WO2025024701A2 - 3d spatial single cell omics by implantation barcoding - Google Patents

Abstract

Described herein are methods, compositions, devices, and kits related to methods for 3-dimensional (3D) spatial single cell omics analysis based on the concept of spatial encoding by positional markers injected with implantation devices (for example, jetlet dispensors). Aspects of the present disclosure provide for at least the following: 1) single cell multiomics: a multiomic profile is generated for each single cell in the sample; and 2) 3D mapping: the single cells can be mapped to 3D spatial locations at cellular resolution.

Description

PATENT Attorney Docket No.079445-1455148-012510PC Client Ref. No. S23-134 3D SPATIAL SINGLE CELL OMICS BY IMPLANTATION BARCODING CROSS-REFERENCE TO RELATED PATENT APPLICATIONS [0001] The present patent application claims benefit of priority to U.S. Provisional Patent Application No. 63/529,017, filed July 26, 2023, which is incorporated by reference for all purposes. STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT [0002] This invention was made with Government support under contracts HG007735 and HG010359 awarded by the National Institutes of Health. The Government has certain rights in the invention. BACKGROUND [0003] Omics methods of a specific modality can measure a certain type of information about the cellular state of the cells in the sample being analyzed. Common omics modalities include: (1) transcriptomics: which aims to measure gene expression for one or a plurality or all genes (e.g., poly-A mRNA); (2) proteomics, which aims to measure protein expression for proteins; and (3) regulomics which aims to measure accessibility of chromosome regions across the genome. Traditionally, the qualification “targeted” is applied to the omics method if the omics method only makes measurement on a pre-determined set of targets. For example, a targeted transcriptomic method will only measure the expression of a pre-selected set of genes. If the method can provide simultaneous data on two or more modalities, it is called a multiomics method. [0004] Spatial omics goes further and combines next generation seq/high level multiplexing with imaging modalities and aims to obtain detailed, molecular-level information about the cells in a tissue sample. Spatial omics can give depth and clarity to our collective insights into spatial distribution of gene expression, cell-state transitions, and cell-cell interactions. [0005] Different spatial omics technologies are differentiated from each other by the approaches they use to encode spatial locations, and the methods they use to deliver the spatial barcodes to the cells. Currently there are two main approaches to spatial omics. The first is based on the sequential application of multiplex single molecule FISH to image the location of gene-specific probes (seqFISH, MERFISH, etc). The second approach is to add oligo spatial barcodes to the molecules of interest (i.e., mRNA, cDNA or oligo tags of antibodies) before sequencing. This can be done in two ways: a) Using a dense grid of spots or immobilized beads to capture nearby target molecules and link them to the spatial barcodes on the spots/beads (10X Visium, Slide-seq, Stereo-Seq), or b) using microfluidics chips to deliver and link x- barcodes and y-barcodes to target molecules fixed in situ on the tissue slide (DBiT-seq). For review and citations of the above methods, see, for example, Box 1 of the review paper (Vandaveyken et al, Methods and applications for single-cell and spatial multi-omics, Nature Review Genetics, 2023). [0006] For the microscopy imaging techniques, these traditional approaches fix the spatial locations of target molecules and then detect (and decode) different species of molecules by sequential single-molecule in-situ hybridization. Microscopy-based methods can achieve excellent subcellular localization (i.e., high spatial resolution) but are usually targeted to a few hundreds of genes/proteins. Such methods also cannot be used to discover new transcripts and proteins, and it can be difficult to image or otherwise analyze large areas. Additionally, current methods are typically 2D in nature, whereas 3D information must be obtained using these methods by analyzing serial sections. [0007] An example of prior approaches of spatial single cell omics is the XYZeq method (see, Lee et al (2021) XYZeq: Spatially resolved single-cell RNA sequencing reveals expression heterogeneity in the tumor microenvironment. Sci Adv. 2021 Apr 21;7(17):eabg4755. doi: 10.1126/sciadv.abg4755), which delivers spatial barcodes by physically partitioning a thin tissue section into a 2D grid of microwells. The oligo can enter intact cells in the microwell and serve as a spatial barcode for those cells. The barcoded cells are then analyzed by standard single cell genomics methods (such those from 10X Genomics) which add cell-specific tags to the sequence reads. The limit of this method is that the center- center distance between the wells is on the order of 200-500 ^m, and each well can contain many cells. The spatial resolution is not sufficient to reveal tissue architecture. It is a 2D method which cannot provide depth (z-coordinate) localization. Furthermore, even if the tissue section only 1 to 2 cell layers thin so that depth is not of interest, the limit in the number and size of the microwells makes it difficult to achieve cellular resolution in the x-y plane. [0008] Another prior example utilized is referred to as “Slide-Tags.” In Slide-Tags (see, Russell et al (2023) Slide-tags: scalable, single-nucleus barcoding for multi-modal spatial genomics. bioRxiv preprint doi: https://doi.org/10.1101/2023.04.01.535228), single nuclei within an intact tissue section are ‘tagged’ with spatial barcode oligonucleotides derived from DNA-barcoded beads with known positions. The beads contain cleavable oligo-barcodes that can enter a nearby intact nucleus. The beads are small and tightly packed (10 ^m center to center). Thus, each nucleus can get multiple bar codes which together can map the position of the nucleus at cellular resolution. The nuclei are then dissociated and collected for single nuclei sequencing (using for example the 10X protocol). The technical challenge in this approach is how to create the spatial barcode on each bead. This was achieved by synthesizing the oligo on beads first. Then, after the beads are put on a surface, the oligo sequence for each bead is determined by imaging during a in-situ sequencing process. This method is limited in that it is 2D in nature. A further limitation is that in such a thin section, a large percentage of cells will have its cell membrane disrupted. The spatial barcodes delivered to the cell may diffuse to neighboring cells, resulting in loss of cellular resolution. Thus, this approach is useful only for analyzing nuclear components. The approach of our invention does not suffer from this limitation. [0009] Currently, there is no spatial single cell method with cellular resolution that can provide direct measurements with information including target ID, cell ID, and 3D spatial location. SUMMARY [0010] Spatial single cell omics methods generate single cell omics data together with an additional “localization data” that can be used to infer some aspects of the spatial location of the cell. To create the localization data, spatial barcodes (usually oligonucleotides) are first delivered to the cells in the tissue sample. Then, standard single cell omics or single cell multiomics methods are used to simultaneously generate i) the single cell omics data of the desired modalities, and ii) the single cell location data (from the spatial codes in the cells). Unlike spatial omics methods, spatial single cell omics methods provide direct measurement of cell-identifying information. [0011] Described herein are methods, compositions, kits, and systems relating to methods of multi-dimensional spatial labeling of biological samples, in particular, differentially labeling or individual cells or other aspects of the biological samples (such as subcellular proteins, for example). [0012] In certain aspects, described herein are methods of multi-dimensional spatial labeling of biological samples. In embodiments, methods as described herein can comprise: using a first implantation devise to implant a first set of positional markers into each unit (e.g. each cell, or each nucleus) in a biological sample comprising a plurality of cells marking positions along a first set of positional axes, where the set can be composed of either one coordinate axis or two coordinate axes, while leaving the units intact after the implantation; dissociating the biological sample into single cells (for ease of reading, hereafter we use “cell” rather than “unit”, but the method will also work if the units are single nuclei); measure and record the first set of positional markers in each of the dissociated single cells; and at the same time also measure and record the desired omics data for each of the dissociated single cells. In some embodiments, the positional marker is an oligonucleotide comprising a barcode sequence specific for the implantation device from which it originates. In some embodiments, individual dissociated cells are placed in partitions with cell-specific barcodes and an assay is performed