector 628b; a third illumination source 623c having a third collector lens 625c, third excitation filter 626c, and a third wavelength-dependent reflector 628c; a fourth illumination source 623d having a fourth collector lens 625d, fourth excitation filter 626d, and a fourth wavelength-dependent reflector 628d; and a fifth illumination source 623e having a fifth collector lens 625e, fifth excitation filter 626e, and a fifth wavelength-dependent reflector 628e. As shown in FIG. 6F, the multi-source illumination devices 620F is arranged in a linear combiner configuration where the optical path of each illumination source shares the same exit path via the field stop 627 (and an output aperture to the extent the illumination device 620F is packaged into a modular housing). In some embodiments, wavelength-dependent reflectors may have varying performance based on the angle at which illumination light is incident on the reflective surface (e.g., performance at varying angles may depend on the coating). As shown in FIG. 6F, the angle between the excitation illumination beam for each illumination source 623a-623e and the optical axis of the field stop 627 is, e.g., 90 degrees (i.e., the angle, a, of wavelength-dependent reflectors is 45 degrees relative to the optical axis of the field stop 627).

[0168] The multi-source illumination device 620F further includes a monolithic heat sink 624 that is thermally coupled to all illumination sources 623a-623e to thereby draw away and diffuse heat generated therefrom. In some embodiments, the heat sink 624 is made from a thermally conductive material, such as a metal (e.g., aluminum, copper, silver, etc.), or other suitable thermally conductive material. In some embodiments, the heat sink 624 includes fins (e.g., skived fins) extending therefrom to improve convective heat transfer to the surrounding air. In some embodiments, the heat sink 624 includes one or more fans configured to force air across the heatsink (e.g., through the fins). In some embodiments, one or more fans are positioned external to the multi-source illumination device 620F and configured to force air over the heat sink 624. In some embodiments, the heat sink 624 may include a vapor chamber. In some embodiments, the vapor chamber includes one or more heat pipes extending vertically from an upper surface of the vapor chamber and through a radiator (e.g., a plurality of thin plates configured to dissipate heat from the one or more heat pipes to ambient air, which may be forced air via the one or more fans).

[0169] FIG. 6G depicts an example of a multi-source illumination device 620G. The multi-source illumination device 620G includes a first illumination source 623a having a first collector lens 625a, first excitation filter 626a, and a first wavelength-dependent reflector 628a; a second illumination source 623b having a second collector lens 625b, second excitation filter 626b, and a second wavelength-dependent reflector 628b; a third illumination source 623c having a third collector lens 625c, third excitation filter 626c, and a third wavelength-dependent reflector 628c; a fourth illumination source 623d having a fourth collectorIOO-I789OIPC lens 625d, fourth excitation filter 626d, and a fourth wavelength-dependent reflector 628d; and a fifth illumination source 623e having a fifth collector lens 625e, fifth excitation filter 626e, and a fifth wavelength-dependent reflector 628e. As shown in FIG. 6G, the multi-source illumination devices 620G is arranged in a linear combiner configuration where the optical path of each illumination source shares the same exit path via the field stop 627 (and an output aperture to the extent the illumination device 620G is packaged into a modular housing). In some embodiments, wavelength-dependent reflectors may have varying performance based on the angle at which illumination light is incident on the reflective surface (e.g., performance at varying angles may depend on the coating). As shown in FIG. 6G, the angle between the excitation illumination beam for each illumination source 623a-623e and the optical axis of the field stop 627 is, e.g., 30 degrees (i.e., the angle, a, of wavelength-dependent reflectors is 60 degrees relative to the optical axis of the field stop 627).

[0170] The multi-source illumination device 620G further includes a first heat sink 624a that is thermally coupled to the first illumination source 623a, third illumination source 623c, and fifth illumination source 623e to thereby draw away and diffuse heat generated therefrom. The illumination device 620G further includes a second heat sink 624b separate from the first heat sink 624a that is thermally coupled to the second illumination source 623b and the fourth illumination source 623d to thereby draw away and diffuse heat generated therefrom. As shown in FIG. 6G, the first heat sink 624a (as well as first illumination source 623a, third illumination source 623c, and fifth illumination source 623e) is positioned on one side of the optical axis of the field stop 627 while the second heat sink 624b (as well as second illumination source 623b and the fourth illumination source 623d) is positioned on an opposite side of the optical axis of the field stop 627. In some embodiments, separating the heatsink into two or more separate heatsinks allows for improved heat transfer away from individual heat sources (and may allow for better air flow to the extent fins and / or fans are used). In some embodiments, the heat sinks 624a, 624b are made from a thermally conductive material, such as a metal (e.g., aluminum, copper, silver, etc.), or other suitable thermally conductive material. In some embodiments, the heat sinks 624a, 624b include fins (e.g., skived fins) extending therefrom to improve convective heat transfer to the surrounding air. In some embodiments, the heat sinks 624a, 624b include one or more fans configured to force air across the heatsink (e.g., through the fins). In some embodiments, one or more fans are positioned external to the multi-source illumination device 620G and configured to force air over the heat sinks. In some embodiments, the heat sinks 624a, 624b may include a vapor chamber. In some embodiments, the vapor chamber includes one or more heat pipes extending vertically from an upper surface of the vapor chamber and through a radiator (e.g., a plurality of thin plates configured to dissipate heat from the one or more heat pipes to ambient air, which may be forced air via the one or more fans).

[0171] FIG. 6H depicts an example of a multi-source illumination device 620H. The multi-source illumination device 620H includes a first illumination source 623a having a first collector lens 625a, first excitation filter 626a, and a first wavelength-dependent reflector 628a; a second illumination source 623b having a second collector lens 625b, second excitation filter 626b, and a second wavelength-dependentIOO-I789OIPC reflector 628b; a third illumination source 623c having a third collector lens 625c, third excitation filter 626c, and a third wavelength-dependent reflector 628c; a fourth illumination source 623d having a fourth collector lens 625d, fourth excitation filter 626d, and a fourth wavelength-dependent reflector 628d; and a fifth illumination source 623e having a fifth collector lens 625e, fifth excitation filter 626e, and a fifth wavelength-dependent reflector 628e. As shown in FIG. 6H, the multi-source illumination devices 620H is arranged in a linear combiner configuration where the optical path of each illumination source shares the same exit path via the field stop 627 (and an output aperture to the extent the illumination device 620G is packaged into a modular housing). In some embodiments, wavelength-dependent reflectors may have varying performance based on the angle at which illumination light is incident on the reflective surface (e.g. , performance at varying angles may depend on the coating). As shown in FIG. 6H, the angle between the excitation illumination beam for each illumination source 623a-623e and the optical axis of the field stop 627 is, e.g., 30 degrees (i.e., the angle, a, of wavelength-dependent reflectors is 60 degrees relative to the optical axis of the field stop 627).

[0172] As shown in FIG. 6H, the third illumination source 623c (with third collector lens 625c and third excitation filter 626c) is generally reflected about the horizontal axis and the corresponding wavelengthdependent reflector 628c is moved away from (out of) the optical axis of the field stop 627. For example, the reflective surface of the wavelength-dependent reflector 628c may be generally perpendicular to the optical axis of the field stop 627. The wavelength-dependent reflector 628c is positioned such that the beam from the second illumination source 623b passes therethrough before being reflected by the wavelengthdependent reflector 628b. This particular arrangement shown in FIG. 6H may allow for a more compact packing of illumination sources 623a-623e in the multi-source illumination device 620H. Optionally, the wavelength-dependent reflector 628e may be removed and the fifth illumination source 623e, fifth collector lens 625e, and fifth excitation filter 626e may be positioned in direct alignment (along and parallel to) the optical axis of the field stop 627, further reducing required components and cost of goods to manufacture the multi-source illumination device 620H. FIGS. 6I-6J illustrate examples where the fifth illumination source 623e, fifth collector lens 625e, and fifth excitation filter 626e are positioned in direct alignment (along and parallel to) the optical axis of the field stop 627.

[0173] In some embodiments, the multi-source illumination device 620H further includes standalone heat sinks (not shown) for each illumination source 623a-623e to thereby draw away and diffuse heat generated therefrom. In some embodiments, the heat sinks are made from a thermally conductive material, such as a metal (e.g., aluminum, copper, silver, etc.), or other suitable thermally conductive material. In some embodiments, the heat sinks include fins (e.g., skived fins) extending therefrom to improve convective heat transfer to the surrounding air. In some embodiments, the heat sinks include one or more fans configured to force air across the heatsink (e.g., through the fins). In some embodiments, one or more fans are positioned external to the multi-source illumination device 620H and configured to force air over the heat sinks. In some embodiments, each of the heat sinks may include a vapor chamber. In some embodiments, the vapor chamber includes one or more heat pipes extending vertically from an upper surface of the vapor chamberIOO-I789OIPC and through a radiator (e.g. , a plurality of thin plates configured to dissipate heat from the one or more heat pipes to ambient air, which may be forced air via the one or more fans).

[0174] FIG. 61 depicts an example of a multi-source illumination device 6201. The multi-source illumination device 6201 is similar to the multi-source illumination device 620H in FIG. 6H and includes a first illumination source 623a having a first collector lens 625a, first excitation filter 626a, and a first wavelength-dependent reflector 628a; a second illumination source 623b having a second collector lens 625b, second excitation filter 626b, and a second wavelength-dependent reflector 628b; a third illumination source 623c having a third collector lens 625c, third excitation filter 626c, and a third wavelength-dependent reflector 628c; a fourth illumination source 623d having a fourth collector lens 625d, fourth excitation filter 626d, and a fourth wavelength-dependent reflector 628d; and a fifth illumination source 623e having a fifth collector lens 625e, fifth excitation filter 626e, and a fifth wavelength-dependent reflector 628e. As shown in FIG. 61, the multi-source illumination devices 6201 is arranged in a linear combiner configuration where the optical path of each illumination source shares the same exit path via the field stop 627 (and an output aperture to the extent the illumination device 6201 is packaged into a modular housing). In some embodiments, wavelength-dependent reflectors may have varying performance based on the angle at which illumination light is incident on the reflective surface (e.g., performance at varying angles may depend on the coating). As shown in FIG. 61, the angle between the excitation illumination beam for each illumination source 623a-623e and the optical axis of the field stop 627 is, e.g., 30 degrees (i.e., the angle, a, of wavelength-dependent reflectors is 60 degrees relative to the optical axis of the field stop 627).

[0175] As shown in FIG. 61, the fifth illumination source 623e, fifth collector lens 625e, and fifth excitation filter 626e is positioned in direct alignment (along and parallel to) the optical axis of the field stop 627, further reducing required components and cost of goods to manufacture the multi-source illumination device 6201.

[0176] In some embodiments, the multi-source illumination device 6201 further includes standalone heat sinks (not shown) for each illumination source 623a-623e to thereby draw away and diffuse heat generated therefrom. In some embodiments, the heat sinks are made from a thermally conductive material, such as a metal (e.g., aluminum, copper, silver, etc.), or other suitable thermally conductive material. In some embodiments, the heat sinks include fins (e.g., skived fins) extending therefrom to improve convective heat transfer to the surrounding air. In some embodiments, the heat sinks include one or more fans configured to force air across the heatsink (e.g., through the fins). In some embodiments, one or more fans are positioned external to the multi-source illumination device 6201 and configured to force air over the heat sinks. In some embodiments, each of the heat sinks may include a vapor chamber. In some embodiments, the vapor chamber includes one or more heat pipes extending vertically from an upper surface of the vapor chamber and through a radiator (e.g., a plurality of thin plates configured to dissipate heat from the one or more heat pipes to ambient air, which may be forced air via the one or more fans).IOO-I789OIPC

[0177] FIG. 6J depicts an example of a multi-source illumination device 620J. The multi-source illumination device 620J is similar to the multi-source illumination devices 620H, 6201 in FIGS. 6H-6I and includes a first illumination source 623a having a first collector lens 625a, first excitation filter 626a, and a first wavelength-dependent reflector 628a; a second illumination source 623b having a second collector lens 625b, second excitation filter 626b, and a second wavelength-dependent reflector 628b; a third illumination source 623c having a third collector lens 625c, third excitation filter 626c, and a third wavelength-dependent reflector 628c; a fourth illumination source 623d having a fourth collector lens 625d, fourth excitation filter 626d, and a fourth wavelength-dependent reflector 628d; and a fifth illumination source 623e having a fifth collector lens 625e, fifth excitation filter 626e, and a fifth wavelength-dependent reflector 628e. As shown in FIG. 6J, the multi-source illumination devices 620J is arranged in a linear combiner configuration where the optical path of each illumination source shares the same exit path via the field stop 627 (and an output aperture to the extent the illumination device 620J is packaged into a modular housing). In some embodiments, wavelength-dependent reflectors may have varying performance based on the angle at which illumination light is incident on the reflective surface (e.g., performance at varying angles may depend on the coating). As shown in FIG. 6J, the angle between the excitation illumination beam for each illumination source 623a-623e and the optical axis of the field stop 627 is, e.g., 30 degrees (i.e., the angle, a, of wavelength-dependent reflectors is 60 degrees relative to the optical axis of the field stop 627).

