Systems and methods for dynamic volumetric imaging

Abstract

Provided herein are methods, systems, and computer program products for dynamic volumetric imaging of an imageable volume including a sample, e.g., a biological sample, with an imaging instrument. A first z-stack of images of a sample is captured by moving an objective of the imaging instrument relative to the sample. Each image of the first z-stack of images has a z- index within the z-stack and a first field of view. A density metric of the first z-stack of images is determined. The density metric corresponds to a point density of one or more target molecule in the first field of view. The density metric is compared with a threshold. A second z-stack is captured, based on the comparison of the metric with the threshold, by moving the objective of the imaging instrument relative to the sample. The second z-stack comprises at least one additional image of the sample within the first field of view, and is interleaved with the first z-stack.

Description

GEI-02225100-179000WO SYSTEMS AND METHODS FOR DYNAMIC VOLUMETRIC IMAGINGCross-Reference to Related Applications

[0001] This application claims the benefit of U.S Provisional Application No. 63 / 738,928, filed December 26, 2024, which is hereby incorporated by reference in its entirety.Field

[0002] The present disclosure is directed to dynamic volumetric imaging of a sample, e.g., a biological sample, during in situ analysis. In particular, the present disclosure describes a method of dynamically adjusting the number of slices in a z-stack of images based on a point density in a previously-captured z-stack of images.Background

[0003] Spatial biology is a field of study that seeks to understand how cells and molecules (e.g., RNA transcripts, DNA, proteins, etc.) are organized and interact within a tissue. In particular, spatial biology enables researchers to understand how different cell types and molecular profiles contribute to tissue function and disease progression by analyzing spatial relationships within 2D and 3D environments. One particular application of spatial biology involves analyzing tissue and molecules within the tissue in their natural positions, also called in situ detection and analysis. In situ detection and analysis methods are emerging from the rapidly developing field of spatial biology (e.g., spatial transcriptomics, spatial proteomics, etc.). 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] During in situ detection and analysis, volumetric imaging is performed on an imageable volume that includes the sample (and sometimes additional volume above and / or below the sample to ensure that target analyte data is not inadvertently missed). To perform volumetric imaging, a plurality of fields of view (FOVs) are imaged and a z-stack of images is obtained at each FOV for each of a plurality of color channels. Each z-stack of images includes a plurality1FH13244839.1GEI-02225100-179000WO of image slices having a delta-z in between each slice. Depending on the assay design (e.g., selected target analytes, probing methods, etc.), some analytes (e.g., highly expressed genes) may fluoresce with higher densities, and information can be missed if the delta-z between each image slice in a z-stack is too large. For example, decoding of detected signal in dense areas of highly expressed transcripts may result in missed transcript calls or low quality transcript calls. Accordingly, there exists a need for dynamic volumetric imaging that can adapt to dense regions of detected signal corresponding to target analytes.Summary

[0005] Aspects of the claimed concepts provided herein provide methods, systems, and computer program products for capturing, with an imaging instrument, volumetric images of a sample.

[0006] In some aspects, provided herein is a method comprising capturing, by moving an objective of an imaging instrument relative to a sample, a first z-stack of images of the sample, the first z-stack of images having a first field of view, each image of the first z-stack of images having a z-index within the z-stack; determining a density metric of the first z-stack of images, wherein the density metric corresponds to a point density of one or more target molecule in the first field of view; comparing the metric with a threshold; and based on the comparison of the metric with the threshold, capturing a second z-stack by moving the objective of the imaging instrument relative to the sample, the second z-stack comprising at least one additional image of the sample within the first field of view, and being interleaved with the first z-stack.

[0007] Moving the objective of the imaging instrument relative to the sample may comprise moving the sample. The first z-stack of images may be captured based on at least one probing cycle of the sample in an imaging instrument. The first z-stack of images may comprise images of the sample stained with one or more fluorescent stain. The one or more fluorescent stains may comprise a nuclear stain. A z spacing of the first z-stack may be at most a micron. The first z-stack and the second z-stack may correspond to a same color channel. A z spacing of the first z-stack may be predetermined. The z spacing may correspond to a minimum movement of the objective of the imaging instrument relative to the sample. The method may further comprise determining a z spacing of the second z-stack based on the point density. The metric may be a brightness. The method may further comprise identifying a plurality of objects in the first field2FH13244839.1GEI-02225100-179000WQ of view, and wherein the metric is a function of a count of the plurality of objects. The method may further comprise determining the metric from a trained machine learning model. A z spacing that corresponds to the second z-stack may be the same as a z spacing of the first z-stack. A z spacing that corresponds to the second z-stack may be smaller than a z spacing of the first z-stack. A z spacing of a combination of the first and the second z-stacks may be at least 50 nm.

[0008] In some aspects, provided herein is a computer program product comprising a computer readable storage medium having program instructions embodied therewith, the program instructions executable by a processor to cause the processor to perform the previous method according the steps described with respect to the previous method.

[0009] In some aspects, provided herein is a system comprising an imaging instrument; and a computing node operatively coupled to the imaging instrument and comprising a computer readable storage medium having program instructions embodied therewith, the program instructions executable by a processor to cause the processor to perform the previous method according the steps described with respect to the previous method.

[0010] In some aspects, provided herein is a method comprising receiving a first set of three-dimensional (3D) positional information of a first plurality of biological molecules within a sample, wherein the first set of 3D positional information is determined from a first z-stack obtained from a first field of view (FOV) of the sample and based on a probing cycle of the sample in an imaging instrument, wherein the first z-stack comprises a first plurality of image slices, wherein the probing cycle comprises generating optical signals corresponding to at least some of the first plurality of biological molecules, wherein the first z-stack defines a first imaging volume; determining a first density metric of at least a portion of the first set of 3D positional information; and when the first density metric is above a first threshold, causing the imaging instrument to capture at least one additional image slice of the sample within the first field of view that is interleaved with the first z-stack.

[0011] The at least one additional image slice is an additional z-stack that may be interleaved with the first z-stack to generate a first combined z-stack. The first z-stack may have a first spacing between adjacent image slices of the first plurality of image slices. The first spacing may be about 500 nm to about 1 pm. The first spacing may be about 750 nm. The first combined z-stack may have a second spacing between adjacent image slices. The second spacing may be about 250 nm to about 1 pm. The second spacing may be about 250 nm to about3FH13244839.1GEI-02225100-179000WQ 500 nm. The second spacing may be less than the first spacing. The method may further comprise performing blob detection on the first z-stack to obtain the first set of 3D positional information. The first set of 3D positional information may comprise blob locations. The method may further comprise dividing the first imaging volume into a plurality of subvolumes. The first density metric may be an average number of blobs in the plurality of subvolumes. Each subvolume of the plurality of subvolumes may have a volume of about 1 m2 to about 100 m2. The first threshold may be about 1 blob per subvolume to about 2 blobs per subvolume. The first threshold may be about 1.5 blobs per subvolume. The first density metric may comprise a brightness for each image in the first plurality of images. The first set of 3D positional information may be obtained from a first color channel during the first probing cycle. The method may further comprise receiving a second set of 3D positional information of a second plurality of biological molecules within the sample, wherein the second set of 3D positional information is determined from a second z-stack obtained from the first FOV of the sample, wherein the second set of 3D positional information is obtained from a second color channel during the first probing cycle. The method may further comprise determining a second density metric of at least a portion of the second set of 3D positional information; and when the second density metric is above a second threshold, causing the imaging instrument to capture at least one additional image slice of the sample within the first field of view that is interleaved with the second z-stack. The first threshold and the second threshold may be equivalent. The first threshold and the second threshold may be different. The first threshold and the second threshold may be dependent on color channel. The first color channel may be different from the second color channel, wherein the first color channel and the second color channel are selected from the group consisting of: red, yellow, green, and blue. The first z-stack may be a first z-stack of a plurality of z-stacks, wherein the plurality of z-stacks of images correspond to a plurality of FOVs. The method may further comprise capturing the first z-stack using the imaging instrument.

[0012] In some aspects, provided herein is a computer program product comprising a computer readable storage medium having program instructions embodied therewith, the program instructions executable by a processor to cause the processor to perform the previous method according the steps described with respect to the previous method.4FH13244839.1GEI-02225100-179000WQ

[0013] In some aspects, provided herein is a system comprising an imaging instrument; and a computing node operatively coupled to the imaging instrument and comprising a computer readable storage medium having program instructions embodied therewith, the program instructions executable by a processor to cause the processor to perform the previous method according the steps described with respect to the previous method.

[0014] In some aspects, provided herein is a method comprising imaging a first z-stack at a first field of view (FOV) of a sample, wherein the first z-stack comprises a first plurality of image slices, wherein the first plurality of image slices comprises a first spacing between adjacent image slices; and imaging a second z-stack at a second FOV of the sample, wherein the second z-stack comprises a second plurality of image slices, wherein the second plurality of image slices comprises a second spacing between adjacent image slices; wherein the first spacing is different from the second spacing based on a comparison of a first density metric and a second density metric to a predetermined threshold, and wherein the first density metric is associated the first z-stack and the second density metric is associated with the second z-stack.

[0015] The second spacing may be smaller than the first spacing when the second density metric is greater than or equal to the predetermined threshold, and the first density metric is less than the predetermined threshold. The first spacing may be a default spacing. The first spacing may be about 500 nm to about 1 pm. The first spacing may be about 750 nm. The second spacing may be about 250 nm to about 1 pm. The second spacing may be about 250 nm to about 500 nm. The imaging the second z-stack at the second FOV may comprise imaging a preliminary z-stack at a plurality of z-heights having the first spacing between adjacent image slices; determining the second density metric of the preliminary z-stack is above the predetermined threshold; and imaging an interleaved z-stack at a plurality of additional z-heights such that combined images from the preliminary z-stack and images from the interleaved z-stack have the second spacing between adjacent image slices. The first density metric may be determined by performing blob detection on the first z-stack and the second density metric may be determined by performing blob detection on the second z-stack. The first z-stack and the second z-stack may define an imageable volume, and the method may further comprise dividing the imageable volume into a plurality of subvolumes. The first density metric and the second density metric may be determined by determining an average number of blobs per subvolume. Each subvolume of the plurality of subvolumes may have a volume of about 1 pm2 to about 100 pm2. The5FH13244839.1GEI-02225100-179000WO predetermined threshold may be about 1 blob per sub volume to about 2 blobs per sub volume. The first threshold may be about 1.5 blobs per subvolume.

[0016] A computer program product may include a computer readable storage medium having program instructions embodied therewith, the program instructions may be executable by a processor to cause the processor to perform any of the methods as described herein.

[0017] A system may include an imaging instrument; and a computing node operatively coupled to the imaging instrument comprising a computer readable storage medium having program instructions embodied therewith, the program instructions being executable by a processor to cause the processor to perform any of the methods as described herein.Brief Description of the Drawings

[0018] 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.

[0019] 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 a detected signal within the imaging volume.

[0020] 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.

[0021] FIGS.4A-4B illustrate cross-sectional views of an optics module in an imaging system, according to some embodiments.

[0022] FIG.5 depicts a computing node according to some embodiments disclosed herein.

[0023] FIG.6 depicts a process for imaging a volume of a sample to generate a plurality of z-stacks of images at a plurality of FOVs, according to various embodiments.

[0024] FIG.7 depicts a process for imaging a volume of a sample to generate a plurality of z-stacks of images including one or more z-stacks of images that are interleaved with another z-stack of images of the same FOV, according to various embodiments.6FH13244839.1GEI-02225100-179000WQ

[0025] FIG.8 depicts a process for imaging a volume of a sample to generate a plurality of z-stacks of images including one or more partial z-stacks of images that are interleaved with another z-stack of images of the same FOV, according to various embodiments.

[0026] FIG.9 depicts steps to dynamically generate a z-stack having additional interleaved slices, such as in FIG. 8, according to various embodiments.

[0027] FIG. 10 is a flowchart illustrating a method of dynamically capturing volumetric images of a sample with an imaging instrument, according to embodiments of the present disclosure.

[0028] FIG. 11 is a flowchart illustrating a method of dynamically capturing volumetric images of a sample with an imaging instrument, according to embodiments of the present disclosure.

[0029] FIG. 12 is a flowchart illustrating a method of dynamically capturing volumetric images of a sample with an imaging instrument, according to embodiments of the present disclosure.