in the partitions to (i) link the cell-specific barcodes to at least one molecule or type of molecule (e.g., RNA or cDNA or gDNA or other cellular nucleic acid) in the cell and (ii) link cell-specific barcodes to positional markers (e.g., oligonucleotides comprising a spatial barcode) in the partition, and then nucleotide sequencing the resulting products. [0013] In embodiments, methods as described herein can comprise a first set of positional markers. The first set of positional markers can comprise a first plurality of nucleic acids comprising barcode sequences specific for the implantation device from which the nucleic acids originate.. In embodiments, each positional marker of the first positional marker set can further comprise one or more of a PCR handle sequence, a unique molecular identifier (UMI) barcode, a sample barcode, or any combination of any thereof. In embodiments, at least one of the one or more subcellular components can comprise an endogenous nucleic acid of each of the plurality of cells. [0014] In embodiments, methods as described herein can further comprise implanting a second set of positional markers into the biological sample along a second set of positional axes using a second implantation device and detecting the second set of positional markers along with the first set of positional markers. In embodiments, the second set of positional markers can further comprise one or more detectable labels linked to each of the positional markers. The second set of positional markers can comprise a second plurality of nucleic acids comprising barcode sequences specific for the implantation device from which the nucleic acids originate. In embodiments, each positional marker of the second positional marker set can further comprise one or more of PCR handle sequence, a unique molecular identifier (UMI) barcode, a sample barcode, or any combination of any thereof. In embodiments, the injection angle of the first implantation device and that of the second implantation device can 45 degree and 135 degree relative to the surface (see FIG.2) of the biological sample. [0015] In embodiments, the two injection angles can be 30 degree and 90 degree respectively. In embodiments, the second set of positional markers can comprise a second plurality of nucleic acids. In some embodiments, each nucleic acid of the first plurality of nucleic acids is different from each nucleic acid of the second plurality of nucleic acids. In embodiments, the implanting can comprise injecting into the biological sample droplets comprising one or more nucleic acids from the first set of positional markers, second set of positional markers, or both. In embodiments, the first implantation device can comprise a two- dimensional (2D) array of injectors (which can be liquid dispensers). In embodiments, a 2D array of the first implantation device is a 100 x 100 to a 1000 x 1000 array. In embodiments, each injector of the 2D array can comprise a unique nucleic acid from the first set of positional markers. In embodiments, a second implantation device can comprise a second two- dimensional (2D) array of injectors (which can be liquid dispensers). In embodiments, the second 2D array of the second implantation device can be a 100 x 100 to a 1000 x 1000 array. In embodiments, each injector of the second 2D array can comprise a unique nucleic acid from the second set of positional markers. In embodiments, each injector of the 2D array can comprise a single or a pair of unique nucleic acids marking a unique position in a 2D array. Thus, the set of barcodes in a cell will define its unique position in the array of injectors. [0016] In embodiments of methods as described herein, the dissociation can comprise one or more of mechanical dissociation, enzymatic dissociation, chemical dissociation, or any combination of any thereof. In embodiments, the dissociation can comprise treating the biological sample with a cell dissociation reagent at 37°C for a period of time; mechanically agitating the sample and reagent mixture; and filtering the agitated mixture to obtain a composition comprising a plurality of single cells. [0017] In embodiments, the detection can be sequencing, polymerase chain reaction (PCR) or next-generation sequencing. [0018] In embodiments, methods as described herein can further comprise reconstructing a multidimensional image using the positional data. [0019] In embodiments, methods as described herein can further comprise analyzing the dissociated single cells using one or more analysis methods, wherein the one or more analysis methods can be one or more of a transcriptomics analysis, a proteomic analysis, a metabolomic analysis, a lipidomic analysis, or any combination of any thereof. In embodiments of methods as described herein, the analysis method can be next-generation sequencing (NGS). [0020] In embodiments, methods as described herein can further comprise, after implanting or linking, rotating the first implantation device, second implantation device, or both, at an angle of about 30 degrees to about 90 degrees, in relation to the first positional axis, second positional axis, or both. In embodiments the rotation angle can be about 45 degrees. In embodiments, the first implantation device, second implantation device, or both, can comprise a laser-actuated supercritical injector (LASI) array. [0021] In some embodiments, the positional markers comprise a plurality of nucleic acids comprising barcode sequences specific for the implantation device from which the nucleic acids originate; and the method further comprises: forming the dissociated cells into different partitions; linking oligonucleotides comprising partition-specific barcodes sequences to cellular DNA, or cDNAs from cellular RNA, and linking the oligonucleotides comprising partition-specific barcodes sequences to the nucleic acids comprising barcode sequences specific for the implantation device. [0022] In some embodiments, the plurality of nucleic acids further comprise a poly A tail sequence and the oligonucleotides comprising partition-specific barcodes sequences comprise a poly T tail sequence and the method comprises performing reverse transcription with the oligonucleotides comprising partition-specific barcodes sequences to form (i) cDNAs from cellular mRNA and (ii) a nucleic acid comprising oligonucleotides comprising partition- specific barcodes sequences linked to the nucleic acids comprising barcode sequences specific for the implantation device. [0023] In some embodiments, the partitions are droplets or microwells. [0024] In some embodiments, the method further comprises nucleotide sequencing products of the linking and associating the partition-specific barcodes with the positional marker to determine a position of the cell in the biological sample. [0025] In certain aspects, described herein are methods of mapping the position of cells of a 3- dimensional multicellular sample. In embodiments, methods according to the present disclosure can comprise the steps of: implanting a first set of positional markers into the sample along a selected injection angle, wherein the set of positional markers is implanted into the sample by a first array of injectors of a first implantation device, wherein each injector of the array delivers droplets comprising a uniquely identifiable positional marker; by a second array of injectors of a second implantation device with an injection angle different from that of the first array, implanting a second set of positional markers into the sample along a second injection trajectory, wherein, wherein each injector of the second array delivers droplets comprising a uniquely identifiable positional marker; dissociating the sample into single cells; performing an analysis of each cell to determine the identity of positional markers present therein; inputting the unique positional marker content of each cell to a computer program, and by the computer program and the known position of each injector in the first and second arrays, determining what was the 3-dimensional position of each cell in the intact sample. In embodiments, injector arrays can comprise one or more LASI devices. In embodiments, each uniquely identifiable positional markers can comprise oligonucleotides of a selected sequence. In embodiments, an analysis to determine the identity of positional markers present in the cells can comprise a sequencing procedure. In embodiments, the sequencing procedure can also be applied to measure the expression level of one or more genes in the cell. In embodiments, the sequencing procedure can further comprise a transcriptomics analysis. In embodiments, one or more additional analyses are performed on each dissociated cell. In embodiments, the one or more additional analyses can be selected from the group consisting of proteomic analysis, metabolomic analysis, lipidomic analysis, and the detection and/or quantification of one or more biomarkers, analytes, pathogens, or other species. BRIEF DESCRIPTION OF THE DRAWINGS [0026] The present application includes the following figures. The figures are intended to illustrate certain embodiments and/or features of the compositions and methods, and to supplement any description(s) of the compositions and methods. The figures do not limit the scope of the compositions and methods, unless the written