[0178] As shown in FIG. 6J, the third illumination source 623c (with third collector lens 625c and third excitation filter 626c) is generally reflected about the horizontal axis and the corresponding wavelengthdependent reflector 628c is moved away from (out of) the optical axis of the field stop 627. For example, the reflective surface of the wavelength-dependent reflector 628c may be generally perpendicular to the optical axis of the field stop 627. The wavelength-dependent reflector 628c is positioned such that the beam from the second illumination source 623b passes therethrough before being reflected by the wavelengthdependent reflector 628b. Additionally, the first illumination source 623a (with first collector lens 625a and first excitation filter 626a) is generally reflected about the horizontal axis and the corresponding wavelengthdependent reflector 628a is moved away from (out of) the optical axis of the field stop 627. For example, the reflective surface of the wavelength-dependent reflector 628a may be generally perpendicular to the optical axis of the field stop 627. The wavelength-dependent reflector 628a is positioned such that the beam from the fourth illumination source 623d passes therethrough before being reflected by the wavelengthdependent reflector 628d. This particular arrangement shown in FIG. 6J may allow for a more compact packing of illumination sources 623a-623e in the multi-source illumination device 620J. Additionally, the fifth illumination source 623e, fifth collector lens 625e, and fifth excitation filter 626e are positioned in direct alignment (along and parallel to) the optical axis of the field stop 627, further reducing required components and cost of goods to manufacture the multi-source illumination device 620J.

[0179] FIG. 6K depicts an example of a multi-source illumination device 620K. The multi-source illumination device 620K is substantially similar to the multi-source illumination device 620F in FIG. 6F and includes a first illumination source 623a having a first collector lens 625a, first excitation filter 626a,IOO-I789OIPC and a first wavelength-dependent reflector 628a; a second illumination source 623b having a second collector lens 625b, second excitation filter 626b, and a second wavelength-dependent reflector 628b; a third illumination source 623c having a third collector lens 625c, third excitation filter 626c, and a third wavelength-dependent reflector 628c; a fourth illumination source 623d having a fourth collector lens 625d, fourth excitation filter 626d, and a fourth wavelength-dependent reflector 628d; and a fifth illumination source 623e having a fifth collector lens 625e, fifth excitation filter 626e, and a fifth wavelength-dependent reflector 628e. In some embodiments, a field lens 629 and / or a condenser lens is positioned after the field stop 627. As shown in FIG. 6K, the multi-source illumination device 620K has been packaged in a modular housing 650 with an output port 652 after the field stop 627. While FIG. 6K shows the general configuration of the multi-source illumination device 620F, any of the multi-source illumination device 620C-620J configurations can be packaged into a similar modular housing 650 as shown in FIG. 6K. In some embodiments, at least a portion of the heatsink 624 (or heatsinks where more than one heat sink is used, for example, where each illumination source has its own standalone heat sink) extends out of the housing 650 (e.g., out of the top of the housing 650). For example, each heat sink 624 may include a skived fin portion that extends out of the housing 650 and is configured to dissipate heat generated by the illumination sources to the ambient air external to the housing 650. In another example, each heat sink 624 may include a radiator portion that extends out of the housing 650 and is configured to dissipate heat generated by the illumination sources to the ambient air external to the housing 650.

[0180] FIG. 6L depicts an example of a multi-source illumination device 620L. The multi-source illumination device 620L is substantially similar to the multi-source illumination device 620F in FIG. 6F and includes a first illumination source 623a having a first collector lens 625a, first excitation filter 626a, and a first wavelength-dependent reflector 628a; a second illumination source 623b having a second collector lens 625b, second excitation filter 626b, and a second wavelength-dependent reflector 628b; a third illumination source 623c having a third collector lens 625c, third excitation filter 626c, and a third wavelength-dependent reflector 628c; a fourth illumination source 623d having a fourth collector lens 625d, fourth excitation filter 626d, and a fourth wavelength-dependent reflector 628d; and a fifth illumination source 623e having a fifth collector lens 625e, fifth excitation filter 626e, and a fifth wavelength-dependent reflector 628e. As shown in FIG. 6L, the multi-source illumination device 620L has been packaged in a modular housing 650 with an output port 652 after the field stop 627 that is perpendicular to the optical path defined by the wavelength-dependent reflectors 628a-628e. To allow for the output port 652 to be in a perpendicular configuration, the third wavelength-dependent reflector 628c is a short pass dichroic, allowing for longer wavelengths (e.g., blue, green, yellow, and red) to be reflected and shorter wavelengths (e.g., nUV) to be transmitted. In some embodiments, a condenser lens and / or a field lens is positioned after the field stop 627. Any of the multi-source illumination device 620C-620J configurations can be packaged into a similar modular housing 650 as shown in FIG. 6L. In some embodiments, at least a portion of the heatsink 624 (or heatsinks where more than one heat sink is used, for example, where each illumination source has its own standalone heat sink) extends out of the housing 650 (e.g., out of the top of the housing 650). ForIOO-I789OIPC example, each heat sink 624 may include a skived fin portion that extends out of the housing 650 and is configured to dissipate heat generated by the illumination sources to the ambient air external to the housing 650. In another example, each heat sink 624 may include a radiator portion that extends out of the housing 650 and is configured to dissipate heat generated by the illumination sources to the ambient air external to the housing 650.

[0181] FIGs 7, 8 and 9 each show a cross-sectional schematic of an optics module (in e.g. a microscope configuration) including an illumination device operating in accordance with a particular illumination scheme. Each optics module 700, 800, 900 includes an illumination device 720, 820, 920 including a single illumination source 723, 823, 923 and collector lens 725, 825, 925, a field stop 727, 827, 927 and a field lens 729, 829, 929. The optics modules 700, 800, 900 also include an objective 702, 802, 902. The operation and structure of these components is the same as described with reference to the corresponding components in FIGs 6A, 6B, 6C and / or 6D unless stated otherwise. The microscopes can also include any, all or none of the other components having structures and functions described with reference to FIGs 6A, 6B, 6C and / or 6D (for example image sensors, imaging dichroics, tube lenses, etc.), however these are omitted from FIGs 7, 8 and 9 so as not to obfuscate the different illumination schemes shown.

[0182] In FIGs 7, 8 and 9, each illumination scheme is illustrated by an associated ray tracing through the optical components of the illumination device and other microscope components. In general terms, the illumination scheme is determined by the relative arrangement of the illumination source, collector lens and field stop. In this sense, since the position of the collector lens relative to the illumination source and field stop and its optical power is determinative, it can be said that the collector lens is arranged to determine the illumination scheme. Depending on the illumination scheme, the collector lens arranged to: a. substantially collimate the illumination light in the space between the collector lens and the field lens (Kohler illumination), or b. form an image of the illumination source at the field stop (critical illumination), or c. form an image of the illumination source between the collector lens and the field lens before or after the field stop (semi-critical illumination).

[0183] The effect of the configuration of the components of the illumination devices 720, 820, 920 on the image of the illumination source and the image of the field stop at different virtual planes throughout the optical system is also shown in each of FIGs 7, 8 and 9 for illustration purposes. Therefore, the description of FIGs 7, 8 and 9 includes details of the spacing between the components of the optics module and the effect this has on the ray paths and image formation throughout optical space in the optics module.

[0184] Embodiments are not restricted to an illumination device for microscope applications and it is envisaged that the illumination devices 720, 820, 920 illustrated in FIGS 7, 8 and 9 can be employed in other illumination applications. Thus, the objective and sample planes in FIGs 7, 8 and 9 can be omitted and / or replaced by other optical components depending on the application. In FIGs 7, 8 and 9, the optical axis hasIOO-I789OIPC no bends or folds. However the present disclosure is not limited to this arrangement and reflectors, combiners or splitters can be provided in any practicable space to fold the optical axis through any practicable angle in order to provide a more compact and / or more useful illumination device or microscope.

[0185] Where the term “lens” is used throughout the present disclosure, this can be replaced with any other type of optic having the same function. Examples include diffraction plates (such as zone plates or spatial light modulators) and reflectors (such as mirrors).

[0186] Although the illumination source, collector lens, field stop and field lens are shown to have a one- to-one relationship (in particular one field lens and one field stop per illuminator source / collector lens), the present disclosure is not limited thereto. For example, as shown in FIGs 6B, 6C and 6D, in an illumination device employing more than one illumination source (and associated collector lens), only one field stop and one field lens may be used. The sharing of a field stop and field lens between multiple illumination source / collector lens combinations (i.e. each combination including one illumination source and one collector lens) provides a more compact and more cost effective system. However, the present disclosure is not limited thereto. For example, each illumination source / collector lens combination can be arranged to illuminate a separate field stop and a combiner (such as the wavelength-dependent reflector plate 628b described with reference to FIGs 6B and 6C) or similar component can be arranged to allow a single field lens to image each field stop. Alternatively, each illumination source / collector lens combination is arranged to illuminate its own field stop and each field stop is imaged by a separate field lens.

[0187] Therefore, in general terms, it may be understood that devices according to the present disclosure can take any of the following three forms, each comprising a first illumination source arranged to output first illumination light; a second illumination source arranged to output second illumination light; a first collector optic (e.g. lens) arranged to collect the first illumination light; and a second collector optic (e.g. lens) arranged to collect the second illumination light:• Form 1 (single field stop, single field optic): The device further comprising: a field stop arranged to provide an aperture for light from the first collector optic and second collector optic; and a field optic arranged to image the field stop. In this form, the first collector optic is arranged to substantially collimate the first illumination light, and the second collector optic is arranged to form an image of the second illumination source between the second collector optic and the field optic, before or after the field stop so that the image of the second illumination source is defocussed at the field stop.• Form 2 (at least two field stops, a corresponding number of field optics): The device further comprising: a first field stop for light from the first collector optic; a second field stop for light from the second collector optic, a first field optic arranged to image the first field stop; a second field optic arranged to image the second field stop. In this form, the first collector optic is arranged to substantially collimate the first illumination light, and the second collector optic is arranged to form an image of the second illumination source between the second collector optic and the second fieldIOO-I789OIPC optic, before or after the second field stop so that the image of the second illumination source is defocussed at the second field stop.• Form 3 (at least two field stops, single field optic): The device further comprising: a first field stop for light from the first collector optic; a second field stop for light from the second collector optic; a field optic arranged to image the first field stop and second field stop. In this form, the first collector optic is arranged to substantially collimate the first illumination light, and the second collector optic is arranged to form an image of the second illumination source between the second collector optic and the field optic, before or after the second field stop so that the image of the second illumination source is defocussed at the second field stop.

[0188] Although the illumination devices 720, 820, 920 are described with only a single illumination source per objective, it is to be understood that the schemes illustrated can be applied to any of the illumination sources described with reference to FIGs 6B, 6C or 6D and any variants thereof, so that different illumination schemes are applied to different illumination sources within the same illumination device. That is, where any illumination source / collector lens combination is described, this can be replaced by the illumination source and collector lens combinations (including their relative arrangement to each other and to the field stop) in FIGs 7, 8 or 9. For example, for a first illumination source having a first luminance uniformity, a Kohler illumination scheme is applied in accordance with FIG. 7. For a second illumination source in the same illumination device (i.e. for illuminating the same sample through the same objective) a semi-critical illumination scheme is applied in accordance with FIG. 9. In some embodiments, the first illumination source has a lower luminance uniformity than the second illumination source.