[0030] FIG. 13A depicts observed and expected density of detected blobs using the methods disclosed herein.

[0031] FIG. 13B depicts observed and expected density of detected blobs using the methods disclosed herein.

[0032] FIG. 14A depicts plots of the number of missed blobs (i.e., the expected but not observed blobs).

[0033] FIG. 14B depicts plots of the number of missed blobs (i.e., the expected but not observed blobs).

[0034] FIG. 15A depicts heat maps for mean local density and maximum local density expected with RNA Q20.

[0035] FIG. 15B depicts heat maps for mean local density and maximum local density expected with RNA Q20.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-Detailed Description

[0036] 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.7FH13244839.1GEI-02225100-179000WQ

[0037] 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 (see FIG. 1), wherein the imaged regions contain target molecules, such as nucleic acids or proteins, and relevant feature data (e.g., morphologic and pathologic data) (see FIG.2).

[0038] Described herein is a spatial transcriptomics solution and technique, which allow for the analysis of the transcriptome within a tissue sample.

[0039] The spatial transcriptomics solution, as described herein may include an in situ capturing technique. This may allow for the transcript to be captured within a tissue sample. After capture of the transcript, sequencing may be performed outside the tissue sample. Such an approach may allow for the tracing back of the transcripts, such as RNA transcripts, to their original location in the tissue sample. The tissue sample may be a particular type of tissue sample such as a fresh-frozen tissue sample or a Formalin Fixed Paraffin Embedded (FFPE) tissue sample.

[0040] The spatial transcriptomics solution may allow for a determination of where in a tissue sample a particular gene is expressed. The spatial transcriptomics solution may allow for a determination of where a cluster of genes that are expressed are located.

[0041] The spatial transcriptomics solution may capture data using slides, which each include one or more (e.g., four) capture areas. Each capture area on a slide may include multiple barcoded spots (e.g., 5000 barcoded spots). Each barcoded spot may include capture oligonucleotides. Each such capture oligonucleotide may bind to RNA in the tissue sample that is applied to the slide. Each barcoded spot may capture the transcripts from a particular number (e.g., 1-10) cells within the tissue sample.

[0042] A tissue sample may be applied (e.g., placed onto) a slide so that information may be captured from the tissue sample via the capture areas of the slide. The tissue sample may be permeabilized. This may allow the tissue sample to release mRNA, to which the capture oligonucleotides, associated with the spots in the slide capture areas, may bind. In addition, the tissue sample may be stained using IF or H&E stains. Image(s) of the stained tissue sample may be taken. The captured mRNA may be synthesized into cDNA. sequencing libraries may be prepared therefrom. The transcriptomics data may contain a spatial barcode from the spot on the slide. This may allow for the linking of the transcriptomics data to the location on the slide (e.g., regions of an image of the biological sample on the slide), as further described herein.

[0043] General terminology:8FH13244839.1GEI-02225100-179000WQ

[0044] 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.

[0045] 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.

[0046] As used herein, the term “amplitude” refers to a signed value (e.g., +1, -1, +0.1, -0.1, +0.01, -0.01, etc.) representing direction of movement of a pixel in an image. In a first example, the amplitude indicates a direction of movement (e.g., towards an attraction basin) using single, discrete values for a positive direction, a negative direction, and no movement along a given dimension (e.g., x-dimension, y-dimension, and / or z-dimension). In some embodiments, the amplitude is a whole integer selected from a set of {-1, 0, +1 } that indicates a direction of motion. In this case, a positive 1 indicates motion in a first direction (e.g., up / +y) along the given dimension (e.g., the vertical dimension / y). A negative value of the amplitude indicates motion in a second, opposite, direction (e.g., down / — y) along the given dimension (e.g., the vertical dimension / y). A zero value indicates no motion in the given dimension. In other embodiments, the amplitude is a signed probability value. In particular, an amplitude on the interval [-1,1] is provided corresponding to a given dimension (e.g., x-dimension, y-dimension, or z-dimension) of an image. In this case, a positive value of the amplitude indicates the9FH13244839.1GEI-02225100-179000WO 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.

[0047] 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.

[0048] With reference to pixels of an image, adjacent pixels are those that share an edge or a corner.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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 for10FH13244839.1GEI-02225100-179000WO convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the claimed subject matter. This applies regardless of the breadth of the range.

[0053] 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.

[0054] 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. As used herein, the term sequencing may include sequencing by synthesis (SBS), sequencing by hybridization (SBH), sequencing by ligation (SBL), sequencing by binding (SBB), and / or any other type of sequencing. SBS may be a DNA sequencing technique in which fluorescently labeled nucleotides are used to sequence clusters on a flow cell surface. SBH may be a DNA sequencing11FH13244839.1GEI-02225100-179000WQ technique in which sets of oligonucleotides are hybridized under conditions that allow detection of complementary sequences in the target nucleic acid. SBL may be a DNA sequencing technique that uses the enzyme DNA ligase to identify the nucleotide present at a given position in a DNA sequence. SBB may be a DNA sequencing technique that involves the examination of a ternary complex that forms between a primer-template nucleic acid hybrid, polymerase and nucleotide triphosphate and acquiring signal that is used to determine nucleic acid base identity without the need for nucleotide incorporation.

[0055] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0056] Barcoding and decoding terminology:

[0057] 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).

[0058] The phrase “barcode diversity” refers to the total number of unique barcode sequences that may be represented by a given set of barcodes.

[0059] 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.

[0060] Physical barcodes can have a variety of different formats. For example, barcodes can include polynucleotide barcodes, random nucleic acid and / or amino acid sequences, and synthetic nucleic acid and / or amino acid sequences. A physical barcode can be attached to an analyte, or to another moiety or structure, in a reversible or irreversible manner. A physical barcode can be added to, for example, a fragment of a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sample before or during sequencing of the sample. In some instances, barcodes can allow for identification and / or quantification of individual sequencing-reads in sequencing -based methods (e.g., a barcode can be or can include a unique molecular identifier or12FH13244839.1GEI-02225100-179000WQ “UMF’). 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.).

[0061] In some instances, barcodes may comprise a series of two or more segments or subbarcodes (e.g., corresponding to “letters” or “code words” in a decoded barcode), each of which may comprise one or more of the subunits or building blocks used to synthesize the physical (e.g., nucleic acid) barcode molecules. For example, a nucleic acid barcode molecule may comprise two or more barcode segments, each of which comprises one or more nucleotides. In some instances, a barcode may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 segments. In some instances, each segment of a barcode molecule may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, or more than 20 subunits or building blocks. For example, each segment of a nucleic acid barcode molecule may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, or more than 20 nucleotides. In some instances, two or more of the segments of a barcode may be separated by non-barcode segments, i.e., the segments of a barcode molecule need not be contiguous.

[0062] 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.13FH13244839.1GEI-02225100-179000WO

[0063] A “designed barcode” (or “designed barcode sequence”) is a barcode (or its digital equivalent; in some instances a designed barcode may comprise a series of code words that can be assigned to gene transcripts and subsequently decoded into a decoded barcode) that meets a specified set of design criteria as required for a specific application. In some instances, a set of designed barcodes may comprise at least 2, at least 5, at least 10, at least 20, at least 40, at least 60, at least 80, at least 100, at least 200, at least 400, at least 600, at least 800, at least 1,000, at least 2,000, at least 4,000, at least 6,000, at least 8,000, at least 10,000, at least 20,000, at least 40,000, at least 60,000, at least 80,000, at least 100,000, at least 200,000, at least 400,000, at least 600,000, at least 800,000, at least 1,000,000, at least 2 x 106, at least 3 x 106, at least 4 x 106, at least 5 x 106, at least 6 x 106, at least 7 x 106, at least 8 x 106, at least 9 x 106, at least 107, at least 108, at least 109, or more than 109unique barcodes. In some instances, a set of designed barcodes may comprise any number of designed barcodes within the range of values in this paragraph, e.g., 1,225 unique barcodes or 2.38 x 106unique barcodes. As noted above for barcodes in general, in some instances designed barcodes may comprise two or more segments (corresponding to two or more code words in a decode barcode). In those cases, the specified set of design criteria may be applied to the designed barcodes as a whole, or to one or more segments (or positions) within the designed barcodes.

[0064] 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.

[0065] 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.

[0066] Probe terminology:

[0067] 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 of14FH13244839.1GEI-02225100-179000WO 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.

[0068] 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.

[0069] In some instances, physical probes may bind or hybridize directly to their target. In some instances, physical probes may bind or hybridize indirectly to their target. For example, in some instances, a secondary probe may bind or hybridize to a primary probe, where the primary probe binds or hybridizes directly to the target analyte. In some instances, a tertiary probe may bind or hybridize to a secondary probe, where the secondary probe binds or hybridizes to a primary probe, and where the primary probe binds or hybridizes directly to the target analyte.

[0070] 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 target15FH13244839.1GEI-02225100-179000WG barcode (e.g., by binding or hybridizing to other probe molecules which itself is bound or hybridized to the target barcode).

[0071] Nucleic acid molecule and nucleotide terminology:

[0072] 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)).

[0073] A nucleic acid can contain nucleotides having any of a variety of analogs of these sugar moieties that are known in the art. A nucleic acid can include natural or non-natural nucleotides. In this regard, a naturally-occurring deoxyribonucleic acid can have one or more bases selected from the group consisting of adenine (A), thymine (T), cytosine (C), or guanine (G), and a ribonucleic acid can have one or more bases selected from the group consisting of uracil (U), adenine (A), cytosine (C), or guanine (G). Useful non-natural bases that can be included in a nucleic acid or nucleotide are known in the art. See, for example, Appella (2009), “Non-Natural Nucleic Acids for Synthetic Biology”, Curr Opin Chem Biol. 13(5-6): 687-696; and Duffy, et al. (2020), “Modified Nucleic Acids: Replication, Evolution, and Next-Generation Therapeutics”, BMC Biology 18:112.

[0074] Samples:

[0075] A sample disclosed herein can be or derived from any biological sample, or other sample, such a hydrogel. Although various embodiments, disclosed herein, are disclosed for use with biological samples, it should be understood that any sample may be used. Methods and compositions disclosed herein may be used for analyzing a biological sample, which may be obtained from a subject using any of a variety of techniques including, but not limited to, biopsy, surgery, and laser capture microscopy (LCM), and generally includes cells and / or other biological material from the subject. In addition to the subjects described above, a biological16FH13244839.1GEI-02225100-179000WQ 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 predisposition to a disease, and / or individuals in need of therapy or suspected of needing therapy.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] 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 system17FH13244839.1GEI-02225100-179000WQ 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.

[0080] In some instances, a substrate herein can be any support that is insoluble in aqueous liquid and which allows for positioning of biological samples, analytes, features, and / or reagents (e.g., probes) on the support. In some instances, a biological sample can be attached to a substrate. Attachment of the biological sample can be irreversible or reversible, depending upon the nature of the sample and subsequent steps in the analytical method. In certain instances, the sample can be attached to the substrate reversibly by applying a suitable polymer coating to the substrate, and contacting the sample to the polymer coating. The sample can then be detached from the substrate, e.g., using an organic solvent that at least partially dissolves the polymer coating. In some instances, the substrate can be coated or functionalized with one or more substances to facilitate attachment of the sample to the substrate. Suitable substances that can be used to coat or functionalize the substrate include, but are not limited to, lectins, poly-lysine, antibodies, and polysaccharides.

[0081] 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.

[0082] Endogenous analytes:

[0083] 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.

[0084] Examples of non-nucleic acid analytes include, but are not limited to, lipids, carbohydrates, peptides, proteins, glycoproteins (N-linked or O-linked), lipoproteins, phosphoproteins, specific phosphorylated or acetylated variants of proteins, amidation variants of proteins, hydroxylation variants of proteins, methylation variants of proteins, ubiquitylation variants of proteins, sulfation variants of proteins, viral coat proteins, extracellular and18FH13244839.1GEI-02225100-179000WQ 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.

[0085] 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.

[0086] 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 large19FH13244839.1GEI-02225100-179000WQ (e.g., RNA greater than 200 nucleic acid bases in length). Examples of small RNAs include 5.8S ribosomal RNA (rRNA), 5S rRNA, tRNA, miRNA, siRNA, snoRNAs, piRNA, tRNA-derived small RNA (tsRNA), and small rDNA-derived RNA (srRNA). The RNA can be double-stranded RNA or single- stranded RNA. The RNA can be circular RNA. The RNA can be a bacterial rRNA (e.g., 16s rRNA or 23s rRNA).