description expressly indicates that such is the case. [0027] FIGs. 1A and 1B illustrate a two-dimensional (2D) embodiment of the present disclosure. As shown in FIG.1A, an injection array (for example, a jetlet dispenser array) can be used to inject one or more spatial barcodes into each cell in a tissue slide. This can be done in a manner that ensures (1) the cells are intact afterwards; and (2) no two cells should share the same set of spatial barcodes. FIG. 1B shows dissociation of the tissue sample in single cells in order to perform single-cell genomics analysis. In these figures, r_i is the gene expression profile of cell i, x_i is the set of spatial codes injected into cell i, and t_i is a cell- identifying barcode created during library preparation for single cell RNA sequencing. [0028] FIG. 2 illustrates a three-dimensional (3D) embodiment according to the present disclosure. As shown in FIG.2, a tissue block (A) is impregnated with spatial barcodes by two implantation devices (B and C). [0029] FIG.3 is another illustration of a 3D embodiment according to the present disclosure, wherein a tissue sample (and each cell therein) impregnated with spatial barcodes utilizing a first and second implantation device (jetlet dispenser arrays according to the present embodiment). [0030] FIGs.4A-4D illustrate aspects of an embodiment of implantation devices according to the present disclosure: a laser-activated supercritical injector (LASI) device. [0031] FIG.5 is a schematic example showing trajectories of 4 jetlets: Each line (a and b) represents a jetlets from the first array and each line (c and d) represents a jetlet from the second array. The 3D details of a single jetlet are provided in FIG.6. FIG.5 shows the projected view from side face 1. From the spatial codes detected in a cell (for example, circled), it can be determined which jetlets have crossed the cell. For example, if a, b are detected from the first array and c, d from the second array, then the 3D location of the cell can be mapped approximately to the intersections of these arrays. [0032] FIG.6 is a schematic of 3D details of a single jetlet. Here J(i,j) denotes the injector in position (i,j) of the injector array. A = circular surface where the jetlet ejected by J(i,j) enters the top face of the tissue slab. B= circular surface where the jetlet exits from the bottom face of the tissue slab. If the spatial barcode carried by this jetlet is detected in a cell, then this cell has nonempty intersection with the slanted cylindrical volume bounded by A [0033] FIG.7A: LASI array with a rectangular grid of channels. The spacing (pitch) of the grid is 10 ^m. Each channel has a depth of 28 ^m and an opening 3 ^m in diameter. FIG.7B: Laser–induced heating of a channel containing a solution will result in the injection and penetration of the solution into a tissue slide facing the channel’s opening. FIG.7C: Confocal image of a human liver tissue slide after injection of fluorescent beads by the LASI device. DETAILED DESCRIPTION I. Introduction [0034] Described herein are methods for multi-dimensional (2D or 3D) spatial single cell omics analysis. Methods as described herein are based on the concept of spatial encoding by using implantation devices to inject one or more spatial barcodes (i.e., positional markers) into individual cells of a multicellular tissue sample. Multi-dimensional spatial single cell omics methods as described herein can generate single-cell omics data but with additional “localization data” associated to each of the single cell profiles. These localization data can be used to infer some aspects of the spatial location of the cell within the tissue sample. In some embodiments, the data is “single cell” in the sense that each piece of measurement data (typically a sequence read) contains enough information to identify the cell under measurement. [0035] To create the localization data, spatial barcodes (usually oligonucleotides, also referred to herein as “positional markers” with oligonucleotides also referred to herein as “nucleic acids”) can first be delivered to the cells in the tissue sample (“delivered” also referred to herein as “injected” or “implanted”). The tissue sample can then be dissociated into single cells, and single cell omics or multi-omics methods can be used to generate: (1) the single cell omics data of the desired modalities, and (2) the single cell location data (from the spatial barcodes in the cells, for example linked to cell-specific barcodes linked during processing of single cells). [0036] Aspects of the present disclosure provide for at least the following: (1) a single cell detection method selected from one or more multiomics wherein one or more multiomic profile is generated for each single cell in the sample; and (2) 2D or 3D mapping, wherein the single cells can be mapped to 2D or 3D spatial locations at cellular resolution. [0037] According to aspects of the present disclosure, one or more spatial barcodes can be implanted in a cell by one of more injector devices in order to provide localization data of that cell. In certain aspects, the spatial barcodes can be oligonucleotides comprising a spatial barcode (i.e., a barcode sequence specific for the implantation device from which it originates). In certain aspects, the oligonucleotides spatial barcode can comprise, for example, DNA and/or RNA or analogs thereof. The barcode sequence can be continuous or discontinuous (e.g., made up of two or more sub-sequences that are not contiguous). The length and complexity of the barcode sequence will vary depending on the number of barcodes needed. In some embodiments, the spatial barcode is between 3-20 bases in length though it will be appreciated other lengths can be used as needed. [0038] The oligonucleotides can further comprise other sequences in addition to the spatial barcode. For example in some aspects, the spatial barcode oligonucleotides can comprise a 5’ PCR handle (i.e., primer site) for amplification, a unique molecular identifier (UMI) sequence, and/or a 3’ tail sequence that can be annealed to a sequence in the cell or a source of call- specific barcodes as discussed below. In some embodiments, the oligonucleotide comprise the spatial barcode sequence and a 3’ poly A tail sequence, for example at least 6 contiguous As such that a subsequence polyT primer in a reverse transcription reaction can anneal to the oligonucleotide and be extended in an reverse transcription reaction to produce complementary copies of spatial barcodes that can be further amplified. [0039] According to aspects of the present disclosure, the spatial barcodes can be implanted into one or more cells of a sample using one or more implantation devices. An implantation device as described herein can inject (i.e., implant) one or more spatial barcodes into each cell in a tissue sample along a first implantation axis. Such implantation can be done in a manner that ensures that (1) the cells are intact afterward; and (2) barcodes are spatially assigned. [0040] Implantation devices as described herein can comprise a first implantation device comprising a 2D array of individual injectors. Implantation devices as described herein can further comprise a second implantation device, which comprises a second array of injectors configured to inject spatial barcodes along a second implantation axis, wherein the second implantation axis is different than the first (i.e., in embodiments they can be opposed to each by an angle of about 30° to about 90°). In embodiments, an injector of an implantation device as described herein can be capable of producing a focused stream of fluid (such as a liquid), called a jetlet, that moves along a linear trajectory, wherein the linear trajectory is an implantation axis described above (also referred to herein as a “positional axis”). Such embodiments of injectors are also referred to herein as “jetlet dispensors.” Following implantation of the spatial barcodes, the tissue sample can then be dissociated into single cells and one or more omics analysis methods applied to each of the single cells. [0041] Following injection, spatial barcodes introduced into any one or more cells can then be linked to an endogenous oligonucleotide, polypeptide, lipid, or any other subcellular molecule inside a cell, or another single cell omics barcode inside a cell (further described in the paragraph below and section V). [0042] After injection (and linking in embodiments), the sample comprising cells into which the spatial barcodes were injected can be dissociated into single cells. Omics analysis can then proceed on the single cells according to methods as known in the art. For example, single cells can be introduced into individual partition such that most cells are the only cell in a partition, allowing for single cell analysis. Examples of partitions without limitation include, droplets (e.g., aqueous droplets in an emulsion with oil) or microwells. Partitions can in addition include partition-specific barcoding polynucleotides that comprise a barcode sequence specific for the partition in which the polynucleotides reside. In some embodiments, such partition-specific barcoding polynucleotides are introduced in multiple copies linked to a bead, e.g., a hydrogel bead. The partition-specific barcoding polynucleotides can subsequently be linked to