[0189] In each of FIGs 7, 8 and 9, an emission plane 731, 831, 931 is located at emitting surface of the illumination source 723, 823, 923. A collector lens front focal plane 732, 832, 932 is formed a distance fCL behind the collector lens, wherein fCL is the front focal length of the collector lens 725, 825, 925. A collector lens plane 733, 833, 933 is located at the optical centre of the collector lens 725, 825, 925. A field stop plane 734, 834, 934 is located at the plane of the aperture of the field stop 727, 827, 927. A field lens plane 735,835, 935 is located at the optical centre of the field lens 729, 829, 929. A field lens back focal plane 736,836, 936 is formed a distance fFL after the field lens 729, 829, 929, wherein fFL is (also) the back focal length of the field lens 729, 829, 929. The microscope 700, 800, 900 is set up so that the front focal plane of the objective 702, 802, 902 is coincident with the field lens back focal plane 736, 836, 936, which lies a distance fMO behind the objective 702, 802, 902, wherein fMO is the front focal length of the objective 702, 802, 902. Therefore, an objective plane 737, 837, 937 located at the optical centre of the objective 702, 802, 902 lies at a distance of fFL+fMO from the field lens 729, 829, 929. An objective back focal plane 738, 838, 938 formed a distance fMO behind the objective 702, 802, 902 wherein fMO is (also) the back focal length of the objective 702, 802, 902. The terms 'in front’ and ‘behind’ refer to the leftward and rightward directions, respectively, in FIGs 7, 8 and 9. Each virtual plane described lies perpendicular to the optical axis.IOO-I789OIPC

[0190] In each of FIGs 7, 8 and 9, a schematic of images of the illumination source and field stop is shown at one or more of the virtual planes, the illumination source intensity profile 751, 851, 951 is shown at the emission plane 731, 831, 931, and the field stop intensity profile 755, 855, 955 is shown at the field stop plane 734, 834, 934. A sample plane illumination profile 757, 857, 957 is shown at the objective focal plane 738, 838, 938. A first image of the illumination source 752, 852, 952, is shown at a position in the optical train which depends on the illumination scheme. The illumination schemes, and hence the position of the first image of the illumination source 752, 852, 952, is different for each of FIGs 7, 8 and 9. A second image of the illumination source 853 is shown at the sample plane 838 in FIG. 8 only.

[0191] FIG. 7 illustrates an illumination device 720 operating in accordance with a Kohler illumination scheme. In this case, the emission plane 731 is coincident (co-planar) with the collector lens front focal plane 732. Thus uncollimated illumination light 741 from the illumination source 723 is collected by the collector lens 725. The collector lens 725 projects collimated illumination light 742 onto the field stop 727, resulting in a substantially uniform field stop intensity profile 755 within the aperture of the field stop. As the field lens 729 is positioned at a distance fFL from the field stop 727 (or the field lens plane 735 is positioned at a distance fFL from the field stop plane 734), the field lens 729 produces collimated field stop light 745 in the infinity space between the field lens 729 and the objective 702, resulting in the field stop image 757, rather than an image of the illumination source, appearing at the sample plane 738. Since the sample plane intensity profile 757 is representative of the field stop intensity profile 755, the sample plane has substantially uniform illumination in the shape of the aperture 754 of the field stop. Therefore, even if the illumination source intensity profile is non-uniform, the sample is substantially uniformly illuminated across the illuminated area. As shown illustrated in the field stop intensity profile 755 in FIG. 7, the collimated illumination light beam cross-section 742x at the field stop 727 is larger than the aperture 754 of the field stop. Thus, the collimated illumination light 742 fills the field stop. This causes the field stop intensity profile 755 across the aperture 754 of the field stop to be substantially uniform in intensity from edge to edge (or from comer to corner, or across the diameter if the aperture 754 is circular). If the collimated illumination light beam cross-section 742x at the field stop 727 is smaller than the aperture 754 of the field stop, the aperture 754 will not be uniformly illuminated from edge to edge.

[0192] FIG. 8 shows a cross-sectional schematic of a microscope including an illumination device operating in accordance with a critical illumination scheme. In this case, the emission plane 831 is set further from the collector lens 825 than the collector lens front focal plane 832. Uncollimated illumination light 841 from the illumination source 823 is collected by the collector lens 825. Unlike in Kohler illumination, field stop plane 834 (and hence the field stop 827) in FIG. 8 is not positioned at the front focal plane of the collector lens, but rather is positioned at the focal position of the focussed illumination light 843. The collector lens 825 focusses illumination light 842 precisely (exactly) at the field stop 827, resulting in the first image of the illumination source 852 appearing in the aperture of the field stop at the field stop plane 834. As in FIG. 7, the field lens 829 in FIG. 8 is positioned at a distance fFL from the field stop 827. In other words, the field lens plane 835 is positioned at a distance fFL from the field stop plane 834. The fieldIOO-I789OIPC lens 829 produces a collimated illumination source light 845 in the infinity space between the field lens 829 and the objective 802, resulting in an image of the illumination source at the sample plane 838. The field stop includes the image of the illumination source and the image of the field stop appears at the sample plane, thereby producing an image of the illumination source at the sample plane 838. That is, since the sample plane intensity profile 857 is representative of the field stop intensity profile 855, the sample plane is illuminated with an intensity profile matching that at the field stop (adjusted for the magnification of the system). Therefore, if the illumination source intensity profile is non-uniform, the sample is not uniformly illuminated across the illuminated area. As shown illustrated in the field stop intensity profile 855 in FIG. 8, even if the image of the illumination source at the field stop 827 is larger than the aperture 854 of the field stop so that the field stop aperture 854 is filled by the image, the field stop intensity profile 855 across the aperture 854 of the field stop will be non-uniform in intensity (across the aperture) if the illumination source luminance is non-uniform.

[0193] FIG. 9 shows a cross-sectional schematic of a microscope including an illumination device operating in accordance with a semi-critical illumination scheme. Like in FIG. 8, in FIG. 9 the emission plane 931 is set further from the collector lens 925 than the collector lens front focal plane 932. Thus uncollimated illumination light 941 from the illumination source 923 is collected by the collector lens 925. Unlike in Kohler illumination, field stop plane 934 (and hence the field stop 927) in FIG. 9 is not positioned at the back focal plane of the collector lens. In addition, unlike in critical illumination, the field stop 927 in FIG. 9 is not positioned at the focal position of the focussed illumination light 942. Instead, the collector lens 925 focusses illumination light 942 between the field stop 927 and the field lens 929, resulting in the first image of the illumination source 952 appearing at an image plane 934i between the field stop 927 (or field stop plane 934) and the field lens 929 (or field lens plane 935). This results in a defocussed image 952d of the field stop appearing at the field stop (e.g. centred at the field stop aperture 954 at the field stop plane 934). As in FIGs 7 and 8, the field lens 929 in FIG. 9 is positioned at a distance fFL from the field stop 927. Therefore, the field lens 929 images the field stop 927. The objective 902 is arranged relative to the field lens 929 so that the image of the field stop appears at the sample plane 938. That is, since the sample plane intensity profile 957 is representative of the field stop intensity profile 955, the sample plane is illuminated with an intensity profile matching that at the field stop (adjusted for the magnification of the system). Since the image of the illumination source is defocussed at the field stop, even if the illumination source intensity profile is non-uniform, the sample can still be have a high degree of uniformity of illuminance across the illuminated area, at least higher than that achievable with a critical illumination scheme. At the same time, the average illuminance intensity at the sample plane 938 can be higher than for the same illumination source employed in a Kohler illumination scheme. As shown in the field stop intensity profile 955 in FIG. 9, if the image of the illumination source at the field stop 927 is larger than the aperture 954 of the field stop, so that the field stop aperture 954 is filled by the defocussed image 952d, the field stop intensity profile 955 across the aperture 954 of the field stop will have a relatively high uniformity (across the aperture, edge to edge or comer to corner or along the diameter) even if the illumination source luminance is non-uniform.IOO-I789OIPC

[0194] The function of semi-critical illumination within the context of the present disclosure is to defocus the image of the illumination source at the field stop so as to increase uniformity of the intensity profile at the field stop compared with critical illumination while also increasing the average intensity at the field stop compared with Kohler illumination. In the example of microscope applications, this can translate to an improved balance between uniformity and average intensity level at the sample plane (in that both can be achieved to desired levels).

[0195] Although the example of semi-critical illumination depicted in FIG. 9 shows that the first image of the illumination source is focussed between the field stop and the field lens, other image positions are possible while still meeting the intended definition of semi-critical illumination and fulfilling the intended function of this illumination scheme as described herein. For example, in semi-critical illumination such as in the example of FIG. 9, the first image of the illumination source can be formed anywhere between the collector lens and the field lens, before or after the field stop. The first image of the illumination source is formed, for example, at least one sixteenth, at least one eighth or at least one quarter of the focal length of the field optic away from the field stop.

[0196] The skilled person can use standard optical design and performance assessment principles such as geometric ray tracing and / or programs such as ZEMAX® optical design program (ZEMAX Development Corporation) to determine an appropriate spacing, optical power, clear aperture and other optical parameters (of e.g. the illumination source, collector lens, field stop and field lens) for achieving / the illumination schemes set out in the present disclosure. The same techniques can be used to assess other characteristics of the devices (e.g. microscope), such as performance characteristics including the uniformity of luminance of the illumination sources and the uniformity and average optical field intensity through the optical train e.g. from each respective illumination source to the sample. Alternatively, image sensors, such as CCD cameras CMOS detectors and the like can be used to assess and / or verify the performance of a real optical setup including illumination devices according to the present disclosure.

[0197] As described herein, embodiments include a device configured so that a first illumination source provides Kohler illumination and a second illumination source provides semi-critical illumination. In some embodiments, the luminance uniformity of each illumination source determines whether a Kohler illumination scheme or semi-critical illumination scheme is employed with that illumination source. In an example, the first illumination source may have a luminance uniformity which is lower than that of the second illumination source. Put another way, the first illumination source may have a luminance nonuniformity which is greater than that of the second illumination source. Optionally, the first illumination source also has a higher average output intensity than that of the second illumination source. The device is configured to provide a Kohler illumination scheme using the first illumination source and a semi-critical illumination scheme using the second illumination source.

[0198] Luminance uniformity LU of the illumination sources can be defined as (Lmin / Lmax). Luminance non-uniformity LNU of the illumination sources can be defined as 1 - (Lmin / Lmax), wherein Lmin is theIOO-I789OIPC minimum luminance across a diagonal (or across the longest dimension) of the light-emitting surface of the illumination source and Lmax is the maximum luminance across the diagonal (or across the longest dimension) of the light-emitting surface. In embodiments, luminance non-uniformities for the illumination source adopted in the semi-critical illumination scheme are: equal to or greater than 0.20; greater than 0.22, greater than 0.24, greater than 0.26, or greater than 0.28.

[0199] In embodiments, the extent to which the image at the field stop in semi-critical illumination is defocussed can determine the extent to which the luminance non-uniformity of the illumination source is translated to an irradiance non-uniformity INU of the field stop by the illumination light. Irradiance uniformity IU can be defined as Emin / Emax. Irradiance non-uniformity can be defined as 1 - (Emin / Emax), wherein Emin is the minimum irradiance across a diagonal (or across the longest dimension) of the aperture of the field stop and Emax is the maximum irradiance across the diagonal (or across the longest dimension) of the aperture of the field stop. In embodiments, irradiance non-uniformities at the field stop aperture in the semi-critical illumination scheme are: less than 0.20, less than 0.18, less than 0.16, less than 0.14, or less than 0.12.

[0200] The extent to which the semi-critical illumination scheme corrects or improves the luminance non- uniformity of the illumination source in the irradiance non-uniformity at the field stop can be defined as a non-uniformity correction metric, NCM, equal to 1 - INU / LNU (1 minus the ratio of the irradiance non- uniformity and the luminance non-uniformity). Thus the higher the NCM, the greater the effect of non- uniformity correction in semi-critical illumination. In embodiments, a Kohler illumination scheme is a applied to a first illumination source and a semi-critical illumination scheme is applied to a second illumination source such that the NCM for the second illumination scheme is greater than zero, greater than 0.05, greater than 0.1, greater than 0.2, greater than 0.3, greater than 0.4, greater than 0.5, or greater than 0.55.