[0087] 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.

[0088] 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.

[0089] Methods and compositions disclosed herein can be used to analyze any number of analytes. For example, the number of analytes that are analyzed can be at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 20, at least about 25, at least about 30, at least about 40, at least about 50, at least about 100, at least about 1,000, at least about 10,000, at least about 100,000 or more different analytes present in a region of the sample or within an individual feature of the substrate.

[0090] 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 singlestranded target sequence is comprised in the same analyte (e.g., nucleic acid) as the first single-20FH13244839.1GEI-02225100-179000WO 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.

[0091] Labelling agents:

[0092] 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.

[0093] 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.

[0094] In the methods and systems described herein, one or more labelling agents capable of binding to or otherwise coupling to one or more features may be used to characterize analytes, cells and / or cell features. In some instances, cell features include cell surface features. Analytes may include, but are not limited to, a protein, a receptor, an antigen, a surface protein, a transmembrane protein, a cluster of differentiation protein, a protein channel, a protein pump, a21FH13244839.1GEI-02225100-179000WO 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.

[0095] In some instances, an analyte binding moiety may include any molecule or moiety capable of binding to an analyte (e.g., a biological analyte, e.g., a macromolecular constituent). A labelling agent may include, but is not limited to, a protein, a peptide, an antibody (or an epitope binding fragment thereof), a lipophilic moiety (such as cholesterol), a cell surface receptor binding molecule, a receptor ligand, a small molecule, a bi-specific antibody, a bispecific T-cell engager, a T-cell receptor engager, a B-cell receptor engager, a pro-body, an aptamer, a monobody, an affimer, a darpin, and a protein scaffold, or any combination thereof. The labelling agents can include (e.g., are attached to) a reporter oligonucleotide that is indicative of the cell surface feature to which the binding group binds. For example, the reporter oligonucleotide may comprise a barcode sequence that permits identification of the labelling agent. For example, a labelling agent that is specific to one type of cell feature (e.g., a first cell surface feature) may have coupled thereto a first reporter oligonucleotide, while a labelling agent that is specific to a different cell feature (e.g., a second cell surface feature) may have a different reporter oligonucleotide coupled thereto.

[0096] 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,22FH13244839.1GEI-02225100-179000WO 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.

[0097] As provided by the invention disclosed herein, one or more features are derived by detecting nuclei, cell membrane, and / or cytoplasm of cells within the input image and / or by extracting features from the detected nuclei, cell membrane, and / or cytoplasm (depending upon the labeling agent(s) utilized within the input image). In some embodiments, features are derived by analyzing cell membrane staining, cell cytoplasm staining, and / or cell nucleus staining.Without being bound by theory or methodology “cytoplasmic staining” may describe a group of pixels arranged in a pattern bearing the morphological characteristics of a cytoplasmic region of a cell. Similarly “membrane staining” may refer to a group of pixels arranged in a pattern bearing the morphological characteristics of a cell membrane, preferably the plasma membrane separating the intracellular environment from the extracellular space; and “nucleus staining” may refer to a group of pixels with strong localized intensity in a pattern bearing the morphological characteristics of a nucleus of the cell. Those of skill in the art will appreciate that the nucleus, cytoplasm, and membrane of a cell have different characteristics and that differently stained tissue samples may reveal different biological features. For example, those of skill would understand that certain cell surface elements and receptors can have staining patterns localized to the membrane or localized to the cytoplasm. Thus, a “membrane” staining pattern may be analytically distinct from a “cytoplasmic” staining pattern. Likewise, a “cytoplasmic” staining pattern and a “nuclear” staining pattern may be analytically distinct.

[0098] 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,23FH13244839.1GEI-02225100-179000WG 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.

[0099] In some embodiments, the labelling described herein may be cell specific (e.g., cell-type specific), thus providing the detection of different cell types within a sample. In some embodiments, the invention disclosed herein, or elements thereof, incorporate identification of cell polarity and / or morphology. Cell polarity may refer to an asymmetry in molecular composition or structure between two sides, thus defining a polarity axis along which cellular processes will be differentially regulated. In some such embodiments, the invention incorporates identifying cellular symmetry, including the distribution of structures and / or organelles within the cells. For example and without limitation, the radial symmetry of labeled structures or organelles relative to other stains, e.g., plasma membrane, cytoplasmic and / or nuclear labels, such as the radial staining pattern of cytoskeletal structures or mitochondria relative to nuclear, cytoplasmic, and / or plasma membrane stains / labels in fibroblastic cell types. Similarly, the polarization of structures or organelles relative to other stains, e.g., labelin 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.

[0100] Exemplary methods for staining tissue structures and guidance in the choice of stains appropriate for various purposes are known in the art and are discussed, for example, in “Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)” and “Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley-Intersciences (1987),” the disclosures of which are incorporated herein by reference. For a description of exemplary labelling agents, reporter oligonucleotides, and methods of use, see, e.g., U.S. Pat. 10,550,429; U.S. Pat. Pub. 20190177800; and U.S. Pat. Pub.20190367969, which are each incorporated by reference herein in their entirety.

[0101] 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 analyte24FH13244839.1GEI-02225100-179000WQ 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).

[0102] 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.

[0103] 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.

[0104] Attachment (coupling) of the reporter oligonucleotides to the labelling agents may be achieved through any of a variety of direct or indirect, covalent or non-covalent associations or attachments. For example, oligonucleotides may be covalently attached to a portion of a labelling agent (such a protein, e.g., an antibody or antibody fragment) using chemical conjugation techniques (e.g., Lightning -Link® antibody labelling kits available from Innova Biosciences), as well as other non-covalent attachment mechanisms, e.g., using biotinylated antibodies and oligonucleotides (or beads that include one or more biotinylated linker, coupled to oligonucleotides) with an avidin or streptavidin linker. Antibody and oligonucleotide biotinylation techniques are available. See, e.g., Fang, et al., “Fluoride-Cleavable Biotinylation25FH13244839.1GEI-02225100-179000WO 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).

[0105] 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.26FH13244839.1GEI-02225100-179000WQ

[0106] 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 labelling agent comprising an analyte binding moiety that binds to the cell surface protein and an analyte binding moiety barcode that identifies that analyte binding moiety. Results of protein analysis in a sample (e.g., a tissue sample or a cell) can be associated with DNA and / or RNA analysis in the sample.

[0107] Assays for in situ detection and analysis:

[0108] 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.

[0109] 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.,27FH13244839.1GEI-02225100-179000WQ DNA) or gene transcript sequences (e.g., mRNA) sequences, and visualized as diffractionlimited spots by fluorescence microscopy (Levsky, et al. (2003) “Fluorescence In situ Hybridization: Past, Present and Future”, Journal of Cell Science 116(14):2833-2838; Raj, et al. (2008) “Imaging Individual mRNA Molecules Using Multiple Singly Labeled Probes”, Nat Methods 5(10): 877-879; Moor, et al. (2016), ibid.). Variations on the smFISH method include, for example, the use of combinatorial labelling schemes to improve multiplexing capability (Levsky, et al. (2003), ibid.), the use of smFISH in combination with super-resolution microscopy (Lubeck, et al. (2014) “Single-Cell In situ RNA Profiling by Sequential Hybridization”, Nature Methods 11(4):360— 361 ).

[0110] MERFISH addresses two of the limitations of earlier in situ hybridization approaches, namely the limited number of target sequences that could be simultaneously identified and the robustness of the approach to readout errors caused by the stochastic nature of the hybridization process (Moor, et al. (2016), ibid.). MERFISH utilizes a binary barcoding scheme in which the probed target mRNA sequences are either fluorescence positive or fluorescence negative for any given imaging cycle (Ke, et al. (2016), ibid.; Moffitt, et al. (2016) “RNA Imaging with Multiplexed Error Robust Fluorescence In situ Hybridization”, Methods Enzymol. 572: 1 49). The encoding probes that contain a combination of target-specific hybridization sequence regions and barcoded readout sequence regions are first hybridized to the target mRNA sequences. In each imaging cycle, a subset of fluorophore-conjugated readout probes is hybridized to a subset of encoding probes. Target mRNA sequences that fluoresce in a given cycle are assigned a value of “1” and the remaining target mRNA sequences are assigned a value of “0”. Between imaging cycles, the fluorescent probes from the previous cycle are photobleached. After, e.g., 14 or 16 rounds of readout probe hybridization and imaging, unique combinations of the detected fluorescence signals generate a 14-bit or 16-bit code that identifies the different gene transcripts. To address the increased error rate for correctly calling the readout codes increases as the number of hybridization and imaging cycles increases, the method may also entail the use of Hamming distances for barcode design and correction of decoding errors (see., e.g., Buschmann, et al. (2013) “Levenshtein Error-Correcting Barcodes for Multiplexed DNA Sequencing”, Bioinformatics 14:272), thereby resulting in an error-robust barcoding scheme.28FH13244839.1GEI-02225100-179000WQ

[0111] 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.).

[0112] 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 micrometersized 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.

[0113] Products of endogenous analytes and / or labelling agents:

[0114]

[0001] 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 ligation29FH13244839.1GEI-02225100-179000WO product, an extension product (e.g., by a DNA or RNA polymerase), a replication product, a transcription / reverse transcription product, and / or an amplification product such as a rolling circle amplification (RCA) product) of a labelling agent that directly or indirectly binds to an analyte in the biological sample is analyzed.

[0115] In some instances, the analyzing comprises using primary probes which comprise a target binding region (e.g., a region that binds to a target such as RNA transcripts) and the primary probes may contain one or more barcodes (e.g., primary barcode). In some instances, the barcodes are bound by detection primary probes, which do not need to be fluorescent, but that include a target-binding portion (e.g., for hybridizing to one or more primary probes) and one or more barcodes (e.g., secondary barcodes). In some instances, the detection primary probe comprises an overhang that does not hybridize to the target nucleic acid but hybridizes to another probe. In some examples, the overhang comprises the barcode(s). In some instances, the barcodes of the detection primary probes are targeted by detectably labeled detection oligonucleotides, such as fluorescently labeled oligos. In some instances, one or more decoding schemes are used to decode the signals, such as fluorescence, for sequence determination.Various probes and probe sets can be used to hybridize to and detect an endogenous analyte and / or a sequence associated with a labelling agent. In some instances, these assays may enable multiplexed detection, signal amplification, combinatorial decoding, and error correction schemes. Exemplary barcoded probes or probe sets may be based on a padlock probe, a gapped padlock probe, a SNAIL (Splint Nucleotide Assisted Intramolecular Ligation) probe set, a PLAYR (Proximity Ligation Assay for RNA) probe set, a PLISH (Proximity Ligation in situ Hybridization) probe set. The specific probe or probe set design can vary

[0116] Hybridization and ligation:

[0117] 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.

[0118] 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 a30FH13244839.1GEI-02225100-179000WO circularizable probe or probe set upon hybridization to a target sequence. In some instances, the ligation product is formed between two or more endogenous analytes. In some instances, the ligation product is formed between an endogenous analyte and a labelling agent. In some instances, the ligation product is formed between two or more labelling agent. In some instances, the ligation product is an intramolecular ligation of an endogenous analyte. In some instances, the ligation product is an intramolecular ligation of a labelling agent, for example, the circularization of a circularizable probe or probe set upon hybridization to a target sequence. The target sequence can be comprised in an endogenous analyte (e.g., nucleic acid such as a genomic DNA or mRNA) or a product thereof (e.g., cDNA from a cellular mRNA transcript), or in a labelling agent (e.g., the reporter oligonucleotide) or a product thereof.

[0119] 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.

[0120] 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.

[0121] 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 variety31FH13244839.1GEI-02225100-179000WQ 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.

[0122] In some instances, the ligation herein is a direct ligation. In some instances, the ligation herein is an indirect ligation. "Direct ligation" means that the ends of the polynucleotides hybridize immediately adjacently to one another to form a substrate for a ligase enzyme resulting in their ligation to each other (intramolecular ligation). Alternatively, "indirect" means that the ends of the polynucleotides hybridize non- adjacently to one another, i.e., 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. In32FH13244839.1GEI-02225100-179000WQ some instances, the ligation herein is preceded by gap filling. In other implementations, the ligation herein does not require gap filling.

[0123] 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.

[0124] 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 basepaired 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.