one or molecule from the cell in the partition, thereby adding a cell-specific barcode to the one or more molecule (because there is only one cell in the partition, the partition-specific barcode becomes a cell-specific barcode sequence). In addition, one or more copy of the partition- specific barcoding polynucleotides can be linked to the spatial barcode oligonucleotide. This latter step will allow one to interpret sequencing reads as originating from a specific cell at a specific location based on the location of the implantation device from which the spatial barcode originated. Optionally, once the cells are located in the partitions, the cells can be lysed to release their contents into the partitions in which they reside. [0043] The cellular molecule(s) to which the partition-specific barcoding polynucleotides are attached can vary as desired by the “omic” method used. In some embodiments, the molecules are DNA from the cell or cDNAs formed by reverse transcription of cellular mRNA. Linkage of the partition-specific barcoding polynucleotides to nucleic acids can comprise ligation, annealing to complementary sequences or both. In some embodiments, the partition-specific barcoding polynucleotides comprise a polyT sequence and are used to primer first strand cDNA synthesis using cellular mRNA as a template. In these embodiments, the oligonucleotides comprising a spatial barcode further comprise a poly A tail end sequence, allowing the reverse transcription reaction to also extend at least some of the partition-specific barcoding polynucleotides that comprise a polyT sequence using the oligonucleotides comprising a spatial barcode and further comprising a poly A tail end sequence as a template, thereby linking the oligonucleotides comprising a spatial barcode (or a complement thereof) to the partition- specific barcoding polynucleotides. [0044] Following cell-specific barcoding of molecules from the cell and of the spatial barcodes, the contents of the partitions can be combined and submitted to nucleotide sequencing or otherwise detected to assess the identity (and for nucleic acid molecules, their sequence) and optionally quantity of the molecules as well. The data can be grouped by cell based on cell-specific barcode sequence. The location of the cell can be determined based on the identity of the spatial barcodes, having originated from one or more particular implantation devices of known location relative to the starting tissue. [0045] In some embodiments, after the data is obtained, positional data from the spatial barcodes can be used to reconstruct a 3D image of the sample, wherein the data from the cells can be tied to the positional data of the spatial barcodes. [0046] The practice of aspects of the present disclosure employs, unless otherwise indicated, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA, which are within the skill of the art. See Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd edition (1989), Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds., (1987)), the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (R. I. Freshney, ed. (1987)). [0047] For nucleic acids, sizes are given in either kilobases (kb), base pairs (bp), or nucleotides (nt). Sizes of single-stranded DNA and/or RNA can be given in nucleotides. These are estimates derived from agarose or acrylamide gel electrophoresis, from sequenced nucleic acids, or from published DNA sequences. For proteins, sizes are given in kilodaltons (kDa) or amino acid residue numbers. Protein sizes are estimated from gel electrophoresis, from sequenced proteins, from derived amino acid sequences, or from published protein sequences. [0048] Oligonucleotides that are not commercially available can be chemically synthesized, e.g., according to the solid phase phosphoramidite triester method first described by Beaucage and Caruthers, Tetrahedron Lett. 22:1859-1862 (1981), using an automated synthesizer, as described in Van Devanter et. al., Nucleic Acids Res. 12:6159-6168 (1984). Purification of oligonucleotides is performed using any art-recognized strategy, e.g., native acrylamide gel electrophoresis or anion-exchange high performance liquid chromatography (HPLC) as described in Pearson and Reanier, J. Chrom.255: 137-149 (1983). The term “oligonucleotide” is not intended to indicate a particular length. In some embodiments, the oligonucleotides are between 15-200 nucleotides long. [0049] Additional aspects of the present disclosure, in particular, relating to methods of single-cell omics and barcodes of methods therein, can be found, for example in: Kim IS. Single-Cell Molecular Barcoding to Decode Multimodal Information Defining Cell States. Mol Cells.2023 Feb 28;46(2):74-85. doi: 10.14348/molcells.2023.2168. Epub 2023 Feb 27. PMID: 36859472; PMCID: PMC9982054.; Vandereyken K, Sifrim A, Thienpont B, Voet T. Methods and applications for single-cell and spatial multi-omics. Nat Rev Genet.2023 Mar 2:1–22. doi: 10.1038/s41576-023-00580-2. Epub ahead of print. PMID: 36864178; PMCID: PMC9979144; and Zilionis R, Nainys J, Veres A, Savova V, Zemmour D, Klein AM, Mazutis L. Single-cell barcoding and sequencing using droplet microfluidics. Nat Protoc.2017 Jan;12(1):44-73. doi: 10.1038/nprot.2016.154. Epub 2016 Dec 8. PMID: 27929523. II. Definitions [0050] Before aspects of the present disclosure are further described, it is to be understood that the present disclosure is not strictly limited to particular embodiments described, as such may of course vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will ultimately only be limited only by the claims. [0051] It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. It should further be understood that as used herein, the term “a” entity or “an” entity refers to one or more of that entity. For example, a nucleic acid molecule refers to one or more nucleic acid molecules. As such, the terms “a”, “an”, “one or more” and “at least one” can be used interchangeably. Similarly, the terms “comprising”, “including” and “having” can be used interchangeably. [0052] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed. [0053] It is appreciated that certain features of the present disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the present disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination were individually and explicitly disclosed. In addition, all sub-combinations are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub-combination were individually and explicitly disclosed herein. [0054] It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation. [0055] As used herein, the term “about” means a range of values including the specified value, which a person of ordinary skill in the art would consider reasonably similar to the specified value. In embodiments, about means within a standard deviation using measurements generally acceptable in the art. In embodiments, about means a range extending to +/- 10% of the specified value (e.g., +/- 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of the specified value). In embodiments, about means the specified value. [0056] As used herein, the term “reverse transcriptase” refers to its plain and ordinary meaning as an enzyme used to generate complementary DNA (cDNA) from an RNA template, a process termed reverse transcription. [0057] As used herein, the terms "complementary" or "complementarity" refers to polynucleotides that are able to form base pairs with one another. Base pairs are typically formed by hydrogen bonds between nucleotide units in an anti-parallel orientation between polynucleotide strands. Complementary polynucleotide strands can base pair in a Watson- Crick manner (e.g., A to T, A to U, C to G), or in any other manner that allows for the formation of duplexes. As persons skilled in the art are aware, when using RNA as opposed to DNA, uracil (U) rather than thymine (T) is the base that is considered to be complementary to adenosine. However, when a uracil is denoted in the context of the present disclosure, the ability to substitute a thymine is implied, unless otherwise stated. "Complementarity" may exist between two RNA strands, two DNA strands, or between a RNA strand and a DNA strand. It is generally understood that two or more polynucleotides may be "complementary" and able to form a duplex despite having less than perfect or less than 100% complementarity. Two sequences are "perfectly complementary" or "100% complementary" if at least a contiguous portion of each polynucleotide sequence, comprising a region of complementarity, perfectly base pairs with the other polynucleotide without any mismatches or interruptions within such region. Two or more sequences are considered "perfectly complementary" or "100% complementary" even if either or both polynucleotides contain additional non-complementary sequences as long as the contiguous region of complementarity within each polynucleotide is able to perfectly hybridize with the other. "Less than perfect" complementarity refers to situations where less than all of the contiguous nucleotides within such region of complementarity are able to base pair with each other. Determining the percentage of complementarity between two polynucleotide sequences is a matter of ordinary skill in the art. [0058] The terms "hybridize” and "hybridization" refer to the formation of complexes between nucleotide sequences which are sufficiently complementary to form duplexes via Watson-Crick base pairing. [0059] The term “nucleic acid,” “nucleotide,” “oligonucleotide,” or “polynucleotide” refers to deoxyribonucleic acids (DNA), ribonucleic acids (RNA) and polymers thereof in either single-, double- or multi-stranded form. The term includes, but is not limited to, single-, double- or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and/or pyrimidine bases or other natural, chemically modified, biochemically modified, non-natural, synthetic or derivatized nucleotide bases. In some embodiments, a nucleic acid can comprise a mixture of DNA, RNA and analogs thereof. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms (SNPs), and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). [0060] The term “gene” means the segment of DNA involved in producing a ribonucleic acid polymer, which in the case of protein coding genes can then be translated into a polypeptide chain. The DNA segment may include regions preceding and following the coding region (leader and trailer) involved in the transcription/translation of the gene product and the regulation of the transcription/translation, as well as intervening sequences (introns) between individual coding segments (exons). [0061] Examples of algorithms that are suitable for determining sequence similarity between a test and a reference sequence are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1977) Nucleic Acids Res. 25: 3389-3402, respectively. Software for performing BLAST analyses is publicly available at the National Center for Biotechnology Information website, ncbi.nlm.nih.gov. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M=1, N=í2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see, e.g., Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)). [0062] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Nat'l. Acad. Sci. USA, 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001. [0063] The terms "polypeptide" and "protein" refer to a polymer of amino acid residues and are not limited to a minimum length. Thus, peptides, oligopeptides, dimers, multimers, and the like, are included within the definition. Both full length proteins and fragments thereof are encompassed by the definition. The terms also include post expression modifications of the polypeptide, for example, glycosylation, acetylation, phosphorylation, hydroxylation, and the like. Furthermore, for purposes of the present disclosure, a "polypeptide" refers to a protein which includes modifications, such as deletions, additions and substitutions to the native sequence, so long as the protein maintains the desired activity. These modifications may be deliberate, as through site directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification. [0064] As used herein, “jetlet” refers both to a narrowly focused stream of liquid that moves along a linear trajectory, and an injection device capable of producing a narrowly focused stream of liquid. [0065] As used herein, a single cell omics method means a method that can generate an omics profile for each cell in a large sample of cells, for example a sample comprising at least 100 cells. [0066] A spatial omics method, as used herein, generates omics data at a set of spatial locations. It is possible that two or more cells may contribute to the omics data at a given location. If the spatial omics method generates data in a two-dimensional grid of locations, it is referred to as a 2D method. 2D methods are typically applied to analyze a very thin slide of tissue whose z-dimension only cover one or two cell layers. If the method can generate data in a 3-dimensional grid where each dimension of the grid can cover multiple cell layers (at least 5), it is referred to as a 3D method. Described herein are 2D and 3D methods. [0067] The term "library" is used according to its common usage in the art, to denote a collection of molecules, optionally organized and/or cataloged in such a way that individual members can be identified. Libraries can include, but are not limited to, combinatorial chemical libraries, natural products libraries, and peptide libraries. The spatial barcodes as described herein can be derived from any one or more oligonucleotide libraries. III. Methods [0068] In certain aspects, described herein are methods relating to spatial determination of cells in a tissue that are also submitted to detection allowing for detection of one or more molecule of a cell, which in some embodiments comprises a single-cell omics analysis. In some embodiments, the single-cell analysis comprises one or more of a transcriptomics analysis (including but not limited to RNAseq) , a proteomic analysis, a metabolomic analysis, a lipidomic analysis, or any combination of any thereof. [0069] Embodiments of methods as described herein can be 2D or 3D, wherein 2D embodiments of methods as described herein may utilize a single array of implantation devices to drive spatial barcodes into cells, and 3D embodiments may utilize two or more arrays of implantation devices (wherein the two or more arrays implant along different relative axes in relation to a tissue sample). [0070] Additional aspects of implantation devices as described herein can be found in section IV below. Additional aspects of spatial barcodes as described herein can be found in section V below. [0071] According to embodiments of methods as described herein, a first implantation device can inject one or more spatial barcodes (also referred to herein as “positional markers”, further describe in section V below), e.g., delivered as part of an oligonucleotide sequence, into different spatially-distinct cells in a tissue slide. Such injection can be performed in a manner that ensures that (1) cells are intact (i.e., not lysed) after injection, and (2) barcodes are in spatially-distinct locations. In some embodiments, the resolution can be a per-cell level, meaning different cells receive different spatial barcodes. After injection of the barcodes, the tissue sample can be dissociated into single cells proceeded by further analysis on each cell (by chemical, enzymatic, or mechanical dissociation, or a combination of any thereof). In 3D embodiments according to the present disclosure, two implantation arrays can be utilized. As an example for 3D embodiments, for a cell with a given 3D coordinate (x, y, z), a first implantation device can provide (x, y) information, and a second implantation device can provide (x, z) or (y, z) information. See, e.g., FIG.2. [0072] In some embodiments, after the spatial barcodes have been injected into the cells, any single cell omics assay can be utilized to analyze one or more molecules of the cells, as long as the assay can add a cell-specific oligonucleotide tag to the sequence reads from that cell (i.e., a barcode, discussed further in section V below). In embodiments, oligonucleotides comprising the spatial barcodes are annealed, extended, and/or ligated or otherwise linked to the cell- specific barcodes utilized in downstream single-cell assays. As noted above, in some embodiments, the single-cell assay detects polyA mRNAs in a cell via reverse transcription using a reverse transcription primer comprising a cell-specific barcode sequence and a poly T 3’ tail that is extended in the reverse transcription assay in the partition. The same reaction can be used to anneal the reveres transcription primer to polyA tail sequences in the spatial barcode oligonucleotides, allowing for a reverse transcription product that comprises the cell-specific and spatial barcodes. In some embodiments, the oligonucleotides comprising the spatial barcodes can be composed of RNA (allowing them to be a template for the reverse transcriptase used to generate cDNAs) or the reverse transcription reaction can also include a polymerase that extends the reverse transcription primer in a DNA-dependent manner. In some embodiments, analysis methods as described herein can include the Single Cell Gene Expression assay and Single Cell Multiome ATAC + Gene expression assay from 10X Genomic, Inc., both operable on the Chromium platform from 10X Genomic, Inc. Nucleotide sequencing of the resulting cDNAs and other products comprising the cell-specific and spatial barcodes is them performed. Any platform can be used for sequencing and in some embodiments the sequencing uses a next-generation sequencing (NGS) platform, for example a massively-parallel sequencing method. [0073] The biological sample (also referred to herein as “tissue sample”) can comprise a plurality of cells from a vertebrate or invertebrate subject. In embodiments, the biological sample can be a biopsy from a subject (wherein the subject is a vertebrate or invertebrate in embodiments, for example, a mammal such as a rodent, non-human primate, or a human). In embodiments, the biological sample can be an organoid formed in vitro from a plurality of immortalized or primary cells (or both). In embodiments, the biological sample can comprise a 3D plurality of cells. Such a sample can be a tissue biopsy from a human subject, for example. Biological samples as described herein may be fixed, for example, in formalin and embedded in paraffin, or may be unfixed. [0074] Methods as described herein can further comprise implanting a second set of positional markers into