[0201] LNU and INU can be sensitive to low luminance sharpness at the edges of the illumination source. This can skew the LNU reading to be unrepresentatively high if there is a roll off in luminance at the edges of the emitting surface of the illumination source, leading to a low Lmin. One way to avoid applying a potentially misleading performance metric is to determine (e.g. measure or model) LNU within the central 95%, 90%, 85%, 80% or 75% of the diagonal or longest dimension of the emitting surface. The same adjustment of the method of determining LNU to account for roll off can be applied to the measurement of INU. That is, the method of determining INU can include determining (e.g. measuring or modelling) INU within the central 95%, 90%, 85%, 80% or 75% of the diagonal or longest dimension of the field stop aperture. However, in embodiments in which the collector lens is arranged so that the illumination light fully fills the field stop aperture, this adjustment of the method of determining INU may not be as useful, since the parameter of interest in some applications can be non-uniformity (or uniformity) over the entire diagonal or longest dimension of the aperture. Therefore, the measurement of INU may be across the whole distance of the longest dimension of the field stop aperture. For example, in microscopy it may be important for the application that the image of the field stop at the sample plane has high uniformity over the entireIOO-I789OIPC irradiated area at the sample plane, and not just in the centre (e.g. central 95%, 90% etc.) of the irradiated area.

[0202] An optional objective of the present disclosure is to maintain high irradiance intensity at the field stop (and, in microscope applications, therefore at the sample plane), while at the same time correcting for the luminance non-uniformity of the illumination source in the irradiance of the field stop (and, in microscope applications, therefore at the sample plane). The inventors have recognised that a parameter of interest in achieving this objective is power efficiency. This parameter is an indication of the optical flux of the illumination light reaching the sample in the context of the optical flux at the light emitting of the illumination source. The power efficiency is an indication of the dilution of the optical flux through the system which takes into account the magnification of the first-formed image of the illumination source in the system (e.g. the first image of the illumination source 752, 852, 952 shown in FIGs 7, 8 and 9) and the clipping of the illumination light by the field stop aperture. The power efficiency is: EE / EL, wherein < E is the total irradiance optical flux of the illumination light at the focal plane of the objective, and whereinEL is the total luminance optical flux of the illumination light at the light emitting surface of the illumination source.

[0203] As described throughout the present disclosure, embodiments include a device including a first and second illumination source, wherein the first illumination source (and associated collector lens) is set up to provide Kohler illumination and the second illumination source is set up to provide semi-critical illumination. In some embodiments, the first illumination source is associated with a first power efficiency and the second illumination source is associated with a second power efficiency. In some embodiments, the power efficiency is defined as the ratio of power of illumination at the sample (e.g., power of illumination after the objective) to the optical power of the illumination source (e.g., LED). In some embodiments, the first power efficiency is about 5% to about 40%. In some embodiments, the first power efficiency is about 10% to about 30%. In some embodiments, the first power efficiency is about 15% to about 30%. In some embodiments, the second power efficiency is about 5% to about 40%. In some embodiments, the second power efficiency is about 10% to about 30%. In some embodiments, the second power efficiency is about 15% to about 30%. In some embodiments, the first power efficiency of the first illumination source (as measured within the illumination device in which it is deployed) is within 30%, 25%, 20%, 15% or 10% of a second power efficiency of the second illumination source (as measured within the illumination device in which it is deployed). In various embodiments, a radiant flux of the first illumination light at the field stop is within 30%, 25%, 20%, 15% or 10% of a radiant flux of the second illumination light at the field stop.

[0204] In various embodiments, the illumination device is configured so that the optical flux provided at the sample by one illumination source is similar to (e.g. is within a threshold difference relative to) the optical flux provided at the sample by one or more other illumination sources of the device. For example, the optical flux provided at the sample by a first illumination source used in one illumination scheme (e.g. a semi-critical illumination scheme) is within 20% (or optionally within 15%, optionally within 10%, optionally within 5%) of the optical flux provided at the sample by a second illumination source used in another illuminationIOO-I789OIPC scheme (e.g. a Kohler illumination scheme). In various embodiments, the illumination sources and collector lenses used in the semi-critical illumination scheme are configured so that the optical flux at the sample (or at the field stop) is within the threshold difference relative to the optical flux at the sample (or at the field stop) provided by the illumination source and collector lens arrangements used in the Kohler illumination scheme.

[0205] Kohler illumination assumes a point source, which is for practical purposes rarely possible. In embodiments, the illumination source is a 2D source with a length and width in the directions perpendicular to the normal of the emitting surface. For example, in some embodiments, a ratio of a largest dimension of an emitting surface of the first illumination source to a clear aperture of the first collector optic is greater than 0.05, optionally greater than 0.1 , optionally greater than 0.15, optionally greater than 0.2. The collector lens should be as large possible so that the illumination scheme is as close as possible to true Kohler illumination. Therefore, the ratio of the largest dimension of the emitting surface of the first illumination source to the clear aperture of the first collector optic is less than 0.5, optionally less than 0.4, optionally less than 0.3, optionally less than 0.2, optionally less than 0.1.

[0206] The power efficiency of the first illumination source can be controlled by controlling the F-number of the collector lens. The F-number, F#, is equal to f / D, wherein f is the distance between the illumination source and collector lens. F# dictates the optical radiant flux of illumination light through the collector lens. Since the first collector lens is necessarily positioned at a distance fCL from the first illumination source, the diameter of the first collector lens and the focal length of the first collector lens are the factors determining F# for the first collector lens. In contrast, in semi-critical illumination, additionally the distance between the second illumination source and the second collector lens can be varied to control F#.

[0207] In both the semi-critical and Kohler illumination schemes, improved illumination can be provided if the defocussed image of the illumination source at the field stop fills the aperture of the field stop entirely. Further improvements are possible if there is overlap between the edge of the defocussed image and the parts of the field stop around at least one (or optionally all) sides of the aperture. This helps to illuminate the effect of roll off in luminance intensity at the edges of the emitting surface of the illumination source. When the illumination device is provided in a microscope setting, for example as described with reference to FIGs 6A-6C, 7, 8 and 9, this can ensure a sharper roll off in illumination at the edges of the illuminated area of the sample plane.

[0208] In embodiments, the size of the field stop is governed by factors other than those central to the design of the illuminations devices according to the present disclosure. Therefore, the size of the field stop is often set and the illumination of the field stop must be governed by the arrangement of the collector lens, its optical power, diameter and its position relative to the illumination source and field stop. Therefore, in embodiments, each of the collector lenses is arranged so that the illumination light fills the field stop entirely, optionally overlapping the field stop around the field stop aperture.IOO-I789OIPC

[0209] In some implementations, such as those where each component of the illumination light corresponds to excitation of respective fluorophores, each component of illumination light includes a respective unique wavelength or spectral range. For instance, the first component of illumination light (e.g., corresponding to both a first illumination source and excitation of a first fluorophore in the sample) has a first peak wavelength and the second illumination light (e.g., corresponding to both a second illumination source and excitation of a second fluorophore in the sample) has a second peak wavelength. In some embodiments, the first peak wavelength corresponds to a wavelength of excitation of the first fluorophore and the second peak wavelength corresponds to a wavelength of excitation of the second fluorophore.General terminology:

[0210] 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.

[0211] It is to be understood that certain terminology is used in the preceding description for convenience and is not limiting. The terms “a”, “an” and “the” should be read as meaning “at least one” unless otherwise specified. The term “comprising” will be understood to mean “including but not limited to” such that systems or method comprising a particular feature or step are not limited to only those features or steps listed but may also comprise features or steps not listed. Similarly, any features included as examples are not to be construed as limiting to the disclosure. Additionally, any combination of features from one implementation with features from one or more other example(s) is to be understood as within the present disclosure. Equally, terms such as “after”, “before”, “in front”, “behind”, “downstream”, “upstream” and so on are used for convenience in interpreting the drawings and are not necessarily to be construed as limiting in absolute terms. Additionally, any method steps which are depicted in the figures as carried out sequentially, without causal connection, may alternatively be carried out in series in any order. Further, any method steps which are depicted as dashed or dotted flowchart boxes are to be understood as being optional.

[0212] As used herein, the term “amplitude” refers to a signed value (e.g., +1, -1, +0.1, -0.1, +0.01, -0.01, etc.) representing direction of movement of a pixel in an image. In a first example, the amplitude indicates a direction of movement (e.g., towards an attraction basin) using single, discrete values for a positive direction, a negative direction, and no movement along a given dimension (e.g., x-dimension, y-dimension, and / or z-dimension). In some embodiments, the amplitude is a whole integer selected from a set of {-1, 0, +1 } that indicates a direction of motion. In this case, a positive 1 indicates motion in a first direction (e.g., up / +y) along the given dimension (e.g., the vertical dimension / y). A negative value of the amplitude indicates motion in a second, opposite, direction (e.g., down / — y) along the given dimension (e.g., the vertical dimension / y). A zero value indicates no motion in the given dimension. In other embodiments, the amplitude is a signed probability value. In particular, an amplitude on the interval [-1,1] is provided corresponding to a given dimension (e.g., x-dimension, y-dimension, or z-dimension) of an image. In thisIOO-I789OIPC case, a positive value of the amplitude indicates the probability of movement in a first direction (e.g., up / +y) along the given dimension (e.g., the vertical dimension / y). A negative value of the amplitude indicates the probability of movement in a second, opposite, direction (e.g., down / -y) along the given dimension (e.g., the vertical dimension / y). A zero value indicates no motion in the given dimension. The magnitude of an amplitude refers to the absolute value of the amplitude, that is the magnitude is without direction. For ease of reference, a pixel having a zero amplitude or an amplitude of low magnitude (i.e., below a given threshold) is referred to as stationary. A pixel having magnitude exceeding that threshold are referred to as moving.

[0213] As used herein, the term “flow” as applied to pixels refers to the piecewise path from a pixel through zero or more intermediate pixels to a basin conforming to the amplitudes of those pixels. For example, a pixel that is adjacent to a basin pixel and has an amplitude indicating movement towards the basin pixel has length one flow to the basin. A piecewise path may be constructed from pixel to pixel according to the movement indicated by each pixel’s amplitude until arrival at a basin.

[0214] With reference to pixels of an image, adjacent pixels are those that share an edge or a corner.

[0215] 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.

[0216] 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.

[0217] 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.

[0218] 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 ofIOO-I789OIPC those included limits are also included in the claimed subject matter. This applies regardless of the breadth of the range.

[0219] 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.

[0220] 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.

[0221] 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:

[0222] 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).

[0223] The phrase “barcode diversity” refers to the total number of unique barcode sequences that may be represented by a given set of barcodes.

[0224] 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.

[0225] 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.IOO-I789OIPCIn 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.).

[0226] In some instances, barcodes may comprise a series of two or more segments or sub-barcodes (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.

[0227] A “digital barcode” (or “digital barcode sequence”) is a representation of a corresponding physical barcode (or target analyte sequence) in a computer-readable, digital format as described above. A digital barcode may comprise one or more “letters” (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, or more than 20 letters) or one or more “code words” (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 code words), where a “code word” comprises, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, or more than 20 letters. In some instances, the sequence of letters or code words in a digital barcode sequence may correspond directly with the sequence of building blocks (e.g., nucleotides) in a physical barcode. In 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.

[0228] 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 least100-178901PC4 x 106, at least 5 x 106, at least 6 x 106, at least 7 x 106, at least 8 x 106, at least 9 x 106, at least 107, at least IO8, at least IO9, or more than IO9unique 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.

[0229] 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.

[0230] A “corrected barcode” (or “corrected barcode sequence”) is a digital barcode sequence derived from a decoded barcode sequence by applying one or more error correction methods.Probe terminology:

[0231] The term “probe” may refer either to a physical probe molecule (e.g. , a nucleic acid probe molecule) or to its representation in a computer-readable, digital format (e.g., as a string of characters representing the sequence of bases in a nucleic acid probe molecule). A “probe” may be, for example, a molecule designed to recognize (and bind or hybridize to) another molecule, e.g. , a target analyte, another probe molecule, etc.