[0125] 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 from each nucleic acid molecule because both nucleic acid molecules lack a single-stranded overhang at the site of ligation.

[0126] Primer extension and amplification:

[0127] 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 or33FH13244839.1GEI-02225100-179000WQ 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.

[0128] 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.

[0129] 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). The34FH13244839.1GEI-02225100-179000WG RCA product is a concatemer containing tens to hundreds of tandem repeats that are complementary to the circular template, and may be used to develop sensitive techniques for the detection of a variety of targets, including nucleic acids (DNA, RNA), small molecules, proteins, and cells (Ali, et al. (2014), ibid.). In some implementations, a primer that hybridizes to the circular probe or circularized probe is added and used as such for amplification. In some instances, the RCA comprises a linear RCA, a branched RCA, a dendritic RCA, or any combination thereof.

[0130] 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.

[0131] 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 (i.e., amplicon) containing multiple copies of the cDNA. Techniques for rolling circle amplification (RCA) are known in the art such as linear RCA, a branched RCA, a dendritic RCA, or any combination thereof. (See, e.g., Baner et al, Nucleic Acids Research, 26:5073-5078, 1998; Lizardi et al, Nature Genetics 19:226, 1998; Mohsen et al., Acc Chem Res.2016 November 15; 49(11): 2540-2550; Schweitzer et al. Proc. Natl Acad. Sci. USA 97:101 13-1 19, 2000; Faruqi et al, BMC Genomics 2:4, 2000; Nallur et al, Nucl. Acids Res. 29:el 18, 2001; Dean et al. Genome Res. 1 1 :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.35FH13244839.1GEI-02225100-179000WQ

[0132] 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.

[0133] In some instances, the RCA template may comprise the target analyte, or a part thereof, where the target analyte is a nucleic acid, or it may be provided or generated as a proxy, or a marker, for the analyte. In some instances, the RCA template may comprise a sequence of the probes and probe sets hybridized to an endogenous analyte and / or a labelling agent. In some instances, the amplification product can be generated as a proxy, or a marker, for the analyte. As noted above, many assays are known for the detection of numerous different analytes, which use a RCA-based detection system, e.g., where the signal is provided by generating a RCP from a circular RCA template which is provided or generated in the assay, and the RCP is detected to detect the analyte. The RCP may thus be regarded as a reporter which is detected to detect the target analyte. However, the RCA template may also be regarded as a reporter for the target analyte; the RCP is generated based on the RCA template, and comprises complementary copies of the RCA template. The RCA template determines the signal which is detected, and is thus indicative of the target analyte. As will be described in more detail below, the 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 (z.e. a reagent in a detection assay), which is used as a reporter for the assay, or a part of a reporter, or signal-generation system. The RCA template used to generate the RCP may thus be a circular (e.g. circularized) reporter nucleic acid molecule, namely from any RCA-based detection assay which uses or generates a circular nucleic acid molecule as a reporter for the assay. Since the RCA template generates the RCP reporter, it may be viewed as part of the reporter system for the assay.36FH13244839.1GEI-02225100-179000WQ

[0134] 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).

[0135] Signal amplification methods:

[0136] In some instances, a method disclosed herein may also comprise one or more signal amplification components and detecting such signals. In some instances, the present disclosure relates to the detection of nucleic acid sequences in situ using probe hybridization and generation of amplified signals associated with the probes. In some instances, the target nucleic acid of a nucleic acid probe comprises multiple target sequences for nucleic acid probe hybridization, such that the signal corresponding to a barcode sequence of the nucleic acid probe is amplified by the presence of multiple nucleic acid probes hybridized to the target nucleic acid. For example, multiple sequences can be selected from a target nucleic acid such as an mRNA, such that a group of nucleic acid probes (e.g., 20-50 nucleic acid probes) hybridize to the mRNA in a tiled fashion. In another example, the target nucleic acid can be an amplification product (e.g., an RCA product) comprising multiple copies of a target sequence (e.g., a barcode sequence of the RCA product).37FH13244839.1GEI-02225100-179000WO

[0137] 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).

[0138] 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 inUS 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.

[0139] 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 / 0376956,38FH13244839.1GEI-02225100-179000WQ WO 2019 / 236841, WO 2020 / 102094, WO 2020 / 163397, and WO 2021 / 067475, all of which are incorporated herein by reference in their entireties.

[0140] In some instances, hybridization chain reaction (HCR) can be used for signal amplification. HCR is an enzyme-free nucleic acid amplification based on a triggered chain of hybridization of nucleic acid molecules starting from HCR monomers, which hybridize to one another to form a nicked nucleic acid polymer. This polymer is the product of the HCR reaction which is ultimately detected in order to indicate the presence of the target analyte. HCR is described in detail in Dirks and Pierce, 2004, PNAS, 101(43), 15275-15278 and in US 7,632,641 and US 7,721,721 (see also US 2006 / 00234261; Chemeris et al, 2008 Doklady Biochemistry and Biophysics, 419, 53-55; Niu et al, 2010, 46, 3089-3091; Choi et al, 2010, Nat. Biotechnol.28(11), 1208-1212; and Song et al, 2012, Analyst, 137, 1396-1401). HCR monomers typically comprise a hairpin, or other metastable nucleic acid structure. In the simplest form of HCR, two different types of stable hairpin monomer, referred to here as first and second HCR monomers, undergo a chain reaction of hybridization events to form a long nicked double-stranded DNA molecule when an “initiator” nucleic acid molecule is introduced. The HCR monomers have a hairpin structure comprising a double stranded stem region, a loop region connecting the two strands of the stem region, and a single stranded region at one end of the double stranded stem region. The single stranded region which is exposed (and which is thus available for hybridization to another molecule, e.g. initiator or other HCR monomer) when the monomers are in the hairpin structure may be known as the “toehold region” (or “input domain”). The first HCR monomers each further comprise a sequence which is complementary to a sequence in the exposed toehold region of the second HCR monomers. This sequence of complementarity in the first HCR monomers may be known as the “interacting region” (or “output domain”). Similarly, the second HCR monomers each comprise an interacting region (output domain), e.g. a sequence which is complementary to the exposed toehold region (input domain) of the first HCR monomers. In the absence of the HCR initiator, these interacting regions are protected by the secondary structure (e.g. they are 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. This39FH13244839.1GEI-02225100-179000WQ 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.

[0141] 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.

[0142] 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 the40FH13244839.1GEI-02225100-179000WQ 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.

[0143] In some instances, detection of nucleic acids sequences in situ includes combination of the sequential decoding methods described herein with an assembly for branched signal amplification. In some instances, the assembly complex comprises an amplifier hybridized directly or indirectly (via one or more oligonucleotides) to a sequence of an oligonucleotide probe described herein. In some instances, the assembly includes one or more amplifiers each including an amplifier repeating sequence. In some aspects, the one or more amplifiers is labeled. Described herein is a method of using the aforementioned assembly, including for example, using the assembly in multiplexed error-robust fluorescent in situ hybridization (MERFISH) applications, with branched DNA amplification for signal readout. In some instances, the amplifier repeating sequence is about 5-30 nucleotides, and is repeated N times in the amplifier. In some instances, the amplifier repeating sequence is about 20 nucleotides, and is repeated at least two times in the amplifier. In some aspects, the one or more amplifier repeating sequence is labeled. For exemplary branched signal amplification, see e.g., U.S. Pat. Pub. No. US20200399689A1 and Xia et al., Multiplexed Detection of RNA using MERFISH and branched DNA amplification. Scientific Reports (2019), each of which is fully incorporated by reference herein.

[0144] 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 a41FH13244839.1GEI-02225100-179000WQ 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).

[0145] Barcoded analytes and detection:

[0146] 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.

[0147] 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.

[0148] 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, oligonucleotidestreptavidin conjugates, modified oligonucleotides, affinity purification, detectable moieties,42FH13244839.1GEI-02225100-179000WQ enzymes, enzymes for detection assays or other functionalities, and / or for detection and identification of the polynucleotide.

[0149] 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).

[0150] 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 to43FH13244839.1GEI-02225100-179000WQ 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.

[0151] Sequential hybridization:

[0152] In some instances, the present disclosure relates to methods and compositions for encoding and detecting analytes in a temporally sequential manner for in situ analysis of an analyte in a biological sample, e.g., a target nucleic acid in a cell in an intact tissue. In some aspects, provided herein is a method for detecting the detectably-labeled probes, thereby generating a signal signature. In some instances, the signal signature corresponds to an analyte of the plurality of analytes. In some instances, the methods described herein are based, in part, on the development of a multiplexed biological assay and readout, in which a sample is first contacted with a plurality of nucleic acid probes comprising one or more probe types (e.g., labelling agent, circularizable probe, circular probe, etc.), allowing the probes to directly or indirectly bind target analytes, which may then be optically detected (e.g., by detectably-labeled probes) in a temporally-sequential manner. In some instances, the probes or probe sets comprising various probe types may be applied to a sample simultaneously. In some instances, the probes or probe sets comprising various probe types may be applied to a sample sequentially. In some aspects, the method comprises sequential hybridization of labelled probes to create a spatiotemporal signal signature or code that identifies the analyte.

[0153] 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 may44FH13244839.1GEI-02225100-179000WO be applied to a sample sequentially. In some instances, the method comprises detecting a plurality of analytes in a sample.

[0154] 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.

[0155] In any of the implementations disclosed herein, the detection of signals can be performed sequentially in cycles, one for each distinct label. In any of the implementations disclosed herein, signals or absence thereof from detectably-labeled probes targeting an analyte in a particular location in the sample can be recorded in a first cycle for detecting a first label, and signals or absence thereof from detectably-labeled probes targeting the analyte in the particular location can be recorded in a second cycle for detecting a second label distinct from the first label. In any of the implementations disclosed herein, a unique signal signature can be generated for each analyte of the plurality of analytes. In any of the implementations disclosed herein, one or more molecules comprising the same analyte or a portion thereof can be associated with the same signal signature.

[0156] 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, in45FH13244839.1GEI-02225100-179000WQ 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.

[0157] 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.

[0158] 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 and46FH13244839.1GEI-02225100-179000WQ 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).

[0159] 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, photobleaching, 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.

[0160] 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.

[0161] 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 that47FH13244839.1GEI-02225100-179000WO 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 (z.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.

[0162] After a signal is inactivated and / or removed, then the sample is re-hybridized in a subsequent round with a subsequent fluorescently labeled oligonucleotide, and the oligonucleotide can be labeled with the same color or a different color as the fluorescently labeled oligonucleotide of the previous cycle. In some instances, as the positions of the analytes, probes, and / or products thereof can be fixed (e.g., via fixing and / or crosslinking) in a sample, the fluorescent spot corresponding to an analyte, probe, or product thereof remains in place during multiple rounds of hybridization and can be aligned to read out a string of signals associated with each target analyte.

[0163] Decoding:

[0164] 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 target48FH13244839.1GEI-02225100-179000WO 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.

[0165] Anchor probes:

[0166] 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.

[0167] 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.

[0168] 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”).49FH13244839.1GEI-02225100-179000WQ 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).

[0169] In various embodiments, the opto-fluidic instrument is configured to analyze one or more target molecules in their naturally occurring place (z.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.

[0170] 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., an50FH13244839.1GEI-02225100-179000WQ 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.

[0171] The biological sample can include any number of macromolecules, for example, cellular macromolecules and organelles (e.g., mitochondria and nuclei). The biological sample can be 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.

[0172] 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.

[0173] 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 required51FH13244839.1GEI-02225100-179000WQ 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.

[0174] 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.

[0175] 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.

[0176] 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 that52FH13244839.1GEI-02225100-179000WQ is then inserted into the SIM of the sample module 160. In some instances, the sample module 160 may also include an X-Y stage onto which the SIM is mounted. The X-Y stage may be configured to move the SIM mounted thereon (e.g., and as such the sample device containing the sample 110 inserted therein) in perpendicular directions along the two-dimensional (2D) plane of the opto-fluidic instrument 120.

[0177] 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.

[0178] 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).

[0179] 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 a53FH13244839.1GEI-02225100-179000WQ 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.

[0180] 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., one or more light source such as LEDs), an objective lens, and / or the like. The optics module 150 may include a fluorescence imaging system that is configured to image the fluorescence emitted by the probes (e.g., oligonucleotides) in the sample 110 after the probes are excited by light from the illumination module of the optics module 150.