the biological sample along a second positional axis using a second implantation device and detecting the second set of positional markers along with the first set of positional markers. See, e.g., FIG. 2 and 5-6. In embodiments, the second set of positional markers comprise oligonucleotides, wherein each oligonucleotide of the second set of positional markers has a unique spatial barcode sequence compared to each oligonucleotide of the first set of positional markers. Each oligonucleotide of the second set of positional markers can further comprise one or more detectable labels (for example, a dye or fluorescent protein such as GFP). In embodiments, the detectable labels of each oligonucleotide of the second set of positional markers can be different or the same as the detectable labels of each oligonucleotide of the first set of positional markers. In embodiments, the implanting can comprise injecting (into the biological sample) one or more nucleic acids from the second set of positional markers. [0075] In embodiments of methods as described herein, the dissociation of cells from the tissue prior to single-cell partition and analysis can comprise one or more of mechanical dissociation, enzymatic dissociation, or chemical dissociation. In embodiments, cells can first be treated with an enzymatic and/or chemical reagent (for example, one such as StemPro Accutase Cell Dissociation Reagent (Thermo Fisher)). The sample can then be incubated at 37°C for 10-15 min with mechanical disturbance, for example, pipetting up and down with a pipette. Single cell suspensions can then be obtained by passing the mixture through a mechanical filter or strainer (for example, a 37 mM cell strainer from STEMCELL Technologies) more than time. Optionally, after measuring the cell concentration, cells can be spun down with a centrifuge (approximately 1 million of cells centrifuged at 300 rcf for 5 min, for example) for further processing or freezing. [0076] In embodiments, following detection and analysis, methods as described herein can further comprise reconstructing a multidimensional image using the positional data. IV. Implantation Devices [0077] Described herein are one or more implantation devices, wherein each implantation device comprises a 2D array of injectors. In embodiments according to the present disclosure, an implantation device can comprise an array of injectors (also referred to as “liquid dispensors” or “jetlet dispensors” in certain embodiments) capable of driving spatial barcodes as described herein through the cell membrane and into a cell without rupturing the membrane (i.e., without killing the cell or otherwise impairing its viability) and by maintaining spatial information (i.e., by the array provided each cell a unique spatial barcode or set of spatial barcodes). Each injector of the implantation device is configured to provide a force that can translocate spatial barcodes as described herein in a fluid vehicle (an aqueous vehicle such as phosphate-buffered saline (PBS), for example) from the injector and into a cell. In embodiments, an injector is capable of injecting a jetlet, i.e., a narrow, focused, liquid stream along a linear trajectory, from the injector and into a cell. In embodiments, an injector can be a jetlet dispensor. In embodiments, implantation devices of the present disclosure can comprise an array of jetlet dispensors. [0078] Implantation devices as described herein comprise a plurality of injectors arranged in an array. In embodiments, implantation devices as described herein can comprise a 2D array of injectors. Each injector of the 2D array can be capable of injecting or otherwise delivering positional markers into a cell of a biological sample. Each injector can be an injector, which, in embodiments, can be a liquid dispenser. Each injector can comprise unique positional markers that are different than those in any other injector in the array. The injector can deliver, to the sample, the positional markers in a liquid droplet, for example. In embodiments, injectors can further comprise injection chambers (also referred to herein as “injector cavities”), which are chambers that are configured to hold a fluid comprising spatial barcodes as described herein. The injection chambers can be operatively linked to the injectors such that each injector injects into a cell the contents of the injector chamber associated with the injector. Each injector can have its own unique injection chamber which materials (such as spatial barcodes) can be loaded into. [0079] In embodiments, an implantation device can comprise an array of about 100 x 100 to about 1000 by 1000 injectors (or any intervening value). In embodiments, implantation devices as described herein can comprise a 100x100, 200x200, 500x500 or 1000x1000 array of injectors, for example. The pitch of each the injections from each injector relative to the sample can be from about 5 micron to about 20 microns. [0080] Two or more implantation devices can be utilized according to aspects and embodiments of the present disclosure (i.e., a first and a second implantation device). In embodiments, a second implantation device can be utilized according to the present disclosure. In embodiments, the second implantation device can be a 2D array of injectors. In embodiments, the second implantation device can be physically identical to the first implantation device. In embodiments, each injector of the second implantation device can comprise unique positional markers that are different than those of the injectors of the first implantation device. [0081] In embodiments, the first implantation device and second implantation can inject the positional markers into the sample along a first positional axis and a second positional axis, respectively. In embodiments, the first and second positional axis are orthogonally opposed to each other. In embodiments, the biological sample is positioned at the intersection of the first and second positional axis. In an embodiment, a first implantation device can inject jetlets at a 45-degree inclination from of the surface of the sample, and the second implantation device can inject jetlets at a 135-degree inclination from the surface of the sample. [0082] In embodiments, after implanting or linking, methods as described herein can further comprise rotating the first implantation device, second implantation device, or both, at an angle of about 30 degrees to about 90 degrees, in relation to the first positional axis, second positional axis, or both. In embodiments, the angle of rotation can be about 45°. [0083] In embodiments, the first implantation device, second implantation device, or both, can comprise a laser-actuated supercritical injector (LASI). FIGs.4A-4D are photos showing aspects of LASI devices (and their use) according to the present disclosure. Additional aspects of LASI devices and their operation can be found, for example, at least in International Patent Cooperation Treaty application no. PCT/US2021/044339 titled “LASER-ACTUATED SUPERCRITICAL INJECTOR” and filed on August 31, 2021, the contents of which are incorporated herein by reference regarding structure and function of the device[s] described therein and operation thereof. [0084] In certain embodiments of implantation devices and injector arrays according to the present disclosure, the LASI device can exploit high-speed fluidic jets (liquid fluidic jets, for example) that are pushed by rapid bubble expansion in a fluid. The bubbles are formed when liquid confined in microcavities or holes (for example, the injectors or injector cavities) are heated up to above the supercritical temperature of the fluid. This leads to the formation of a short but ultra-high vapor pressure (supercritical) fluid that ejects the fluid (and any cargo contained therein) out through microchannels (i.e., injectors or jetlet dispensors). This liquid stream penetrates a cell, organ or tissue juxtaposed to a surface containing the microchannels and provides sufficient force to penetrate into the cell, tissue, or organ leading to effective delivery of a cargo. [0085] In an embodiment of an injector array, a spatial resolution of 10 ^m can be utilized. To analyze a tissue slab of dimension 1mm x 1mm x 100^m at a resolution of 10 ^m, for example, a 2D array of injectors can be prepared in which the center-to-center distance between neighboring injectors is 10 ^m. This can mean that the array can contain 100x100=10,000 injectors. To obtain depth information, at least two different injector arrays can be utilized (in the sense that two different set of spatial barcodes are used in the two arrays) to inject into the tissue slab at different angles. Thus, in an embodiment, a 100x100 array of jetlets can be utilized that can penetrate to a distance of about 150 ^m into the tissue slab. In other embodiments, the resolution can be improved to 5 ^m or even 3 ^m by modifying the spatial geometry of the array (the density of injectors, injector to injector distance, injector diameter, etc.). [0086] In embodiments, 8” wafers can be used for the production of the injector arrays. Each individual chip can have a 2D grid (i.e., array) of injectors with radius R of about 1um, about 2um or about 3um. The distance D between two injectors can be larger than 2R. For example, if the chip size is 1mm*1mm, R = 1um and D = 4um, then the array will have about 200*200 injectors. In embodiments, the injectors on the injector array can be manufactured by the deep reactive-ion etching (DRIE) method, which uses a highly anisotropic etching process to achieve deep penetration and create steep-sided holes and trenches in wafers/substrates with high aspect ratios. In such embodiments, the etching can be done in silicon to create the holes and trenches. In certain aspects, areas of the silicon (in particular areas in/around the holes and trenches that form the injectors) can be doped or otherwise modified to withstand heat better than naïve silicon (heat from a laser, for example). [0087] Disclosed herein are materials, compositions, and methods that can be used for, can be used in conjunction with, or can be used in preparation for the disclosed embodiments. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutations of these compositions may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a method is disclosed and discussed, and a number of modifications that can be made to a number of molecules included in the method are discussed, each and every combination and permutation of the method, and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Likewise, any subset or combination of these is also specifically contemplated and disclosed. This concept applies to all aspects of this disclosure including, but not limited to, steps in methods using the disclosed compositions. Thus, if there are various additional steps that can be performed, it is understood that each of these additional steps can be performed with any specific method steps or combination of method steps of the disclosed methods, and that each such combination or subset of combinations is specifically contemplated and should be considered disclosed. [0088] Publications cited herein and the material for which they are cited are hereby specifically incorporated by reference in their entireties. The following description provides further non-limiting examples of the disclosed compositions and methods. V. Spatial barcodes [0089] Described herein are compositions comprising spatial barcodes (i.e., positional markers). In embodiments, spatial barcodes as described herein can comprise oligonucleotides. In embodiments, spatial barcodes as described herein can comprise an oligonucleotide sequence with a poly-T tail on the 3’ end. [0090] In embodiments, the first set of positional markers can comprise a plurality of spatial barcodes, each specific for the implantation device from which they originate. [0091] In embodiments, the second set of positional markers can comprise a plurality of spatial barcodes, each specific for the implantation device from which they originate. [0092] Spatial barcodes as described herein can be loaded into each injector (and/or injector cavities) of the first implantation device, second implantation device, or both. Spatial barcodes can be loaded according to methods of the present disclosure so that each injector (and/or injector cavity) comprises a plurality of spatial barcodes, wherein each oligonucleotide of the plurality of spatial barcodes in each injector (and/or injector cavity) of each implantation device comprises spatial barcodes having oligonucleotide sequences that are unique compared to spatial barcodes loaded into any other injector (and/or injector cavity). In some embodiments, an implantation device can include more than one spatial barcode sequence (rather than each being identical in a particular implantation device) but nevertheless oligonucleotide sequences are unique compared to spatial barcodes loaded in any other injector (and/or injector cavity). [0093] In embodiments, oligonucleotides comprising spatial barcodes as described herein can comprise a polymerase chain reaction (i.e., PCR) handle (i.e., primer site). The PCR handle can be an oligonucleotide sequence on the 5’ end of a spatial barcode oligonucleotide, for example, that can be used to design complementary primers to and amplify, e.g., using PCR. [0094] In embodiments, spatial barcodes as described herein can further comprise one or more unique molecular identifiers (UMIs). [0095] In embodiments, each oligonucleotide comprising spatial barcode comprises a poly- A tail on the 3’ end of the spatial barcode (or otherwise end opposite the PCR handle). [0096] In embodiments, to load and create the oligonucleotides comprising spatial barcodes into injectors of implantation devices described herein, electric field-guided loading of oligo x-barcodes can be used for each column and oligo y-barcodes for each row in the array. Alternatively, in-situ DNA synthesis can be utilized to create a different oligo within each injection chamber of the implantation device, for example, an injector chamber. [0097] In embodiments, oligonucleotides comprising spatial barcodes as described herein can further comprise a detectable label as known in the art, for example (and without intending to be limiting), a fluorescent reporter such as green fluorescent protein (GFP) or a dye (such as 6-FAM, TET, VIC, HEX, NED, PET, for example). It would be within the ordinary skill in the art for the skilled artisan to find and utilize a reporter for spatial barcodes as described herein, for example. Each oligonucleotide of the first set of positional markers (i.e., spatial barcodes) can comprise the same or a different detectable label than each oligonucleotide of the second set of positional markers (i.e., spatial barcodes). [0098] According to embodiments of methods of the present disclosure, the spatial barcodes as described herein can further integrate with and interact with existing single-cell omic assays and their own respective barcodes. In embodiments, such barcodes of the existing single-cell omic assays can comprise a polymerase chain reaction (i.e., PCR) handle (i.e., primer site), and an oligonucleotide barcode. In embodiments, spatial barcodes as described herein can further comprise one or more unique molecular identifiers (UMIs). In embodiments, each existing single-cell omic assay barcode can comprise a poly-A or poly-T tail on the 3’ end of the spatial barcode (or otherwise end opposite the PCR handle). Such barcodes can also comprise additional features, for example, anchors that are interspersed with the barcode elements described above, additional barcode sequences, detectable labels, and the like. [0099] Additional aspects relating to the design of barcodes (e.g., spatial or cell-specific barcodes) as described herein can be found, for example, in Johnson MS, Venkataram S, Kryazhimskiy S. Best Practices in Designing, Sequencing, and Identifying Random DNA Barcodes. J Mol Evol.2023 Jun;91(3):263-280. doi: 10.1007/s00239-022-10083-z. Epub 2023 Jan 18. PMID: 36651964; PMCID: PMC10276077, which is incorporated by reference in its entirety as if fully set forth herein. EXAMPLES [0100] The following examples are offered to illustrate, but not to limit, aspects of the present disclosure. EXAMPLE 1 – PROPHETIC 3D SPATIAL SINGLE-CELL OMICS BY JETLET BARCODING [0101] When a high-speed focused stream of water hits a tissue slab, it can penetrate many layers (10 layers, for example) of cells into the slab. After a jetlet has passed through a cell, part of the liquid in the jetlet will stay behind in the cell. If the diameter of the jetlet is small (1 ^m, for example) compared to that of the cell (20 ^m, for example), the opening the jetlet made in the cell membrane will re-seal and the cell will remain intact. Furthermore, there may be minimal lateral spreading as the jetlet penetrates into the tissue sample as well. [0102] As described herein, implantation devices, such as jetlet dispensors, can deliver spatial barcodes to cells in a 3D tissue sample. Methods as described herein can then use these codes to compute or to map the regions occupied by each cell. Specifically, the spatial barcode delivered by a jetlet to a cell that it has entered is just the barcode of this jetlet. In methods according to the present disclosure, the jetlet’s orientation and location of entrance to the jetlet will be known, and partial information can be obtained on the cell’s location from the barcode. If the cell is hit by multiple jetlets from different angles, the cell’s 3D location can then be approximated. [0103] For example, if a tissue sample is provided which is cubic in shape and has linear dimension 0.2 mm. Assuming an average cell diameter of 10 ^m, such a sample may contain as many as 8,000 cells. To deliver spatial barcodes to this sample, an ejection array can be constructed and utilized which, such as a 2D array of nozzles for jetlet ejection. For example, the array may have a pitch of 3 ^m (center of one nozzle to the next) and a nozzle diameter of 1 ^m. As shown in FIG. 2, the region highlighted in black in tissue block A represents the region occupied by a cell in this tissue sample. Nozzle array B shoots jetlets into the topmost face of the tissue block. Nozzle array C shoots jetlets into the rightmost face of the tissue block. [0104] With FIG.2 in mind, when this tissue sample is analyzed by a single cell omics assay (i.e., sc omics assay), the barcodes of the jetlets in array B that had crossed the highlighted cell region can be detected in the assay, for example linked to cell-specific barcode sequences. The