[0232] 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.

[0233] 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 secondary100-178901PC 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.

[0234] 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 another molecule, detection probes may be labeled with a fluorophore or other detectable tag). In some instances, a probe may be designed to recognize and bind (or hybridize) to a physical barcode sequence (or segments thereof). In some instances, a probe may be used to detect and decode a barcode, e.g., a nucleic acid barcode. In some instances, a probe may bind or hybridize directly to a target barcode. In some instances, a probe may bind or hybridize indirectly to a target barcode (e.g., by binding or hybridizing to other probe molecules which itself is bound or hybridized to the target barcode).Nucleic acid molecule and nucleotide terminology:

[0235] 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)).

[0236] 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 nonnatural bases that can be included in a nucleic acid or nucleotide are known in the art. See, for example, Appella (2009), “Non-Natural Nucleic Acids for Synthetic Biology”, Curr Opin Chem Biol. 13(5-6): 687- 696; and Duffy, et al. (2020), “Modified Nucleic Acids: Replication, Evolution, and Next-Generation Therapeutics”, BMC Biology 18:112.Samples:

[0237] 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 usingIOO-I789OIPC any of a variety of techniques including, but not limited to, biopsy, surgery, and laser capture microscopy (LCM), and generally includes cells and / or other biological material from the subject. In addition to the subjects described above, a biological sample can be obtained from a prokaryote such as a bacterium, an archaea, a virus, or a viroid. A biological sample can also be obtained from non-mammalian organisms (e.g. , a plant, an insect, an arachnid, a nematode, a fungus, or an amphibian). A biological sample can also be obtained from a eukaryote, such as a tissue sample, a patient derived organoid (PDO) or patient derived xenograft (PDX). A biological sample from an organism may comprise one or more other organisms or components therefrom. For example, a mammalian tissue section may comprise a prion, a viroid, a virus, a bacterium, a fungus, or components from other organisms, in addition to mammalian cells and non-cellular tissue components. Subjects from which biological samples can be obtained can be healthy or asymptomatic individuals, individuals that have or are suspected of having a disease (e.g., a patient with a disease such as cancer) or a pre-disposition to a disease, and / or individuals in need of therapy or suspected of needing therapy.

[0238] 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.

[0239] 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.

[0240] 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.

[0241] 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.

[0242] 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 theIOO-I789OIPC 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.

[0243] A variety of steps can be performed to prepare or process a biological sample for and / or during an assay. Except where indicated otherwise, the preparative or processing steps described below can generally be combined in any manner and in any order to appropriately prepare or process a particular sample for and / or analysis.Endogenous analytes:

[0244] 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.

[0245] 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, ubiquity lation 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.IOO-I789OIPC

[0246] 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.

[0247] Examples of nucleic acid analytes also include RNA analytes such as various types of coding and non-coding RNA. Examples of the different types of RNA analytes include messenger RNA (mRNA), including a nascent RNA, a pre -mRNA, a primary-transcript RNA, and a processed RNA, such as a capped mRNA (e.g., with a 5’ 7-methyl guanosine cap), a polyadenylated mRNA (poly-A tail at the 3’ end), and a spliced mRNA in which one or more introns have been removed. Also included in the analytes disclosed herein are non-capped mRNA, a non-polyadenylated mRNA, and a non-spliced mRNA. The RNA analyte can be a transcript of another nucleic acid molecule (e.g., DNA or RNA such as viral RNA) present in a tissue sample. Examples of a non-coding RNAs (ncRNA) that is not translated into a protein include transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs), as well as small non-coding RNAs such as microRNA (miRNA), small interfering RNA (siRNA), Piwi-interacting 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).

[0248] 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.

[0249] 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.

[0250] Methods and compositions disclosed herein can be used to analyze any number of analytes. For example, the number of analytes that are analyzed can be at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 20, at least about 25, at least about 30, at least about 40, at least about 50, at least about 100, at least about 1,000, at leastIOO-I789OIPC 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.

[0251] 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 single-stranded target sequence is comprised in a different analyte (e.g., nucleic acid) from the first single-stranded target sequence.Labelling agents:

[0252] In some instances, provided herein are methods and compositions for analyzing endogenous analytes (e.g., RNA, ssDNA, and cell surface or intracellular proteins and / or metabolites) in a sample using one or more labelling agents. In some instances, an analyte labelling agent may include an agent that interacts with an analyte (e.g., an endogenous analyte in a sample). In some instances, the labelling agents can comprise a reporter oligonucleotide that is indicative of the analyte or portion thereof interacting with the labelling agent. For example, the reporter oligonucleotide may comprise a barcode sequence that permits identification of the labelling agent. In some cases, the sample contacted by the labelling agent can be further contacted with a probe (e.g., a single-stranded probe sequence), that hybridizes to a reporter oligonucleotide of the labelling agent, in order to identify the analyte associated with the labelling agent. In some instances, the analyte labelling agent comprises an analyte binding moiety and a labelling agent barcode domain comprising one or more barcode sequences, e.g., a barcode sequence that corresponds to the analyte binding moiety and / or the analyte. An analyte binding moiety barcode includes to a barcode that is associated with or otherwise identifies the analyte binding moiety. In some instances, by identifying an analyte binding moiety by identifying its associated analyte binding moiety barcode, the analyte to which the analyte binding moiety binds can also be identified. An analyte binding moiety barcode can be a nucleic acid sequence of a given length and / or sequence that is associated with the analyte binding moiety. An analyte binding moiety barcode can generally include any of the variety of aspects of barcodes described herein.

[0253] 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.

[0254] 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,IOO-I789OIPC 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.

[0255] In some instances, an analyte binding moiety may include any molecule or moiety capable of binding to an analyte (e.g., a biological analyte, e.g., a macromolecular constituent). A labelling agent may include, but is not limited to, a protein, a peptide, an antibody (or an epitope binding fragment thereof), a lipophilic moiety (such as cholesterol), a cell surface receptor binding molecule, a receptor ligand, a small molecule, a bi-specific antibody, a 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.

[0256] Accordingly, terms such as “stain”, “staining”, “labeling”, and the like, may be used interchangeably to refer to elements, complexes, and macromolecules that allow a substance, structure, organelle, and / or component in a sample to be more easily detected than if said substance, structure, organelle, and / or component had not been stained or stained. For example, a tissue sample treated with a DNA dye such as DAPI (4',6-diamidino-2-phenylindole) makes the nucleus of a cell more visible and makes detection or quantification of such cells easier than if they were not stained. Without being bound by theory or methodology, the labeling described herein may be used to mark a cell, structure, particle, or other target, and may be useful in discovering, determining expression, localization, confirmation, quantification, or measuring properties within a sample. Without limitation, labeling agents disclosed herein include stains, dyes, ligands, antibodies, particles, and other substances that may bind to or 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.

[0257] As provided by the invention disclosed herein, one or more features are derived by detecting nuclei, cell membrane, and / or cytoplasm of cells within the input image and / or by extracting features from the detected nuclei, cell membrane, and / or cytoplasm (depending upon the labeling agent(s) utilized within the input image). In some embodiments, features are derived by analyzing cell membrane staining, cellIOO-I789OIPC cytoplasm staining, and / or cell nucleus staining. Without being bound by theory or methodology “cytoplasmic staining” may describe a group of pixels arranged in a pattern bearing the morphological characteristics of a cytoplasmic region of a cell. Similarly “membrane staining” may refer to a group of pixels arranged in a pattern bearing the morphological characteristics of a cell membrane, preferably the plasma membrane separating the intracellular environment from the extracellular space; and “nucleus staining” may refer to a group of pixels with strong localized intensity in a pattern bearing the morphological characteristics of a nucleus of the cell. Those of skill in the art will appreciate that the nucleus, cytoplasm, and membrane of a cell have different characteristics and that differently stained tissue samples may reveal different biological features. For example, those of skill would understand that certain cell surface elements and receptors can have staining patterns localized to the membrane or localized to the cytoplasm. Thus, a “membrane” staining pattern may be analytically distinct from a “cytoplasmic” staining pattern. Likewise, a “cytoplasmic” staining pattern and a “nuclear” staining pattern may be analytically distinct.

[0258] 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.

[0259] In some embodiments of the invention, the labelling described herein may be cell specific (e.g. , celltype 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.IOO-I789OIPC

[0260] Exemplary methods for staining tissue structures and guidance in the choice of stains appropriate for various purposes are known in the art and are discussed, for example, in “Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)” and “Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley-Intersciences (1987),” the disclosures of which are incorporated herein by reference. Lor 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.

[0261] 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). In some instances in which the plurality of analytes includes a single species of analyte, the analyte binding moieties of the plurality of analyte labelling agents are the same. In some instances in which the plurality of analytes includes a single species of analyte, the analyte binding moieties of the plurality of analyte labelling agents are the different (e.g., members of the plurality of analyte labelling agents can have two or more species of analyte binding moieties, wherein each of the two or more species of analyte binding moieties binds a single species of analyte, e.g., at different binding sites). In some instances, the plurality of analytes includes multiple different species of analyte (e.g., multiple different species of polypeptides).

[0262] 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.

[0263] 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.

[0264] 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. Lor 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 biotinylatedIOO-I789OIPC linker, coupled to oligonucleotides) with an avidin or streptavidin linker. Antibody and oligonucleotide biotinylation techniques are available. See, e.g., Fang, et al., “Fluoride-Cleavable Biotinylation Phosphoramidite for 5'-end-Labelling and Affinity Purification of Synthetic Oligonucleotides,” Nucleic Acids Res. Jan. 15, 2003; 31 (2):708-715, which is entirely incorporated herein by reference for all purposes. Likewise, protein and peptide biotinylation techniques have been developed and are readily available. See, e.g., U.S. Pat. No. 6,265,552, 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).

[0265] 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.

[0266] In some instances, multiple different species of analytes (e.g., polypeptides) from the biological sample can be subsequently associated with the one or more physical properties of the biological sample. For example, the multiple different species of analytes can be associated with locations of the analytes in the biological sample. Such information (e.g., proteomic information when the analyte binding moiety(ies) recognizes a polypeptide(s)) can be used in association with other spatial information (e.g., genetic information from the biological sample, such as DNA sequence information, transcriptome information (i.e., sequences of transcripts), 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 labellingIOO-I789OIPC 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:

[0267] 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.

[0268] Various methods can be used for in situ detection and analysis of target analytes, e.g. , sequencing by synthesis (SBS), sequencing by ligation (SBL), sequencing by hybridization (SBH). Non-limiting examples of in situ hybridization techniques include single molecule fluorescence in situ hybridization (smFISH) and multiplexed error-robust fluorescence in situ hybridization (MERFISH). smFISH enables in situ detection and quantification of gene transcripts in tissue samples at the locations where they reside by making use of libraries of multiple short oligonucleotide probes (e.g., approximately 20 base pairs (bp) in length), each labeled with a fluorophore. The probes are sequentially hybridized to gene sequences (e.g., DNA) or gene transcript sequences (e.g., mRNA) sequences, and visualized as 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 smLISH method include, for example, the use of combinatorial labelling schemes to improve multiplexing capability (Levsky, et al. (2003), ibid.), the use of smLISH in combination with super-resolution microscopy (Lubeck, et al. (2014) “Single -Cell In situ RNA Profiling by Sequential Hybridization”, Nature Methods 11(4):360— 361).

[0269] MERLISH 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.). MERLISH 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 Eluorescence 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” andIOO-I789OIPC 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.

[0270] 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.).

[0271] 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.

[0272] Products of endogenous analytes and / or labelling agents:

[0273] In some instances, provided herein are methods and compositions for analyzing one or more products of an endogenous analyte and / or a labelling agent in a biological sample. In some instances, an endogenous analyte (e.g., a viral or cellular DNA or RNA) or a product (e.g., a hybridization product, a ligation product, an extension product (e.g., by a DNA or RNA polymerase), a replication product, a transcription / reverse transcription product, and / or an amplification product such as a rolling circle amplification (RCA) product) thereof is analyzed. In some instances, a labelling agent that directly or indirectly binds to an analyte in the biological sample is analyzed. In some instances, a product (e.g., a hybridization product, a ligation product, an extension product (e.g., by a DNA or RNA polymerase), aIOO-I789OIPC 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.