[0181] 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.

[0182] 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.54FH13244839.1GEI-02225100-179000WQ

[0183] 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.

[0184] 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 an 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 light 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.

[0185] 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.

[0186] 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 microfluidic55FH13244839.1GEI-02225100-179000WO 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.

[0187] 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).

[0188] 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.

[0189] The one or more materials used to fabricate sample carrier devices for the disclosed systems (e.g., substrates configured to receive a sample, microscope slides, substrates comprising one or more etched microfluidic channels, flow cells or microfluidic devices comprising one or more microfluidic channels or sample chambers, etc.) can be optically transparent to facilitate use with spectroscopic or imaging-based detection techniques. In some instances, the entire sample carrier device can be optically transparent. Alternatively, in some instances, only a portion of the sample carrier device (e.g., an optically transparent “window”) can be optically transparent.

[0190] The sample carrier devices for the disclosed systems (e.g., substrates configured to receive a sample, microscope slides, substrates comprising one or more etched microfluidic56FH13244839.1GEI-02225100-179000WQ 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).

[0191] 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. 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).

[0192] 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. In57FH13244839.1GEI-02225100-179000WQ various embodiments, the one or more emission filters 265 are configured to filter light from the sample (e.g., emitted from one or more fluorophores, autofluorescence, etc.) for a predetermined range of wavelengths (e.g., each filter has one or more blocking band(s) and / or transmission band(s) that may be different or may overlap at least in part). In various embodiments, the emission filters 265 align (e.g., via motorized translation) with optics and / or the sensor array. In various embodiments, the sample 230 is probed with fluorescent probes configured to bind to a target (e.g., DNA or RNA) that, when illuminated with a particular wavelength (or range of wavelengths) of light, emit light signals that can be detected by the sensor array 260. In various embodiments, the sample 230 is repeatedly probed with two or more (e.g., two, three, four, five, six, etc.) different sets of probes. In various embodiments, each set of probes corresponds to a specific color (e.g., blue, green, yellow, or red) such that, when illuminated by that color, probes bound to a target emit light signals. In some embodiments, the sensor array 260 is aligned with the optical axis 251 of the objective lens 220 (i.e., the optical axis of the camera is coincident with and parallel to the optical axis of the objective lens 220). In various embodiments, the sensor array 260 is positioned perpendicularly to the objective lens 220 (i.e., the optical axis of the camera is perpendicular to and intersects the optical axis of the objective lens 220). In various embodiments, a tube lens 261 is mounted in the optical path to focus light on the sensor array 260 thereby allowing for image formation with infinity-corrected objectives. Descriptions of optical modules and illumination assemblies for use in opto-fluidic instruments can be found in U.S. 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.

[0193] In various embodiments, the sample is illuminated with one or more wavelengths configured to induce fluorescence in the sample. In various embodiments, the sample is probed during one or more probing cycles with one or more fluorescent probes configured to bind to one or more target analytes. In various embodiments, the one or more wavelengths are selected to induce fluorescence in a subset of the one or more fluorescent probes. In various embodiments, each probing cycle includes illumination with two or more (e.g., four) colors of light. In various embodiments, the sample is treated with a fluorescent stain configured to illuminate one or more structures within the sample. In various embodiments, the sample is contacted with a nuclear stain. In various embodiments, the sample is contacted with 4',6-diamidino-2-phenylindole (“DAPI”) configured to bind to adenine-thymine-rich regions in DNA. In various embodiments,58FH13244839.1GEI-02225100-179000WQ 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.

[0194] 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.

[0195] 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.

[0196] In computing node 10 there is a computer system / server 12, which is operational with numerous other general purpose or special purpose computing system environments or configurations. Examples of well-known computing systems, environments, and / or configurations that may be suitable for use with computer system / server 12 include, but are not limited to, personal computer systems, server computer systems, thin clients, thick clients, handheld or laptop devices, multiprocessor systems, microprocessor-based systems, set 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.

[0197] Computer system / server 12 may be described in the general context of computer systemexecutable instructions, such as program modules, being executed by a computer system.59FH13244839.1GEI-02225100-179000WQ 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.

[0198] 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.

[0199] 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.

[0200] 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.

[0201] System memory 28 can include computer system readable media in the form of volatile memory, such as random access memory (RAM) 30 and / or cache memory 32. Computer system / server 12 may further include other removable / non-removable, volatile / non-volatile computer system storage media. By way of example only, storage system 34 can be provided for reading from and writing to a non-removable, non-volatile magnetic media (not shown and typically called a “hard drive”). Although not shown, a magnetic disk drive for reading 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,60FH13244839.1GEI-02225100-179000WO 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.

[0202] 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 as described herein.

[0203] 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.

[0204] The present invention may be a system, a method, and / or a computer program product. 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 invention.

[0205] 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 specific61FH13244839.1GEI-02225100-179000WQ examples of the computer readable storage medium includes the following: a portable computer diskette, a hard disk, a random access memory (RAM), a read-only memory (ROM), an erasable programmable read-only memory (EPROM or Flash memory), a static random access memory (SRAM), a portable compact disc read-only memory (CD-ROM), a digital versatile disk (DVD), a memory stick, a floppy disk, a mechanically encoded device such as punch-cards or raised structures in a groove having instructions recorded thereon, and any suitable combination of the foregoing. A computer readable storage medium, as used herein, is not to be construed as being transitory signals per se, such as radio waves or other freely propagating electromagnetic waves, electromagnetic waves propagating through a waveguide or other transmission media (e.g., light pulses passing through a fiber-optic cable), or electrical signals transmitted through a wire.

[0206] 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.

[0207] Computer readable program instructions for carrying out operations of the present invention 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 computer62FH13244839.1GEI-02225100-179000WG (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 invention.

[0208] Aspects of the present invention are described herein with reference to flowchart illustrations and / or block diagrams of methods, apparatus (systems), and computer program products according to embodiments. 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.

[0209] These computer readable program instructions may be provided to a processor of a general purpose computer, special purpose computer, or other programmable data processing apparatus to produce a machine, such that the instructions, which execute via the processor of the computer or other programmable data processing apparatus, create means for implementing the functions / acts specified in the flowchart and / or block diagram block or blocks. These computer readable program instructions may also be stored in a computer readable storage medium that can direct a computer, a programmable data processing apparatus, and / or other devices to function in a particular manner, such that the computer readable storage medium having instructions stored therein comprises an article of manufacture including instructions which implement aspects of the function / act specified in the flowchart and / or block diagram block or blocks.

[0210] 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.

[0211] Dynamic Volumetric Imaging based on Detected Target Analyte Density

[0212] Without being bound by theory or methodology, in situ detection and decoding are processes including a plurality of decoding cycles in each of which a different set of barcode probes (e.g., fluorescently-labeled oligonucleotides) is contacted with target analytes (e.g.,63FH13244839.1GEI-02225100-179000WG mRNA sequences) or with target barcodes (e.g., nucleic acid barcodes) associated with the target analytes present in a sample (e.g., a tissue sample) under conditions that promote hybridization. One or more images (e.g., fluorescence images) are acquired in each decoding cycle, and the images are processed to detect the presence and locations of one or more barcode probes in each cycle. The presence and locations of one or more target analyte sequences or associated barcode sequences are then inferred from corresponding code words that are determined based on the set of, e.g., fluorescence signals detected in each decoding cycle of the decoding process.

[0213] Immunohistochemistry (IHC) and in situ hybridization (ISH) methodologies are well known in the art and have been multiplexed to a degree corresponding to the number of dyes that can be differentiated during observation, e.g., microscopy. However, performing spatial molecular measurements at the “omic” level by simply increasing the number of dyes is not possible. Some of the key objectives in spatial omics are to detect, quantify, and map genes and their activity to specific regions in a tissue sample at cellular or sub- cellular resolution.Innovative methods for performing spatial omic analyses on tissue samples may rely on ex situ technologies, such as next generation sequencing (NGS) and combinatorial barcoding to assign biomolecules to their spatial locations. Direct imaging-based methods, such as in-situ sequencing (IS S), detect biomolecules through, for example, hybridization of labeled probes and are identified according to patterns of fluorescence signal, thereby allowing for precise spatial localization in tissue, cells, and / or subcellular spaces. In situ genome sequencing (IGS) employs DNA sequencing directly within the biological sample, essentially combining ISS and NGS techniques to localize paired-end sequences in their endogenous biological space. However, these spatial sequencing systems ultimately rely on fluorescence microscopy to detect the presence or absence of clusters of fluorescent dye molecules on biological samples. The time spent imaging these dye-clusters (blobs) may represent a limitation to the throughput of such a system. Therefore, it is desirable to maximize the efficiency of the imaging data collected.

[0214] Methods, systems, and computer program products are provided herein for maximizing the efficiency of the imaging data collected when performing spatial molecular measurements on, for example and without limitation, prepared tissue sections. Such molecular measurement methodologies may include in-situ sequencing (IGS) systems and ex-situ next-generation sequencing (NGS) systems and in-situ sequencing systems (e.g., in-situ sequencing (ISS) and in-situ genome sequencing (IGS)).64FH13244839.1GEI-02225100-179000WG

[0215] For example, for NGS techniques and systems, which sequence nucleic acids introduced in solution form, the instrument, assay, and optical systems (e.g., opto-fluidic instrument) are designed to maximize throughput. Such throughput maximization is often achieved by limiting the size of the blobs to the minimum size resolvable by the optical system and / or by positioning the target nucleic acid molecules to produce blobs as tightly spaced as possible using a template. A combination of such size minimization and density maximization may result in high efficiency imaging data and high throughput.

[0216] As another example, for ISS and IGS techniques and systems, which sequence nucleic acids that may be immobilized in a 3 -dimensional matrix of material, the size of the blobs is often controlled. However, the spatial context of the blobs in in-situ sequencing is relevant to the analysis. Thus, the spatial context of the blobs in ISS and IGS cannot be disturbed or manipulated in service of maximizing throughput as is done in NGS.

[0217] In ISS and IGS techniques and systems, blobs may be immobilized in a 3-D matrix. This means that volumetric (or 3-D) imaging may be required to localize the blobs in space. In ISS and IGS techniques and systems, blob density may be modulated by design of the assay. For a typical assay, if all target nucleic acids were tagged dye molecules at the same time, the object density would exceed the resolving power of the optical system. To avoid this, the assay may be designed to image all the target molecules across several imaging -cycles, with only a fraction of target molecules hybridized with dye-molecules in a given imaging-cycle. Using this approach, there may be variability in the blob-density for a given sample due to biological variation and the limitations of assay design. Reducing the pixel size may increase the blob density that can be resolved. For a z-stack of images, X and Y pixel sizes may be defined by the design of the optical system (e.g., an opto-fluidic instrument) and selection of components (e.g., a camera), however, the Z pixel size may be defined by the objective movement between successive images (z-slices) taken to form an imaging volume. As used herein, a point density may be a number of points or clusters in a given area. For example, a point density may be the number of blobs in an image corresponding to a particular field of view.

[0218] FIG.6 depicts a process 600 for imaging a volume of a sample, such as a biological sample or hydrogel, to generate a plurality of z-stacks of images at a plurality of FOVs. As shown in FIG. 6, z-stacks of images of a sample are acquired by an imaging instrument or retrieved from another source (e.g., a local or remote database). As used herein, a z-stack of65FH13244839.1GEI-02225100-179000WO images includes two or more image slices, each slice representing a focal plane having a different z-height from the substrate on which the sample sits. In various embodiments, each slice is assigned a z-index (e.g., the first image slice away from the substrate surface is assigned a z-index of 1, the next image slice). In various embodiments, the z-stack of images is acquired during each of a plurality of probing cycles of an imaging instrument (where emitted light from fluorescently labelled rolling circle products (RCPs) is imaged). For example, the predetermined number of probing cycles may be 15 probing cycles. In another example, where a large number of target analytes are detected, e.g., for whole transcriptome analysis, the number of cycles may be 20, 30, 40, 50, 60, 70, 75, 80, 90, 100, 110, 120, etc.

[0219] In various embodiments, a ZCYX imaging procedure is followed. Specifically, a z-stack of images is obtained at a fixed FOV (with fixed x- and y-values) for each color channel of a plurality of color channels. Then the fixed x- and / or y-values are changed to obtain images at a new FOV, repeating the z-stack imaging for each color channel, and this process continues for the entire tissue sample (or for a selected subset of FOVs). One benefit of this technique is that no image registration may be needed. Also, moving first in the z-direction, without changing the x- or y-values, can help minimize error since moving in the x- and y-directions can produce more error than moving in the z-direction.

[0220] In various embodiments, a ZYXC imaging procedure is followed. Specifically, a z-stack of images is obtained at a fixed FOV (with fixed x- and y-values) for a single color channel before changing the x- and / or y-values to obtain images at a new FOV in the same color channel. After all FOVs are imaged for the entire tissue sample (or for a selected subset of FOVs) in the same color channel, the color channel is switched to the next color channel and a z-stack of images is obtained at all FOVs in the next color channel. One benefit of this method may be that the reduced number of switches between color channels results in less time elapsed for the entire procedure. Switching from one color channel to another color channel can take longer than moving in the z-direction through the sample. Also, moving first in the z-direction, without changing the x- or y-values, can help minimize error since moving in the x- and y-directions can produce more error than moving in the z-direction. Image registration may be required for FOVs across all color channels due to thermal drift in the sample and / or mechanical drift in the motion systems (e.g., z-stage, xy stage, etc.).66FH13244839.1GEI-02225100-179000WO

[0221] Referring back to FIG. 6, in various embodiments, at 602, an imaging instrument captures a z-stack (representing a volume) of images of a FOV of a sample in one (or more) color channel. In various embodiments, the z-stack of images includes a plurality of z-slices (2D images) with a predefined spacing (e.g., delta-z) between adjacent slices in the Z direction (e.g., in the direction along the optical axis of the objective lens, or a direction that is perpendicular to the substrate surface on which the sample is positioned). In various embodiments, the predefined spacing is approximately an axial resolution of the objective associated with the imaging instrument. In various embodiments, the predefined spacing is about 0.20 m to about 2.0 pm. In various embodiments, the predefined spacing is about 0.50 pm to about 1.0 pm. In various embodiments, the predefined spacing is about 0.75 pm. In various embodiments, the predefined spacing is about 0.50 pm. In various embodiments, at 604, the sample is moved in the imaging instrument in the X and / or Y direction to the next FOV to image the next FOV in the one (or more) color channel. In various embodiments, the steps performed at 602 as well as 604 are repeated until, at 606, all FOVs, which include the full sample area (or a predefined subset of FOVs that may be selected by a user or automatically determined by the instrument as having non-detached sample), are imaged / scanned in the one (or more) color channel. In various embodiments, the color channel is changed to a different color channel (e.g., red illumination channel is switched to yellow illumination channel, yellow illumination channel is switched to green illumination channel, green illumination channel is switched to blue illumination channel, blue illumination channel is switched to nUV illumination channel, or vice versa), and 602 is repeated, to image the same set of FOVs (imaged in the first color channel) in the different color channel. In various embodiments, the steps performed at 602 as well as 604 are repeated until, at 606, all FOVs, which include the full sample area (or a predefined subset of FOVs that may be selected by a user or automatically determined by the instrument as having non-detached sample), are imaged / scanned in the one or more additional color channels. In various embodiments, the color channels include two color channel (e.g., red and green, blue and yellow), three color channels, four color channels (e.g., red, yellow, green, and blue), or five color channels (e.g., red, yellow, green, blue, and near ultraviolet).

[0222] FIG.7 depicts a process 700 for imaging an imageable volume including a sample, such as a biological sample or a hydrogel, to generate a plurality of z-stacks of images including one or more z-stacks of images that are interleaved with another z-stack of images of the same FOV.67FH13244839.1GEI-02225100-179000WO As shown in FIG. 7, in various embodiments, at 702, an imaging instrument captures a z-stack (representing a volume) of images of a FOV of a sample in one (or more) color channel. In various embodiments, the z-stack of images includes a plurality of z-slices (2D images) with a first predefined spacing (e.g., delta-z) between adjacent slices in the Z direction (e.g., in the direction along the optical axis of the objective lens, or a direction that is perpendicular to the substrate surface on which the sample is positioned). In various embodiments, the first predefined spacing is approximately an axial resolution of the objective associated with the imaging instrument. In various embodiments, the first predefined spacing is about 0.20 m to about 2.0 pm. In various embodiments, the first predefined spacing is about 0.30 pm to about 1.0 pm. In various embodiments, the first predefined spacing is about 0.75 pm. In various embodiments, the first predefined spacing is about 0.50 pm. In various embodiments, the first predefined spacing is about 0.375 pm. In various embodiments, a motor, controlled by the imaging instrument, and attached to an objective lens (e.g., an objective having a numerical aperture of about 1.0), which is used to capture the plurality of z-stacks of images, is successively moved in the Z direction based on the first predefined spacing and according to the locations / z-indices to be imaged in the z-direction. Alternatively, in various embodiments, the objective lens is stationary and the stage on which the sample is positioned is moved in the Z direction by the predetermined spacing to move the focal plane of the objective through the sample to obtain the z-stacks of images. In various embodiments, at 704, a density metric (e.g., a point density, volume integrated brightness, and / or other metric correlated with the density) is determined based on at least one of the images (z-slices) of the first z-stack captured at 702. In some embodiments, each slice in the z-stack of images is processed individually to determine a density metric for that slice. In some embodiments, the entire z-stack of images is processed to determine a density metric for the FOV from which the z-stack of images is obtained. In various embodiments, a point density is a number of points (e.g., blobs detected from light emissions by tagged molecules, such as RCPs) or clusters of points or diffuse region in a given area. For example, a point density may be the number of blobs detected (using a blob detection algorithm) in a single image of the z-stack of images corresponding to a particular FOV. In various embodiments, the metric is a volume-integrated intensity, a brightness metric (e.g., binned pixel brightness values and a number of pixels in one or more of the bins), a function of the number of blobs in the first FOV, and / or determined based on a machine learning model trained using 68FH13244839.1GEI-02225100-179000WQ known point density information for various tissues and various target analytes. In various embodiments, a mathematical inverse of the imaging process is performed for each z-slice of the first z-stack or for the entirety of the first z-stack, based on the point spread function, to obtain an estimate of the image intensity and / or brightness. In various embodiments, an estimate of the image intensity and / or brightness is based on a frequency space analysis (e.g., a Fourier transform such as a fast Fourier transform) of each z-slice of the first z-stack or for the entirety of the first z-stack. In various embodiments, at 706, the density metric and / or other metric, for either a z-slice of the first z-stack (in a particular FOV) or for the entire first z-stack, is compared to a threshold value (e.g., if the density metric exceeds the threshold value). In various embodiments, if the density metric and / or other metric is beyond (e.g., exceeds) the threshold value, process 700 proceeds to 710. In various embodiments, if the density metric and / or other metric is not beyond (e.g., does not exceed) the threshold value, process 700 proceeds to 708. In various embodiments, at 708, the sample (or the objective lens) is moved in the imaging instrument in the X and / or Y direction to the next FOV to image the next FOV in the same one (or more) color channel. In various embodiments, the steps performed at 702 as well as 708 are repeated until, at 712, all FOVs (or a predefined subset of FOVs that may be selected by a user or automatically determined by the instrument as having non-detached sample), which may include the full sample area, are imaged / scanned for the one (or more) color channel.

[0223] In various embodiments, at 710, the imaging instrument images returns to the first FOV corresponding to the same X and Y location as at 702 to capture a second z-stack (representing a volume) of images of the sample in the same one (or more) color channel as at 702. In various embodiments, the second z-stack of images includes a plurality of z-slices (2D images) with a second predefined spacing (e.g., delta-z) between adjacent slices at locations that are interwoven in the Z direction with the images of the first z-stack captured at 702. In various embodiments, the second predefined spacing is the same as the first predefined spacing. In various embodiments, the second z-stack of images that is interwoven with the first z-stack of images has slices that are about halfway between the slices of the first z-stack of images. Put another way, the z-heights of the slices in the second z-stack of images are offset from the z-heights of the first z-stack of images by about one half of the first predefined spacing. In various embodiments, the z-indices of the slices within the second z-stack of images is halfway between the z-indices of the first z-stack of images. For example, the second z-stack of images may have z-indices of 0.5,69FH13244839.1GEI-02225100-179000WQ 1.5, 2.5, .. 30.5, etc. while the z-indices of the first z-stack are 1, 2, 3, ..., 30, 31, etc. In various embodiments, a motor, controlled by the imaging instrument, and attached to an objective lens, which is used to capture the plurality of z-stacks of images, is successively moved in the Z direction based on the second predefined spacing and according to the locations to be imaged in the Z direction. In various embodiments, the first z-stack of images and the second z-stack of images is combined into a combined single z-stack (representing a volume) of images. In various embodiments, the resulting spacing (delta-z) between adjacent slices in the combined z-stack of images is one half the first predefined spacing. In various embodiments, the combined z-stack of images represents the same volume as the first z-stack of images. In various embodiments, the combined z-stack of images represents a volume that is larger (e.g., 1 or two slices larger) than the original volume represented by the first z-stack of images. In various embodiments, this combined single z-stack includes a finer z-pixel size (the distance between slices is decreased).

[0224] FIG.8 depicts a process 800 for imaging an imageable volume including a sample, such as a biological sample or a hydrogel, to generate a plurality of z-stacks of images including one or more partial z-stacks of images that are interleaved with another z-stack of images of the same FOV. As shown in FIG. 8, in various embodiments, at 802, an imaging instrument captures a z-stack (representing a volume) of images of a FOV of a sample in one (or more) color channel. In various embodiments, the z-stack of images includes a plurality of z-slices (2D images) with a first predefined spacing (e.g., delta-z) between adjacent slices in the Z direction (e.g., in the direction along the optical axis of the objective lens, or a direction that is perpendicular to the substrate surface on which the sample is positioned). In various embodiments, the first predefined spacing is approximately an axial resolution of the objective associated with the imaging instrument. In various embodiments, the first predefined spacing is about 0.20 m to about 2.0 pm. In various embodiments, the first predefined spacing is about 0.30 pm to about 1.0 pm. In various embodiments, the first predefined spacing is about 0.375 pm to about 0.75 pm. In various embodiments, the first predefined spacing is about 0.75 pm. In various embodiments, the first predefined spacing is about 0.75 pm. In various embodiments, the first predefined spacing is about 0.50 pm. In various embodiments, the first predefined spacing is about 0.375 pm. In various embodiments, a motor, controlled by the imaging instrument, and70FH13244839.1GEI-02225100-179000WQ attached to an objective lens (e.g., an objective having a numerical aperture of about 1.0), which is used to capture the plurality of z-stacks of images, is successively moved in the Z direction based on the first predefined spacing and according to the locations / z-indices to be imaged in the z-direction. Alternatively, in various embodiments, the objective lens is stationary and the stage on which the sample is positioned is moved in the Z direction by the predetermined spacing to move the focal plane of the objective through the sample to obtain the z-stacks of images. In various embodiments, at 804, a density metric (e.g., a point density, volume integrated brightness, and / or other metric correlated with the density) is determined based on at least one of the images (z-slices) of the first z-stack captured at 702. In some embodiments, each slice in the z-stack of images is processed individually to determine a density metric for that slice. In some embodiments, the entire z-stack of images is processed to determine a density metric for the FOV from which the z-stack of images is obtained. In various embodiments, a point density is a number of points (e.g., blobs detected from light emissions by tagged molecules, such as RCPs) or clusters of points or diffuse region in a given area. For example, a point density may be the number of blobs detected (using a blob detection algorithm) in a single image of the z-stack of images corresponding to a particular FOV. In various embodiments, the metric is a volume-integrated intensity, a brightness metric (e.g., binned pixel brightness values and a number of pixels in one or more of the bins), a function of the number of blobs in the first FOV, and / or determined based on a machine learning model trained using known point density information for various tissues and various target analytes. In various embodiments, a mathematical inverse of the imaging process is performed for each z-slice of the first z-stack or for the entirety of the first z-stack, based on the point spread function, to obtain an estimate of the image intensity and / or brightness. In various embodiments, an estimate of the image intensity and / or brightness is based on a frequency space analysis (e.g., a Fourier transform such as a fast Fourier transform) of each z-slice of the first z-stack or for the entirety of the first z-stack. In various embodiments, at 806, the density metric and / or other metric either for a z-slice of the first z-stack (in a particular FOV) or for the entire first z-stack is compared to a threshold value (e.g., if the density metric exceeds the threshold value). In various embodiments, if the density metric and / or other metric is beyond (e.g., exceeds) the threshold value, process 800 proceeds to 810. In various embodiments, if the density metric and / or other metric is not beyond (e.g., does not exceed) the threshold value, process 800 proceeds to 808. In various embodiments, at 808, the sample (or71FH13244839.1GEI-02225100-179000WQ the objective lens) is moved in the imaging instrument in the X and / or Y direction to the next FOV to image the next FOV in the same one (or more) color channel. In various embodiments, the steps performed at 802 as well as 808 are repeated until, at 812, all FOVs (or a predefined subset of FOVs that may be selected by a user or automatically determined by the instrument as having non-detached sample), which may include the full biological sample area, are / scanned for the one (or more) color channel.