[0274] 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 PL A YR (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:

[0275] Various probes and probe sets can be hybridized to an endogenous analyte and / or a labelling agent and each probe may comprise one or more barcode sequences. The specific probe or probe set design can vary. In some instances, the hybridization of a primary probe or probe set (e.g., a circularizable probe or probe set) to a target nucleic acid analyte and may lead to the generation of a rolling circle amplification (RCA) template. In some instances, the assay uses or generates a circular nucleic acid molecule which can be the RCA template.

[0276] 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)IOO-I789OIPC 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.

[0277] 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 situ hybridization (PLISH) probe set. See, e.g., U.S. Pat. Pub. 2020 / 0224243 which is hereby incorporated by reference in its entirety.

[0278] 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.

[0279] In some instances, the enzymatic ligation involves use of a ligase. In some aspects, the ligase used herein comprises an enzyme that is commonly used to join polynucleotides together or to join the ends of a single polynucleotide. An RNA ligase, a DNA ligase, or another variety of ligase can be used to ligate two nucleotide sequences together. Ligases comprise ATP-dependent double-strand polynucleotide ligases, NAD-i-dependent double-strand DNA or RNA ligases and single-strand polynucleotide ligases, for example any of the ligases described in EC 6.5.1.1 (ATP-dependent ligases), EC 6.5.1.2 (NAD+-dependent ligases), EC 6.5.1.3 (RNA ligases). Specific examples of ligases comprise bacterial ligases such as E. coli DNA ligase, Tth DNA ligase, Thermococcus sp. (strain 9° N) DNA ligase (9°N™ DNA ligase, New England Biolabs), Taq DNA ligase, Ampligase™ (Epicentre Biotechnologies) and phage ligases such as T3 DNA ligase, T4 DNA ligase and T7 DNA ligase and mutants thereof. In some instances, the ligase is a T4 RNA ligase. In some instances, the ligase is a splintR ligase. In some instances, the ligase is a single stranded DNA ligase. In some instances, the ligase is a T4 DNA ligase. In some instances, the ligase is a ligase that has an DNA-splinted DNA ligase activity. In some instances, the ligase is a ligase that has an RNA-splinted DNA ligase activity.

[0280] In some instances, the ligation herein is a direct ligation. In some instances, the ligation herein is an indirect ligation. "Direct ligation" means that the ends of the polynucleotides hybridize immediately adjacently to one another to form a substrate for a ligase enzyme resulting in their ligation to each other (intramolecular ligation). Alternatively, "indirect" means that the ends of the polynucleotides hybridize non-IOO-I789OIPC 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, the gap of one or more nucleotides between the hybridized ends of the polynucleotides may be "filled" by one or more "gap" (oligo)nucleotide(s) which are complementary to a splint, padlock probe, or target nucleic acid. The gap may be a gap of 1 to 60 nucleotides or a gap of 1 to 40 nucleotides or a gap of 3 to 40 nucleotides. In specific implementations, the gap may be a gap of about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more nucleotides, of any integer (or range of integers) of nucleotides in between the indicated values. In some instances, the gap between said terminal regions may be filled by a gap oligonucleotide or by extending the 3' end of a polynucleotide. In some cases, ligation involves ligating the ends of the probe to at least one gap (oligo)nucleotide, such that the gap (oligo)nucleotide becomes incorporated into the resulting polynucleotide. In some instances, the ligation herein is preceded by gap filling. In other implementations, the ligation herein does not require gap filling.

[0281] 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.

[0282] 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.

[0283] 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 incorporated herein by reference). A wide variety of different methods can be used for proximity ligating nucleic acid molecules, including (but not limited to) “sticky-end” and “blunt- end” ligations. Additionally, single-stranded ligation can be used to perform proximity ligation on a 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 fromIOO-I789OIPC each nucleic acid molecule because both nucleic acid molecules lack a single-stranded overhang at the site of ligation.Primer extension and amplification:

[0284] 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.

[0285] A primer is generally a single-stranded nucleic acid sequence having a 3’ end that can be used as a substrate for a nucleic acid polymerase in a nucleic acid extension reaction. RNA primers are formed of RNA nucleotides, and are used in RNA synthesis, while DNA primers are formed of DNA nucleotides and used in DNA synthesis. Primers can also include both RNA nucleotides and DNA nucleotides (e.g., in a random or designed pattern). Primers can also include other natural or synthetic nucleotides described herein that can have additional functionality. In some examples, DNA primers can be used to prime RNA synthesis and vice versa (e.g., RNA primers can be used to prime DNA synthesis). Primers can vary in length. For example, primers can be about 6 bases to about 120 bases. For example, primers can include up to about 25 bases. A primer, may in some cases, refer to a primer binding sequence. A primer extension reaction generally refers to any method where two nucleic acid sequences become linked (e.g., hybridized) by an overlap of their respective terminal complementary nucleic acid sequences (i.e., for example, 3’ termini). Such linking can be followed by nucleic acid extension (e.g. , an enzymatic extension) of one, or both termini using the other nucleic acid sequence as a template for extension. Enzymatic extension can be performed by an enzyme including, but not limited to, a polymerase and / or a reverse transcriptase.

[0286] 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 Soc Rev. 43(10):3324-3341). The RCA product is a concatemer containing tens to hundreds of tandem repeats that are complementary to the circular template, and may be used to develop sensitive techniques for the detection of a variety of targets, including nucleic acids (DNA, RNA), small molecules, proteins, and cells (Ali, et al. (2014), ibid.). In some implementations, a primer thatIOO-I789OIPC 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.

[0287] In some instances, the amplification is performed at a temperature between or between about 20°C and about 60°C. In some instances, the amplification is performed at a temperature between or between about 30°C and about 40°C. In some aspects, the amplification step, such as the rolling circle amplification (RCA) is performed at a temperature between at or about 25°C and at or about 50°C, such as at or about 25°C, 27°C, 29°C, 31°C, 33°C, 35°C, 37°C, 39°C, 41°C, 43°C, 45°C, 47°C, or 49°C.

[0288] 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-isothermal amplification. In some instances, after the formation of the hybridization complex and association of the amplification probe, the hybridization complex is rolling-circle amplified to generate a cDNA nanoball (z.e., amplicon) containing multiple copies of the cDNA. Techniques for rolling circle amplification (RCA) are known in the art such as linear RCA, a branched RCA, a dendritic RCA, or any combination thereof. (See, e.g., Baner et al, Nucleic Acids Research, 26:5073-5078, 1998; Lizardi et al, Nature Genetics 19:226, 1998; Mohsen et al., Acc ChemRes. 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 :1095- 1099, 2001; Schweitzer et al, Nature Biotech. 20:359-365, 2002; U.S. Patent Nos. 6,054,274, 6,291,187, 6,323,009, 6,344,329 and 6,368,801). Exemplary polymerases for use in RCA comprise DNA polymerase such phi29 (cp29) polymerase, Klenow fragment, Bacillus 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.

[0289] 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, or a 7-Deaza-7-Propargylamino-dATP moiety modification.

[0290] In some instances, the RCA template may comprise the target analyte, or a part thereof, where the target analyte is a nucleic acid, or it may be provided or generated as a proxy, or a marker, for the analyte. In some instances, the RCA template may comprise a sequence of the probes and probe sets hybridized to100-178901PC 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.

[0291] In some instances, an assay may detect a product herein that includes a molecule or a complex generated in a series of reactions, e.g., hybridization, ligation, extension, replication, transcription / reverse transcription, and / or amplification (e.g., rolling circle amplification), in any suitable combination. For example, a product comprising a target sequence for a probe disclosed herein (e.g., a bridge probe or L- probe) may be a hybridization complex formed of a cellular nucleic acid in a sample and an exogenously added nucleic acid probe. The exogenously added nucleic acid probe may comprise an overhang that does not hybridize to the cellular nucleic acid but hybridizes to another probe (e.g., a detection probe). The exogenously added nucleic acid probe may be optionally ligated to a cellular nucleic acid molecule or another exogenous nucleic acid molecule. In other examples, a product comprising a target sequence for a probe disclosed herein (e.g., an anchor probe) may be an RCP of a circularizable probe or probe set which hybridizes to a cellular nucleic acid molecule (e.g., genomic DNA or mRNA) or product thereof (e.g., a transcript such as cDNA, a DNA-templated ligation product of two probes, or an RNA-templated ligation product of two probes). In other examples, a product comprising a target sequence for a probe disclosed herein (e.g., a bridge probe or L-probe) may be a probe hybridizing to an RCP. The probe may comprise an overhang that does not hybridize to the RCP but hybridizes to another probe (e.g., a detection probe).Signal amplification methods:

[0292] 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 targetIOO-I789OIPC 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).

[0293] Alternatively or additionally, amplification of a signal associated with a barcode sequence of a nucleic acid probe can be amplified using one or more signal amplification strategies off of an oligonucleotide probe that hybridizes to the barcode sequence. In some aspects, amplification of the signal associated with the oligonucleotide probe can reduce the number of nucleic acid probes needed to hybridize to the target nucleic acid to obtain a sufficient signal-to-noise ratio. For example, the number of nucleic acid probes to tile a target nucleic acid such as an mRNA can be reduced. In some aspects, reducing the number of nucleic acid probes tiling a target nucleic acid enables detection of shorter target nucleic acids, such as shorter mRNAs. In some instances, no more than one, two, three, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18. 19, or 20 nucleic acid probes may be hybridized to the target nucleic acid. In instances wherein the target nucleic acid is an amplification product, signal amplification off of the oligonucleotide probes may reduce the number of target sequences required for detection (e.g., the length of the RCA product can be reduced).

[0294] 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.

[0295] 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.

[0296] 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 acidIOO-I789OIPC molecules starting from HCR monomers, which hybridize to one another to form a nicked nucleic acid polymer. This polymer is the product of the HCR reaction which is ultimately detected in order to indicate the presence of the target analyte. HCR is described in detail in Dirks and Pierce, 2004, PNAS, 101(43), 15275-15278 and in US 7,632,641 and US 7,721,721 (see also US 2006 / 00234261; Chemeris et al, 2008 Doklady Biochemistry and Biophysics, 419, 53-55; Niu et al, 2010, 46, 3089-3091; Choi et al, 2010, Nat. Biotechnol. 28(11), 1208-1212; and Song et al, 2012, Analyst, 137, 1396-1401). HCR monomers typically comprise a hairpin, or other metastable nucleic acid structure. In the simplest form of HCR, two different types of stable hairpin monomer, referred to here as first and second HCR monomers, undergo a chain reaction of hybridization events to form a long nicked double-stranded DNA molecule when an “initiator” nucleic acid molecule is introduced. The HCR monomers have a hairpin structure comprising a double stranded stem region, a loop region connecting the two strands of the stem region, and a single stranded region at one end of the double stranded stem region. The single stranded region which is exposed (and which is thus available for hybridization to another molecule, e.g. initiator or other HCR monomer) when the monomers are in the hairpin structure may be known as the “toehold region” (or “input domain”). The first HCR monomers each further comprise a sequence which is complementary to a sequence in the exposed toehold region of the second HCR monomers. This sequence of complementarity in the first HCR monomers may be known as the “interacting region” (or “output domain”). Similarly, the second HCR monomers each comprise an interacting region (output domain), e.g. a sequence which is complementary to the exposed toehold region (input domain) of the first HCR monomers. In the absence of the HCR initiator, these interacting regions are protected by the secondary structure (e.g. they are 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 a nicked chain of alternating units of the first and second monomer species. The presence of the HCR initiator is thus required in order to trigger the HCR reaction by hybridization to and invasion of a first HCR monomer. The first and second HCR monomers are designed to hybridize to one another are thus may be defined as cognate to one another. They are also cognate to a given HCR initiator sequence. HCR monomers which interact with one another (hybridize) may be described as a set of HCR monomers or an HCR monomer, or hairpin, system.IOO-I789OIPC

[0297] 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.