[0225] In various embodiments, at 810, the imaging instrument images returns to the first FOV corresponding to the same X and Y location as at 802 to capture a second z-stack (representing a volume) of images of the sample in the same one (or more) color channel as at 802. In various embodiments, the second z-stack of images includes a plurality of z-slices (2D images) with a second predefined spacing (e.g., delta-z) between adjacent slices at locations that are interleaved in the Z direction with the images of the first z-stack captured at 802. In various embodiments, the second predefined spacing is the same as the first predefined spacing. In various embodiments, the second z-stack of images that is interleaved with the first z-stack of images has slices that are about halfway between the slices of the first z-stack of images. Put another way, the z-heights of the slices in the second z-stack of images are offset from the z-heights of the first z-stack of images by about one half of the first predefined spacing. In various embodiments, the z-indices of the slices within the second z-stack of images is halfway between the z-indices of the first z-stack of images. For example, the second z-stack of images may have z-indices of 0.5, 1.5, 2.5, ..., 30.5, etc. while the z-indices of the first z-stack are 1, 2, 3, ..., 30, 31, etc. In various embodiments, a motor, controlled by the imaging instrument, and attached to an objective lens, which is used to capture the plurality of z-stack of images, is successively moved in the Z direction based on the second predefined spacing and according to the locations to be imaged in the Z direction. In various embodiments, the second z-stack is based on the assessment performed at 804. In various embodiments, the second z-stack includes z-slices (2D images) in positions adjacent to at least a portion of the z-slice positions of the first z-stack, where the portion of z-slices of the first z-stack of images is assessed to have a density metric and / or other metric beyond (e.g., exceeds) the threshold value. As a result, in various embodiments, the second z-stack generated by technique 800 includes a different number of z-slices (e.g., fewer z-slices), than the second z-stack generated by technique 700. For example, where the first 10 z-slices of the first z-stack of images (which may have 30-40 total slices at a72FH13244839.1GEI-02225100-179000WQ 0.75 m delta-z spacing between slices) are determined to have density metrics that are above the threshold density, the imaging system obtains interleaved slices between adjacent slices of only the first 10 slices and does not image interleaved slices in between the remaining slices of the first z-stack of images. In various embodiments, the interleaved slices may include any suitable number of slices that is less than or equal to the number of slices in the first z-stack of images. In various embodiments, where the delta-z between adjacent slices of the first z-stack of images is large, two or more interweaving slices are obtained between adjacent slices of the first z-stack of images. In various embodiments, the first z-stack of images and the second z-stack of images is combined into a combined single z-stack (representing a volume) of images. In various embodiments, the resulting spacing (delta-z) between adjacent slices in the combined z-stack of images is one half the first predefined spacing. In various embodiments, the combined z-stack of images represents the same volume as the first z-stack of images. In various embodiments, the combined z-stack of images represents a volume that is larger (e.g., 1 or two slices larger) than the original volume represented by the first z-stack of images. In various embodiments, this combined single z-stack includes a finer z-pixel size (the distance between slices is decreased).

[0226] FIG.9 depicts steps to dynamically generate a z-stack having additional interleaved slices, such as technique 800 in FIG. 8. In various embodiments, if there is a-priori knowledge about the density of target analytes within a sample, the z-spacing of the first z-stack as described with reference to FIG.8 is modulated according to the density of the target analytes. For example, if the density of target analytes is relatively low (e.g., as compared to the threshold value from 806), the delta-z of the z-slices of the first z-stack may be made larger. As another example, if the density of target analytes is relatively high (e.g., as compared to the threshold value from 806), the z-spacing of the z-slices of the first z-stack may be made smaller. In various embodiments, where a sample has one or more structures, such as a biological sample that has identifiable regions and / or structures within its 3D volume where a high density of target analytes is expected, the predefined spacing of the first z-stack can be assigned based on the structures (and, therefore, the expected density of the target analytes) of the sample. In various embodiments, such an approach does not need to interweave a first z-stack with a second z-stack as in process 700 of FIG. 7 or process 800 of FIG. 8. Instead, in various embodiments, a73FH13244839.1GEI-02225100-179000WO different predefined spacing is used for each imaging volume (e.g., z-stack captured at a particular FOV and with respect to a particular color channel).

[0227] In various embodiments, the systems and methods presented herein, such as those depicted in FIGS.7 and 8 require that the precision / repeatability of the Z-stage position to be about one third of the minimum z-slice spacing. For example, if minimum z-slice spacing is 300nm, the methods presented herein may require lOOnm accuracy for Z-stage movement (e.g., when the Z-stage settles after a movement to a suitable resting location, the Z-stage resting location is within lOOnm of the target location). In various embodiments, a supplemental high-density scan (to produce the second z-stack of images to be combined with a first z-stack of images for a single FOV in FIGS.7 or 8) is performed. In various embodiments, the supplemental high-density scan follows obtaining of the first z-stack of images in the single FOV, rather than after a scan of the full sample tissue (i.e., after all FOVs are scanned), which may not provide the precision in Z (or X and / or Y) that may be required for optimal instrument performance.

[0228] FIG. 10 is a flowchart 1000 illustrating a first method of capturing, with an imaging instrument, volumetric images of a sample, according to embodiments of the present disclosure. At 1002, by moving an objective of an imaging instrument relative to a sample, a first z-stack of images of the sample may be captured, the first z-stack of images having a first field of view, each image of the first z-stack of images having a z-index within the z-stack. At 1004, a density metric of the first z-stack of images may be determined. The density metric may correspond to a point density of one or more target molecule in the first field of view. At 1006, the metric may be compared with a threshold. At 1008, based on the comparison of the metric with the threshold, a second z-stack may be captured by moving the objective of the imaging instrument relative to the sample. The second z-stack may comprise at least one additional image of the sample within the first field of view, and being interleaved with the first z-stack.

[0229] FIG. 11 is a flowchart 1100 illustrating a second method of capturing, with an imaging instrument, volumetric images of a sample, according to embodiments of the present disclosure. At 1102, a first set of three-dimensional (3D) positional information of a first plurality of biological molecules within a sample may be received. The first set of 3D positional information may be determined from a first z-stack obtained from a first field of view (FOV) of the sample and based on a probing cycle of the sample in an imaging instrument. The first z-stack may74FH13244839.1GEI-02225100-179000WQ comprise a first plurality of image slices. The probing cycle may comprise generating optical signals corresponding to at least some of the first plurality of biological molecules. The first z-stack may define a first imaging volume. At 1104, a first density metric of at least a portion of the first set of 3D positional information may be determined. At 1106, when the first density metric is above a first threshold, the imaging instrument may be caused to capture at least one additional image slice of the sample within the first field of view that is interleaved with the first z-stack.

[0230] FIG. 12 is a flowchart 1200 illustrating a third method of capturing, with an imaging instrument, volumetric images of a sample, according to embodiments of the present disclosure. At 1202, a first z-stack at a first field of view (FOV) of a sample may be imaged. The first z-stack may comprise a first plurality of image slices. The first plurality of image slices may comprise a first spacing between adjacent image slices. At 1204, a second z-stack may be imaged at a second FOV of the sample. The second z-stack may comprise a second plurality of image slices. The second plurality of image slices may comprise a second spacing between adjacent image slices. The first spacing may be different from the second spacing based on a comparison of a first density metric and a second density metric to a predetermined threshold. The first density metric may be associated the first z-stack and the second density metric is associated with the second z-stack.

[0231] The flowchart and block diagrams in the Figures illustrate the architecture, functionality, and operation of possible implementations of systems, methods, and computer program products according to various embodiments of the present invention. In this regard, each block in the flowchart or block diagrams may represent a module, segment, or portion of instructions, which comprises one or more executable instructions for implementing the specified logical function(s). In some alternative implementations, the functions noted in the block may occur out of the order noted in the figures. For example, two blocks shown in succession may, in fact, be executed substantially concurrently, or the blocks may sometimes be executed in the reverse order, depending upon the functionality involved. It will also be noted that each block of the block diagrams and / or flowchart illustration, and combinations of blocks in the block diagrams and / or flowchart illustration, can be implemented by special purpose hardware-based systems that perform the specified functions or acts or carry out combinations of special purpose hardware and computer instructions.75FH13244839.1GEI-02225100-179000WG

[0232] The descriptions of the various embodiments of the present invention have been presented for purposes of illustration, but are not intended to be exhaustive or limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The terminology used herein was chosen to best explain the principles of the embodiments, the practical application or technical improvement over technologies found in the marketplace, or to enable others of ordinary skill in the art to understand the embodiments disclosed herein.

[0233] EXAMPLES

[0234] Example 1 : Modelling shows that Local Density of Decoded RCPs closely matches Expected Local Density of RCPs when using Dynamic Volumetric Imaging

[0235] FIGS. 13A-B show four graphs for each of three modelled experiments. Referring now to FIG. 13 A, the first row of graphs represents a 4-color channel model of a standard protocol performed on an in situ analysis instrument (without dynamic volumetric imaging as described herein) with a mouse brain + add on gene panel on a mouse brain sample. The second row of graphs represents a 4-color channel model of a 5k human multi gene assay performed on an in situ analysis instrument (with dynamic volumetric imaging) with a human liver sample (5k genes will have significantly more signal detected within the imageable volume).

[0236] Referring now to FIG. 13B, the row of graphs represents a 4-color channel model of a 5k human multi gene assay performed on an in situ analysis instrument (with dynamic volumetric imaging) with a human kidney sample (5k genes will have significantly more signal detected within the imageable volume). The dashed diagonal line represents a local density of decoded RCPs with a Q20 score (x-axis) that equals the modelled local density of RCPs that exist (y-axis). In the standard protocol (top row) that include 15 imaging cycles, the solid line sharply moves upwards and away from the dashed line, meaning that the local density of decoded RCPs with a Q20 score is increasing at a slower rate (or staying roughly the same, as shown in the yellow channel) while the modelled local density of RCPs increases. This implies that RCPs are being missed during decoding (or are being decoded at lower Q scores) as modelled RCP density increases. In the bottom row of FIG 13A and the top row of FIG. 13B that include 35 imaging cycles, the solid line remains relatively close to the dashed line and only starts to move upwards and away from the dashed line at significantly greater densities of RCPs than in the standard protocol, meaning that the local density of decoded RCPs with a Q20 score is increasing at a76FH13244839.1GEI-02225100-179000WG slower rate (or staying roughly the same, as shown in the yellow channel) while the modelled local density of RCPs increases. This implies that the methods described herein for dynamic volumetric imaging increase the robustness of decoding (allow for more RCPs to be decoded correctly to a gene) for higher densities of modelled RCPs.

[0237] Example 2: Missed Blobs are Decreased when using Dynamic Volumetric Imaging

[0238] FIGS. 14A-B show four graphs for each of three modelled experiments. Referring now to FIG. 14A, the first row of graphs represents a 4-color channel model of a standard protocol performed on an in situ analysis instrument (without dynamic volumetric imaging as described herein) with a mouse brain + add on gene panel on a mouse brain sample. The second row of graphs represents a 4-color channel model of a 5k human multi gene assay performed on an in situ analysis instrument (with dynamic volumetric imaging) with a human liver sample (5k genes will have significantly more signal detected within the imageable volume).

[0239] Referring now to FIG. 14B, the row of graphs represents a 4-color channel model of a 5k human multi gene assay performed on an in situ analysis instrument (with dynamic volumetric imaging) with a human kidney sample (5k genes will have significantly more signal detected within the imageable volume). Each of the graphs show the fraction of missing blobs after blob detection is performed (y-axis) relative to the modelled local density of RCPs that exist (x-axis). In the standard protocol (top row of FIG. 14A) that include 15 imaging cycles, the solid line shows that 25% of RCPs are missing from localities for densities ranging from about 70 blobs / 100 pmA2 to about 110 blobs / 100 pmA2. Blob detection algorithms can detect the most blobs in the blue color channel (left-most graph) and green color channel (second from left graph) which begin to miss 25% of blobs at a local density of about 100-110 blobs / 100 pmA2, followed by the yellow color channel (second from right graph) and the red color channel (rightmost graph), which begin to miss 25% of blobs at a local density of about 70-90 blobs / 100 pmA2. One possible explanation for this is diffraction limited imaging because the wavelength of blue is shorter than green is shorter than yellow is shorter than red. Thus, blue light should have a higher optical resolution and be able to discern between two closely arranged point light sources. In the second row of FIG. 14A and the row of FIG. 14A that include 35 imaging cycles, the solid line remains substantially under the 25% cutoff of missing blobs in the blue color channel (left-most graph) and green color channel (second from left graph) for higher densities of up to 250-310 blobs / 100 mA2, and the missing blobs do not equal 25% until significantly77FH13244839.1GEI-02225100-179000WO higher densities of 180-250 blobs / 100 mA2 in the yellow color channel (second from right graph) and the red color channel (right most graph). This implies that the methods described herein for dynamic volumetric imaging increase the robustness of blob detection (allow for more individual RCPs to be detected) for higher densities of modelled RCPs.

[0240] Example 3: Increased Maximum Q20 Decoded Local Density when using Dynamic Volumetric Imaging

[0241] As shown in FIGS. 15A-B, the methods described herein improve the maximum local density of RCPs that are decoded with a Q20 score. In particular, using a standard protocol on an in situ analysis instrument (without dynamic volumetric imaging as described herein) with a mouse brain + add on gene panel on a mouse brain sample, the expected maximum local density of RCPs decoded with a Q20 score shows a consistent range between about 100 blobs / 100 mA2 to about 200 blobs / 100 mA2 in all four color channels (z.e., red, yellow, green, and blue). When applying the methods described herein on the same in situ analysis instrument using a 5k human multi gene assay (with dynamic volumetric imaging) with a human liver sample (5k genes will have significantly more signal detected within the imageable volume), the expected maximum local density of RCPs decoded with a Q20 score shows an increase in the range to between about 100 blobs / 100 mA2 to about 300 blobs / 100 mA2 in all four color channels (z.e., red, yellow, green, and blue). Similarly, when applying the methods described herein on the same in situ analysis instrument using a 5k human multi gene assay (with dynamic volumetric imaging) with a human kidney sample (5k genes will have significantly more signal detected within the imageable volume), the expected maximum local density of RCPs decoded with a Q20 score shows an significant increase in the range to between about 200 blobs / 100 mA2 to above 300 blobs / 100 mA2 in all four color channels (z.e., red, yellow, green, and blue).78FH13244839.1

Claims

1. GEI-02225100-179000WO CLAIMSWhat is claimed is:

1. A method comprising :capturing, by moving an objective of an imaging instrument relative to a sample, a first z- stack of images of the sample, the first z-stack of images having a first field of view, each image of the first z-stack of images having a z-index within the z-stack;determining a density metric of the first z-stack of images, wherein the density metric corresponds to a point density of one or more target molecule in the first field of view;comparing the metric with a threshold; andbased on the comparison of the metric with the threshold, capturing a second z-stack by moving the objective of the imaging instrument relative to the sample, the second z-stack comprising at least one additional image of the sample within the first field of view, and being interleaved with the first z-stack.

2. The method of claim 1 , wherein moving the objective of the imaging instrument relative to the sample comprises moving the sample.

3. The method of claim 1, wherein the first z-stack of images is captured based on at least one probing cycle of the sample in an imaging instrument.

4. The method of claim 1, wherein the first z-stack of images comprises images of the sample stained with one or more fluorescent stain.

5. The method of claim 4, wherein the one or more fluorescent stains comprises a nuclear stain.

6. The method of claim 1, wherein a z spacing of the first z-stack is at most a micron.

7. The method of claim 1, wherein the first z-stack and the second z-stack correspond to a same color channel.

8. The method of claim 1, wherein a z spacing of the first z-stack is predetermined.79FH13244839.1GEI-02225100-179000WO9. The method of claim 8, wherein the z spacing corresponds to a minimum movement of the objective of the imaging instrument relative to the sample.

10. The method of claim 1 further comprising determining a z spacing of the second z-stack based on the point density.

11. The method of claim 1, wherein the metric is a brightness.

12. The method of claim 1, further comprising identifying a plurality of objects in the first field of view, and wherein the metric is a function of a count of the plurality of objects.

13. The method of claim 1, further comprising determining the metric from a trained machine learning model.

14. The method of claim 1, wherein a z spacing that corresponds to the second z-stack is the same as a z spacing of the first z-stack.

15. The method of claim 1, wherein a z spacing that corresponds to the second z-stack is smaller than a z spacing of the first z-stack.

16. The method of claim 1, wherein a z spacing of a combination of the first and the second z- stacks is at least 50 nm.

17. A computer program product comprising a computer readable storage medium having program instructions embodied therewith, the program instructions executable by a processor to cause the processor to perform a method according to any one of claims 1-16.

18. A system comprising:an imaging instrument; anda computing node operatively coupled to the imaging instrument and comprising a80FH13244839.1GEI-02225100-179000WO computer readable storage medium having program instructions embodied therewith, the program instructions executable by a processor to cause the processor to perform a method according to any one of claims 1-16.

19. A method comprising:receiving a first set of three-dimensional (3D) positional information of a first plurality of biological molecules within a sample, wherein the first set of 3D positional information is determined from a first z-stack obtained from a first field of view (FOV) of the sample and based on a probing cycle of the sample in an imaging instrument, wherein the first z-stack comprises a first plurality of image slices, wherein the probing cycle comprises generating optical signals corresponding to at least some of the first plurality of biological molecules, wherein the first z-stack defines a first imaging volume;determining a first density metric of at least a portion of the first set of 3D positional information; andwhen the first density metric is above a first threshold, causing the imaging instrument to capture at least one additional image slice of the sample within the first field of view that is interleaved with the first z-stack.

20. The method of claim 19, wherein the at least one additional image slice is an additional z- stack that is interleaved with the first z-stack to generate a first combined z-stack.

21. The method of claim 19 or claim 20, wherein the first z-stack has a first spacing between adjacent image slices of the first plurality of image slices.

22. The method of claim 21, wherein the first spacing is about 500 nm to about 1 pm.

23. The method of claim 21, wherein the first spacing is about 750 nm.

24. The method of any one of claims 20 to 23, wherein the first combined z-stack has a second spacing between adjacent image slices.

25. The method of claim 24, wherein the second spacing is about 250 nm to about 1 pm.81FH13244839.1GEI-02225100-179000WO26. The method of claim 24, wherein the second spacing is about 250 nm to about 500 nm.

27. The method of any one of claims 24 to 26, wherein the second spacing is less than the first spacing.

28. The method of any one of claims 19 to 27, further comprising performing blob detection on the first z-stack to obtain the first set of 3D positional information.

29. The method of any one of claims 19 to 28, wherein the first set of 3D positional information comprises blob locations.

30. The method of any one of claims 19 to 29, further comprising dividing the first imaging volume into a plurality of subvolumes.

31. The method of claim 30, wherein the first density metric is an average number of blobs in the plurality of subvolumes.

32. The method of claim 30 or claim 31, wherein each subvolume of the plurality of subvolumes has a volume of about 1 pm2to about 100 pm2.

33. The method of any one of claims 19 to 32, wherein the first threshold is about 1 blob per subvolume to about 2 blobs per sub volume.

34. The method of any one of claims 19 to 32, wherein the first threshold is about 1.5 blobs per subvolume.

35. The method of any one of claims 19 to 30, wherein the first density metric comprises a brightness for each image in the first plurality of images.82FH13244839.1GEI-02225100-179000WO 36. The method of any one of claims 19 to 35, wherein the first set of 3D positional information is obtained from a first color channel during the first probing cycle.

37. The method of claim 36, further comprising receiving a second set of 3D positional information of a second plurality of biological molecules within the sample, wherein the second set of 3D positional information is determined from a second z-stack obtained from the first FOV of the sample, wherein the second set of 3D positional information is obtained from a second color channel during the first probing cycle.

38. The method of claim 37, further comprising determining a second density metric of at least a portion of the second set of 3D positional information; andwhen the second density metric is above a second threshold, causing the imaging instrument to capture at least one additional image slice of the sample within the first field of view that is interleaved with the second z-stack.

39. The method of claim 38, wherein the first threshold and the second threshold are equivalent.

40. The method of claim 38, wherein the first threshold and the second threshold are different.

41. The method of claim 38, wherein the first threshold and the second threshold are dependent on color channel.

42. The method of any one of claims 37 to 41, wherein the first color channel is different from the second color channel, wherein the first color channel and the second color channel are selected from the group consisting of: red, yellow, green, and blue.

43. The method of any one of claims 19 to 42, wherein the first z-stack is a first z-stack of a plurality of z-stacks, wherein the plurality of z-stacks of images correspond to a plurality of FOVs.83FH13244839.1GEI-02225100-179000WO 44. The method of any one of claims 19 to 43, further comprising capturing the first z-stack using the imaging instrument.

45. A computer program product comprising a computer readable storage medium having program instructions embodied therewith, the program instructions executable by a processor to cause the processor to perform a method according to any one of claims 19 to 43.

46. A system comprising:an imaging instrument; anda computing node operatively coupled to the imaging instrument and comprising a computer readable storage medium having program instructions embodied therewith, the program instructions executable by a processor to cause the processor to perform a method according to any one of claims 19-43.

47. A method comprising:imaging a first z-stack at a first field of view (FOV) of a sample, wherein the first z-stack comprises a first plurality of image slices, wherein the first plurality of image slices comprises a first spacing between adjacent image slices; andimaging a second z-stack at a second FOV of the sample, wherein the second z-stack comprises a second plurality of image slices, wherein the second plurality of image slices comprises a second spacing between adjacent image slices,wherein the first spacing is different from the second spacing based on a comparison of a first density metric and a second density metric to a predetermined threshold, and wherein the first density metric is associated the first z-stack and the second density metric is associated with the second z-stack.

48. The method of claim 47, wherein the second spacing is smaller than the first spacing when:the second density metric is greater than or equal to the predetermined threshold, andthe first density metric is less than the predetermined threshold.84FH13244839.1GEI-02225100-179000WO 49. The method of claim 47 or claim 48, wherein the first spacing is a default spacing.

50. The method of any one of claims 47 to 49, wherein the first spacing is about 500 nm to about 1 pm.

51. The method of any one of claims 47 to 49, wherein the first spacing is about 750 nm.

52. The method of any one of claims 47 to 51, wherein the second spacing is about 250 nm to about 1 pm.

53. The method of any one of claims 47 to 51, wherein the second spacing is about 250 nm to about 500 nm.

54. The method of any one of claims 47 to 53, wherein imaging the second z-stack at the second FOV comprises:imaging a preliminary z-stack at a plurality of z-heights having the first spacing between adjacent image slices;determining the second density metric of the preliminary z-stack is above the predetermined threshold; andimaging an interleaved z-stack at a plurality of additional z-heights such that combined images from the preliminary z-stack and images from the interleaved z-stack have the second spacing between adjacent image slices.

55. The method of any one of claims 47 to 54, wherein the first density metric is determined by performing blob detection on the first z-stack and the second density metric is determined by performing blob detection on the second z-stack.

56. The method of any one of claims 47 to 55, wherein the first z-stack and the second z-stack define an imageable volume, and the method further comprising dividing the imageable volume into a plurality of subvolumes.85FH13244839.1GEI-02225100-179000WO 57. The method of claim 56, wherein the first density metric and the second density metric are determined by determining an average number of blobs per subvolume.

58. The method of claim 56 or claim 57, wherein each subvolume of the plurality of subvolumes has a volume of about 1 pm2to about 100 pm2.

59. The method of any one of claims 47 to 58, wherein the predetermined threshold is about 1 blob per subvolume to about 2 blobs per subvolume.

60. The method of any one of claims 47 to 58, wherein the first threshold is about 1.5 blobs per subvolume.86FH13244839.1

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