[0298] 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, the plurality of LO-HCR monomers may not comprise a metastable secondary structure. In some instances, the LO-HCR polymer may not comprise a branched structure. In some instances, performing the linear oligo hybridization chain reaction comprises contacting the target nucleic acid molecule with the initiator to provide the initiator hybridized to the target nucleic acid molecule. In any of the instances herein, the target nucleic acid molecule and / or the analyte can be an RCA product.

[0299] In some instances, detection of nucleic acids sequences in situ includes combination of the sequential decoding methods described herein with an assembly for branched signal amplification. In some instances, the assembly complex comprises an amplifier hybridized directly or indirectly (via one or more oligonucleotides) to a sequence of an oligonucleotide probe described herein. In some instances, the assembly includes one or more amplifiers each including an amplifier repeating sequence. In some aspects, the one or more amplifiers is labeled. Described herein is a method of using the aforementioned assembly, including for example, using the assembly in multiplexed error-robust fluorescent in situ hybridization (MERFISH) applications, with branched DNA amplification for signal readout. In some instances, the amplifier repeating sequence is about 5-30 nucleotides, and is repeated N times in the amplifier. In some instances, the amplifier repeating sequence is about 20 nucleotides, and is repeated at least two times in the amplifier. In some aspects, the one or more amplifier repeating sequence is labeled. For exemplary branchedIOO-I789OIPC 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.

[0300] In some instances, an oligonucleotide probe described herein can be associated with an amplified signal by a method that comprises signal amplification by performing a primer exchange reaction (PER). In various instances, a primer with domain on its 3’ end binds to a catalytic hairpin, and is extended with a new domain by a strand displacing polymerase. For example, a primer with domain 1 on its 3’ ends binds to a catalytic hairpin, and is extended with a new domain 1 by a strand displacing polymerase, with repeated cycles generating a concatemer of repeated domain 1 sequences. In various instances, the strand displacing polymerase is Bst. In various instances, the catalytic hairpin includes a stopper which releases the strand displacing polymerase. In various instances, branch migration displaces the extended primer, which can then dissociate. In various instances, the primer undergoes repeated cycles to form a concatemer primer (see e.g., U.S. Pat. Pub. No. US20190106733, which is incorporated herein by reference, for exemplary molecules and PER reaction components).Barcoded analytes and detection:

[0301] 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.

[0302] 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.

[0303] 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.100-178901PC

[0304] 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).

[0305] 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. Pub. 20190055594 and WO2019199579A1, which are hereby incorporated by reference in their entirety.Sequential hybridization:

[0306] 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 bindIOO-I789OIPC 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.

[0307] In some aspects, provided herein is a method involving a multiplexed biological assay and readout, in which a sample is first contacted with a plurality of nucleic acid probes, allowing the probes to directly or indirectly bind target analytes, which may then be optically detected (e.g., by detectably-labeled probes) in a temporally sequential manner. The plurality of nucleic acid probes themselves may be detectably-labeled and detected; in other words, the nucleic acid probes themselves serve as the detection probes. In other implementations, a nucleic acid probe itself is not directly detectably-labeled (e.g., the probe itself is not conjugated to a detectable label); rather, in addition to a target binding sequence (e.g., a sequence binding to a barcode sequence in an RCA product), the nucleic acid probe further comprises a sequence for detection which can be recognized by one or more detectably-labeled detection probes. In some instances, the probes or probe sets comprising various probe types may be applied to a sample simultaneously. In some instances, the probes or probe 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.

[0308] 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.

[0309] In any of the implementations disclosed herein, the detection of signals can be performed sequentially in cycles, one for each distinct label. In any of the implementations disclosed herein, signals or absence thereof from detectably-labeled probes targeting an analyte in a particular location in the sample can be recorded in a first cycle for detecting a first label, and signals or absence thereof from detectably- labeled probes targeting the analyte in the particular location can be recorded in a second cycle for detecting a second label distinct from the first label. In any of the implementations disclosed herein, a unique signal signature can be generated for each analyte of the plurality of analytes. In any of the implementationsIOO-I789OIPC disclosed herein, one or more molecules comprising the same analyte or a portion thereof can be associated with the same signal signature.

[0310] 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.

[0311] 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.

[0312] In some instances, detection of an optical signal signature comprises several rounds of detectably- labeled probe hybridization (e.g., contacting a sample with detectably-labeled probes), detectably-labeled probe detection, and detectably-labeled probe removal. In some instances, a sample is contacted with plurality first detectably-labeled probes, and said probes are hybridized to a plurality of nucleic acid analytes within the sample in decoding hybridization round 1. In some instances, a first detection round is performed following detectably-labeled probe hybridization. After hybridization and detection of a first plurality of detectably-labeled probes, probes are removed, and a sample may be contacted with a second plurality round of detectably-labeled probes targeting the analytes targeted in decoding hybridization round 1. The second plurality of detectably-labeled probes may hybridize to the same nucleic acid(s) as the first plurality of detectably-labeled probes (e.g. , hybridize to an identical or hybridize to new nucleic acid sequence within the same nucleic acid), or the second plurality of detectably-labeled probes may hybridize to different nucleic acid(s) compared to the first plurality of detectably-labeled probes. Following m rounds of contacting a sample with a plurality of detectably-labeled probes, probe detection, and probe removal, ultimately a unique signal signature to each nucleic acid is produced that may be used to identify and quantify said nucleic acids and the corresponding analytes (e.g., if the nucleic acids themselves are not the analytes of interest and each is used as part of a labelling agent for one or more other analytes such as protein analytes and / or other nucleic acid analytes).IOO-I789OIPC

[0313] 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.

[0314] In some examples, removal of a probe (e.g., un-hybridizing the entire probe), signal modifications (e.g., quenching, masking, photo-bleaching, signal enhancement (e.g., via FRET), signal amplification, etc.), signal removal (e.g., cleaving off or permanently inactivating a detectable label) can be performed. Inactivation may be caused by removal of the detectable label (e.g., from the sample, or from the probe, etc.), and / or by chemically altering the detectable label in some fashion, e.g., by photobleaching the detectable label, bleaching or chemically altering the structure of the detectable label, e.g., by reduction, etc.). In some instances, the fluorescently labeled oligonucleotide and / or the intermediate probe hybridized to the fluorescently labeled oligonucleotide (e.g., bridge probe or L-probe) can be removed. In some instances, a fluorescent detectable label may be inactivated by chemical or optical techniques such as oxidation, photobleaching, chemically bleaching, stringent washing or enzymatic digestion or reaction by exposure to an enzyme, dissociating the detectable label from other components (e.g., a probe), chemical reaction of the detectable label (e.g., to a reactant able to alter the structure of the detectable label) or the like. For instance, bleaching may occur by exposure to oxygen, reducing agents, or the detectable label could be chemically cleaved from the nucleic acid probe and washed away via fluid flow.

[0315] In some instances, removal of a signal comprises displacement of probes with another reagent (e.g., probe) or nucleic acid sequence. For example, a given probe (e.g., detectably-labeled probes and / or the intermediate probe hybridized to the fluorescently labeled oligonucleotide (e.g., bridge probe or E-probe)) may be displaced by a subsequent probe that hybridizes to an overlapping region shared between the binding sites of the probes. In some cases, a displacement reaction can be very efficient, and thus allows for probes to be switched quickly between cycles, without the need for chemical stripping (or any of the damage to the sample that is associated therewith). In some instances, a sequence for hybridizing the subsequent or displacer probe (i.e. a toehold sequence) may be common across a plurality of probes capable of hybridizing to a given binding site. In some aspects, a single 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.

[0316] 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 someIOO-I789OIPC instances, as the positions of the analytes, probes, and / or products thereof can be fixed (e.g. , via fixing and / or crosslinking) in a sample, the fluorescent spot corresponding to an analyte, probe, or product thereof remains in place during multiple rounds of hybridization and can be aligned to read out a string of signals associated with each target analyte.Decoding:

[0317] 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:

[0318] In some instances, the present methods may further involve contacting the target analyte, e.g., a nucleic acid molecule, or proxy thereof with an anchor probe. In some instances, the anchor probe comprises a sequence complementary to an anchor probe binding region, which is present in all target nucleic acid molecules (e.g., in primary or secondary probes), and a detectable label. The detection of the anchor probe via the detectable label confirms the presence of the target nucleic acid molecule. The target nucleic acid molecule may be contacted with the anchor probe prior to, concurrently with, or after being contacted with the first set of detection probes. In some instances, the target nucleic acid molecule may be contacted with the anchor probe during multiple decoding cycles. In some instances, multiple different anchor probes comprising different sequences and / or different reporters may be used to confirm the presence of multiple different target nucleic acid molecules. The use of multiple anchor probes is particularly useful when detection of a large number of target nucleic acid molecules is required, as it allows for optical crowding to be reduced and thus for detected target nucleic acid molecules to be more clearly resolved.IOO-I789OIPC

[0319] 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.

[0320] 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).

[0321] 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.

[0322] 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.IOO-I789OIPC

[0323] 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.

[0324] 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.

[0325] 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.

[0326] It is to be understood that the above description is intended to be illustrative, and not restrictive. Many other implementations will be apparent to those of skill in the art upon reading and understanding the above description. Although the present disclosure has been described with reference to specific example implementations, it will be recognized that the disclosure is not limited to the implementations described but can be practiced with modification and alteration within the scope of the appended claims. Accordingly, theIOO-I789OIPC 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.

[0327] Also described herein are the following numbered embodiments:Embodiment 1. A device for a microscope, the device comprising: a first illumination source arranged to output first illumination light; a second illumination source arranged to output second illumination light; a first collector optic arranged to collect the first illumination light; a second collector optic arranged to collect the second illumination light; a first field stop for light from the first collector optic; a second field stop for light from the second collector optic a first field optic arranged to image the first field stop; a second field optic arranged to image the second field stop; wherein the first collector optic is arranged to substantially collimate the first illumination light, and the second collector optic is arranged to form an image of the second illumination source between the second collector optic and the second field optic, before or after the second field stop so that the image of the second illumination source is defocussed at the second field stop.Embodiment 2. The device of embodiment 1 , wherein a luminance uniformity of the first illumination source is lower than a luminance uniformity of the second illumination source.Embodiment 3. The device of embodiment 1 or 2, wherein a ratio of a largest dimension of an emitting surface of the first illumination source to a clear aperture of the first collector optic is greater than 0.05, optionally greater than 0.1, optionally greater than 0.15, optionally greater than 0.2.Embodiment 4. The device of any preceding embodiment, wherein the second collector optic is arranged such that an irradiance uniformity at the second field stop is greater than the luminance uniformity of the second illumination source.Embodiment 5. The device of embodiment 4, wherein the irradiance uniformity is:Emin / Emax,IOO-I789OIPC wherein Emin is the minimum irradiance across an entire diagonal or longest dimension of the aperture of the second field stop and Emax is the maximum irradiance across the entire diagonal or longest dimension of the aperture of the second field stop, and wherein the luminance uniformity is:Lmin / Lmax, wherein Lmin is the minimum luminance across a central 100%, 95%, 90%, 85%, 80% or 75% of a diagonal or longest dimension of the light-emitting surface of the second illumination source and Lmax is the maximum luminance across the diagonal of the light-emitting surface.Embodiment 6. The device of embodiment 4 or 5, wherein the irradiance uniformity is greater than 0.80, optionally greater than 0.82, optionally greater than 0.84, optionally greater than 0.86, optionally greater than 0.88.Embodiment 7. The device of any of embodiments 2-6, wherein the luminance uniformity is less than or equal to than 0.80, optionally less than 0.78, optionally less than 0.76, optionally less than 0.74, optionally less than 0.72.Embodiment 8. The device of any preceding embodiment, wherein a first power efficiency of the first illumination source is within 20% of a second power efficiency of the second illumination source.Embodiment 9. The device of embodiment 8, further comprising an objective disposed to receive the first illumination light from the first field optic and second illumination light from the second field optic, wherein the first power efficiency is: E1 / L1 wherein OEl is the total irradiance optical flux of the first illumination light at the focal plane of the objective, and wherein OLl is the total luminance optical flux of the first illumination light at the light emitting surface of the first illumination source, and wherein the second power efficiency is:&E2 / &L2 wherein OE2 is the total irradiance optical flux of the second illumination light at the focal plane of the objective, and wherein OL2 is the total luminance optical flux of the second illumination light at the light emitting surface of the second illumination source.Embodiment 10. The device of any preceding embodiment, wherein the second collector optic is arranged to form an image of the second illumination source between the second field stop and the second field optic.IOO-I789OIPCEmbodiment 11. The device of embodiment 10, wherein the second collector optic is arranged to focus the image of the second illumination source at least one sixteenth, at least one eighth or at least one quarter of the focal length of the second field optic away from the second field stop.Embodiment 12. The device of any preceding embodiment, wherein the second collector optic is arranged so that the second field stop aperture is fully illuminated by the second illumination light.Embodiment 13. The device of any preceding embodiment, wherein the first collector optic is arranged so that the first field stop aperture is fully illuminated by the first illumination light.Embodiment 14. The device of any preceding embodiment, further comprising an objective disposed to receive the first illumination light from the first field optic and the second illumination light from the second field optic, wherein the first field stop is positioned at the front focal plane of the first field optic, the second field stop is positioned at the front focal plane of the second field optic, an optical separation between the first field optic and the objective is equal to the sum of the back focal length of the first field optic and the front focal length of the objective, and an optical separation between the second field optic and the objective is equal to the sum of the back focal length of the second field optic and the front focal length of the objective.Embodiment 15. The device of any preceding embodiment, wherein the distance between the first field stop and the first collector optic is equal to the front focal length of the first collector optic.Embodiment 16. The device of any preceding embodiment, wherein the distance between the second illumination source and the second collector optic is greater than the back focal length of the second collector optic.Embodiment 17. The device of any preceding embodiment, wherein a first optical distance between the first collector optic and the first field stop is less than a second optical distance between the second collector optic and the second field stop.Embodiment 18. The device of any preceding embodiment, further comprising: a third illumination source arranged to output third illumination light; a third collector optic arranged to collect the third illumination light; a third field stop for light from the third collector optic; a third field optic arranged to image the third field stop; wherein the third field stop is arranged to provide an aperture for light from the third collector optic, the third collector optic is arranged to substantially collimate the third illumination light, andIOO-I789OIPC optionally a luminance uniformity of the third illumination source is lower than a luminance uniformity of the second illumination source.Embodiment 19. The device of embodiment 18, wherein a first optical distance between the first collector optic and the first field stop is less than a second optical distance between the second collector optic and the second field stop, and wherein a third optical distance between the third collector optic and the third field stop is less than the first optical distance and less than the second optical distance.Embodiment 20. The device of embodiment 19, wherein a difference between the first optical distance and the third optical distance is less than the difference between the first optical distance and the second optical distance.Embodiment 21. The device of any preceding embodiment, further comprising: a fourth illumination source arranged to output fourth illumination light; and a fourth collector optic arranged to collect the fourth illumination light; a fourth field stop for light from the fourth collector optic; a fourth field optic arranged to image the fourth field stop; wherein the fourth field stop is arranged to provide an aperture for light from the fourth collector optic, the fourth collector optic is arranged to form an image of the fourth illumination source between the fourth collector optic and the fourth field optic, before or after the fourth field stop so that the image of the fourth illumination source is defocussed at the fourth field stop, and optionally a luminance uniformity of the fourth illumination source is higher than a luminance uniformity of the first illumination source.Embodiment 22. The device of embodiment 21, wherein a fourth optical distance between the fourth collector optic and the fourth field stop is greater than a second optical distance between the second collector lens and the second field stop.Embodiment 23. The device of embodiment 22, wherein a difference between the fourth optical distance and the second optical distance is less than the difference between the first optical distance and the second optical distance.Embodiment 24. The device of any of embodiments 21-23, wherein the fourth collector optic is arranged to form an image of the fourth illumination source between the fourth field stop and the fourth field optic.IOO-I789OIPCEmbodiment 25. The device of any preceding embodiment, further comprising a first dichroic arranged between the first collector optic and the first field stop and a second dichroic arranged between the second collector optic and the second field stop, wherein the first dichroic is arranged to reflect the first illumination light and transmit the second illumination light, and the second dichroic is arranged to reflect the second illumination light.Embodiment 26. The device of any preceding embodiment, wherein the first illumination light has a first wavelength and the second illumination light has a second wavelength,Embodiment 27. The device of embodiment 26, wherein the first wavelength is shorter than the second wavelength.Embodiment 28. The device of embodiment 26, wherein the second wavelength is shorter than the first wavelength.

[0328] Also described herein are the following numbered clauses:Clause 1. A device for a microscope, the device comprising: a first illumination source arranged to output first illumination light; a second illumination source arranged to output second illumination light; a first collector optic arranged to collect the first illumination light; a second collector optic arranged to collect the second illumination light; a first field stop for light from the first collector optic; a second field stop for light from the second collector optic a field optic arranged to image the first field stop and second field stop; wherein the first collector optic is arranged to substantially collimate the first illumination light, and the second collector optic is arranged to form an image of the second illumination source between the ...

Claims

IOO-I789OIPCCLAIMS1. A device comprising: a first illumination source arranged to output first illumination light; a second illumination source arranged to output second illumination light; a first collector optic arranged to collect the first illumination light; a second collector optic arranged to collect the second illumination light; a field stop arranged to provide an aperture for light from the first collector optic and second collector optic; and a field optic arranged to image the field stop; wherein: the first collector optic is arranged to substantially collimate the first illumination light, and the second collector optic is arranged to form an image of the second illumination source between the second collector optic and the field optic, before or after the field stop so that the image of the second illumination source is defocussed at the field stop.

2. The device of claim 1, wherein a luminance uniformity of the first illumination source is lower than a luminance uniformity of the second illumination source.

3. The device of claim 1 or 2, wherein a ratio of a largest dimension of an emitting surface of the first illumination source to a clear aperture of the first collector optic is greater than 0.05, optionally greater than 0.1, optionally greater than 0.15, optionally greater than 0.2.

4. The device of any preceding claim, wherein the second collector optic is arranged such that an irradiance uniformity at the field stop is greater than the luminance uniformity of the second illumination source.

5. The device of claim 4, wherein the irradiance uniformity is:Emin / Emax, wherein Emin is the minimum irradiance across an entire diagonal or longest dimension of the aperture of the field stop and Emax is the maximum irradiance across the entire diagonal or longest dimension of the aperture of the field stop, and wherein the luminance uniformity is:Lmin / Lmax, wherein Lmin is the minimum luminance across a central 100%, 95%, 90%, 85%, 80% or 75% of a diagonal or longest dimension of the light-emitting surface of the second illumination source and Lmax is the maximum luminance across the diagonal of the light-emitting surface.IOO-I789OIPC6. The device of claim 4 or 5, wherein the irradiance uniformity is greater than 0.80, optionally greater than 0.82, optionally greater than 0.84, optionally greater than 0.86, optionally greater than 0.88.

7. The device of any of claims 2-6, wherein the luminance uniformity is less than or equal to than 0.80, optionally less than 0.78, optionally less than 0.76, optionally less than 0.74, optionally less than 0.72.

8. The device of any preceding claim, wherein a first power efficiency of the first illumination source is within 20% of a second power efficiency of the second illumination source.

9. The device of claim 8, further comprising an objective disposed to receive the first and second illumination light from the field optic, wherein the first power efficiency is: E1 / L1 wherein OEl is the total irradiance optical flux of the first illumination light at the focal plane of the objective, and wherein OLl is the total luminance optical flux of the first illumination light at the light emitting surface of the first illumination source, and wherein the second power efficiency is:&E2 / &L2 wherein OE2 is the total irradiance optical flux of the second illumination light at the focal plane of the objective, and wherein OL2 is the total luminance optical flux of the second illumination light at the light emitting surface of the second illumination source.

10. The device of any preceding claim, wherein the second collector optic is arranged to form an image of the second illumination source between the field stop and the field optic.

11. The device of claim 10, wherein the second collector optic is arranged to focus the image of the second illumination source at least one sixteenth, at least one eighth or at least one quarter of the focal length of the field optic away from the field stop.

12. The device of any preceding claim, wherein the second collector optic is arranged so that the field stop aperture is fully illuminated by the second illumination light.

13. The device of any preceding claim, wherein the first collector optic is arranged so that the field stop aperture is fully illuminated by the first illumination light.

14. The device of any preceding claim, further comprising an objective disposed to receive the first and second illumination light from the field optic, wherein the field stop is positioned at the front focal plane ofIOO-I789OIPC the field optic, and an optical separation between the field optic and the objective is equal to the sum of the back focal length of the field optic and the front focal length of the objective.

15. The device of any preceding claim, wherein the distance between the field stop and the first collector optic is equal to the front focal length of the first collector optic.

16. The device of any preceding claim, wherein the distance between the second illumination source and the second collector optic is greater than the back focal length of the second collector optic.

17. The device of any preceding claim, wherein a first optical distance between the first collector optic and the field stop is less than a second optical distance between the second collector optic and the field stop.

18. The device of any preceding claim, further comprising a third illumination source arranged to output third illumination light; a third collector optic arranged to collect the third illumination light; wherein the field stop is arranged to provide an aperture for light from the third collector optic, the third collector optic is arranged to substantially collimate the third illumination light, and optionally a luminance uniformity of the third illumination source is lower than a luminance uniformity of the second illumination source.

19. The device of claim 18, wherein a first optical distance between the first collector optic and the field stop is less than a second optical distance between the second collector optic and the field stop, and wherein a third optical distance between the third collector optic and the field stop is less than the first optical distance and less than the second optical distance.

20. The device of claim 19, wherein a difference between the first optical distance and the third optical distance is less than the difference between the first optical distance and the second optical distance.

21. The device of any preceding claim, further comprising a fourth illumination source arranged to output fourth illumination light; and a fourth collector optic arranged to collect the fourth illumination light; wherein the field stop is arranged to provide an aperture for light from the fourth collector optic, the fourth collector optic is arranged to form an image of the fourth illumination source between the fourth collector optic and the field optic, before or after the field stop so that the image of the fourth illumination source is defocussed at the field stop, and optionally a luminance uniformity of the fourth illumination source is higher than a luminance uniformity of the first illumination source.IOO-I789OIPC22. The device of claim 21, wherein a fourth optical distance between the fourth collector optic and the field stop is greater than a second optical distance between the second collector lens and the field stop.

23. The device of claim 22, wherein a difference between the fourth optical distance and the second optical distance is less than the difference between the first optical distance and the second optical distance.

24. The device of any of claims 21-23, wherein the fourth collector optic is arranged to form an image of the fourth illumination source between the field stop and the field optic.

25. The device of any preceding claim, further comprising a first dichroic arranged between the first collector optic and the field stop and a second dichroic arranged between the second collector optic and the field stop, wherein the first dichroic is arranged to reflect the first illumination light and transmit the second illumination light, and the second dichroic is arranged to reflect the second illumination light.

26. The device of any preceding claim, wherein the first illumination light has a first wavelength and the second illumination light has a second wavelength,27. The device of claim 26, wherein the first wavelength is shorter than the second wavelength.

28. The device of claim 26, wherein the second wavelength is shorter than the first wavelength.

29. A device comprising: a first illumination source arranged to output first illumination light; a second illumination source arranged to output second illumination light; a first collector optic arranged to collect the first illumination light; a second collector optic arranged to collect the second illumination light; a first field stop for light from the first collector optic; a second field stop for light from the second collector optic a first field optic arranged to image the first field stop; a second field optic arranged to image the second field stop; wherein the first collector optic is arranged to substantially collimate the first illumination light, and the second collector optic is arranged to form an image of the second illumination source between the second collector optic and the second field optic, before or after the second field stop so that the image of the second illumination source is defocussed at the second field stop.IOO-I789OIPC30. A device comprising: a first illumination source arranged to output first illumination light; a second illumination source arranged to output second illumination light; a first collector optic arranged to collect the first illumination light; a second collector optic arranged to collect the second illumination light; a first field stop for light from the first collector optic; a second field stop for light from the second collector optic a field optic arranged to image the first field stop and second field stop; wherein the first collector optic is arranged to substantially collimate the first illumination light, and the second collector optic is arranged to form an image of the second illumination source between the second collector optic and the field optic, before or after the second field stop so that the image of the second illumination source is defocussed at the second field stop.

Images