barcodes for jetlets in array C that had crossed the cell can also be detected. The localization data associated with a cell will can comprise two parts: (1) the coordinates of the jetlets in array B that had crossed the cell, and (2) the coordinates of the jetlets in array C that had crossed the cell. The first part can be used to compute (approximately) the projection of the cell onto the x-y plane. The second part can be used to compute the projection of the cell onto the x-z plane. Although this does not provide an exact reconstruction of the 3D region occupied the cell, it will bound it by the intersection of two cylinders, the centroid of this intersection will approximate the centroid of the cell with an error of the order of the maximal diameter of the cell. [0105] Note that orientation of arrays B and C relative to the tissue block A can be different from that shown in FIG.2. For example, both arrays can be rotated along the y-axis by 45° (or about 30° to about 90°). Then, the jetlets of array B and the jetlets of array C will both enter the top surface of the tissue block at an inclined angle, but they are still perpendicular to each other. This arrangement will allow us to analyze tissue blocks with larger x and y dimensions. [0106] The jetlet array needed to implement methods as described herein can be produced using existing approaches such as the LASI (Laser Activated Supercritical Injector) device[s] and method referenced throughout the present disclosure (and discussed further in section V above). 3D image reconstruction from multiple projections [0107] To obtain a more accurate estimate of cell locations, methods as described herein can be formulated as a 3D image reconstruction problem. The tissue block can be partitioned into a 3D array of voxels. The trajectory of each jetlet from array B can be represented as a vertical one-dimensional (1D) column of voxels, where the tissue block is just the union of these trajectories. If the 3D region occupied by the cell is specified as a connected set of voxels, it can be used to compute the set of trajectories from array B that intersect with this cell region, and similarly for array C. By comparing these with the actually observed set of trajectories that had crossed the cell, the error for this specification of the cell region can be computed by, for example, defining the total loss as the sum of these errors over all cell regions. This total loss is a function of the joint specification of cell regions. The localization of the cells can then be considered as an optimization problem where this total loss function is minimized to obtain a joint specification of the cell regions. The bounds on the cell regions (discussed in the previous paragraph) can be used as constraints to help with the minimization. [0108] This 3D image reconstruction approach can be extended to cover the case when more than two jetlet arrays are used, with each array oriented differently relative to the tissue block. Each array will provide a different 2D projection of the cell region. If enough projects are obtained, the cell region can be reconstructed with high accuracy. 3D localization with the help of reflectance confocal microscopy [0109] An alternative and powerful method is to use reflectance confocal microscopy to build a 3D model of the tissue block, which can provide the location and boundary for each cell. This can be done as long as the imaging depth needed is not larger than about 250 ^m. After the microscopy, the tissue block will still be intact and can be subjected to methods and analyses as described herein to obtain omics profiles and jetlet barcodes for each single cell. To map a single cell omics profile to this 3D model, the cell regions in the 3D model can be searched for a region that is most consistent (using the above error) with the location data associated with this profile. The microscopy-based 3D model not only can greatly simply the spatial mapping of the single cell profiles, but can also provide highly accurate determination of cell boundaries and morphologies. If confocal microscopy is available, this can be a preferred method for mapping the cell locations over other microscopy modalities. Example 2 – Injection of an array of jetlets into a slab of human liver tissue [0110] Injection of an array of jetlets into a slab of human liver tissue was tested using the LASI method depicted in FIG. 7A. The LASI array (FIG. 7A) is a silicon chip with a 2- dimensional grid of deep channels etched into it. The channels were loaded with fluorescent nanobeads suspended in solution. The array was scanned with a laser to heat up the liquid at the deep end of the channel, which then causes the liquid to shoot out of the channel at high speed as a jetlet (FIG. 7B). The jetlet ejected by a channel then hit and penetrated into a slide of human liver tissue facing the LASI array, at a location corresponding to the location of the channel on the LASI array. The performance of the LASI injections was assessed by using a confocal microscope to measure the distribution of fluorescent beads left behind by the jetlets (FIG. 7C). The jetlets from adjacent channels remained clearly separated from each other, suggesting that there was minimal lateral spread of a jetlet after it entered the tissue sample. Using this version of the array, the penetration can reach 33^m, which can cover several cell layers. With a higher aspect ratio for the channel and with suitable changes in the semiconductor to achieve better delivery of laser energy, it will be possible, for example to increase the penetration to more than 100 ^m. [0111] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. The disclosure encompasses all combinations of the particular embodiments recited herein, as if each combination had been individually and laboriously recited. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

WHAT IS CLAIMED IS: 1. A method of multi-dimensional spatial labeling of a biological sample, comprising: implanting a first set of positional markers in a biological sample comprising a plurality of cells along a first positional axis using a first implantation device; dissociating the biological sample into single cells; isolating the first set of positional markers from the dissociated single cells; and detecting the first set of positional markers to obtain positional data. The method of claim 1, wherein the first set of positional markers comprises a first plurality of nucleic acids comprising barcode sequences specific for the implantation device from which the nucleic acids originate. 3. The method of claim 2, wherein the each of the nucleic acids further comprise a PCR handle sequence, a unique molecular identifier (UMI) barcode, a sample barcode, or any combination of any thereof. 4. The method of any one of claims 1 to 3, further comprising implanting a second set of positional markers into the biological sample along a second positional axis using a second implantation device and detecting the second set of positional markers along with the first set of positional markers. 5. The method of claim 4, wherein the second set of positional markers comprises a second plurality of nucleic acids comprising barcode sequences specific for the implantation device from which the nucleic acids originate. 6. The method of claims 5, wherein the positional markers of the second set of positional markers further comprises a PCR handle sequence, a unique molecular identifier (UMI) barcode, a sample barcode, or any combination of any thereof. The method of any one of claims 4 to 6, where the first positional axis and second positional axis each are about a 45-degree and 135-degree inclination to the top surface of the biological sample. 8. The method of any one of claim 4 to 6, wherein the first positional axis and second positional axis are orthogonally opposed to each other. 9. The method of any one of claim 4 to 8, wherein the second set of positional markers comprises a second plurality of nucleic acids, wherein each nucleic acid of the first plurality of nucleic acids is different from each nucleic acid of the second plurality of nucleic acids. 10. The method of any one of claims 1 to 9, wherein the implanting comprises injecting into the biological sample droplets comprising one or more nucleic acids from the first set of positional markers, second set of positional markers, or both. 11. The method of any one of claims 1 to 10, wherein the first implantation device comprises a two-dimensional (2D) array of injectors. 12. The method of claim 11, wherein the 2D array of the first implantation device is a 100 x 100 to a 1000 x 1000 array. 13. The method of claims 11 or 12, wherein each injector of the 2D array comprises a unique nucleic acid from the first set of positional markers. 14. The method of any one of claims 4 to 13, wherein the second implantation device comprises a second two-dimensional (2D) array of injectors.

15. The method of any one of claims 4 to 14, wherein the second 2D array of the second implantation device a 100 x 100 to a 1000 x 1000 array. 16. The method of claims 14 or 15, wherein each position of the second 2D array comprises a unique nucleic acid from the second set of positional markers. 17. The method of any one of claims 1 to 16, wherein the dissociation comprises one or more of mechanical dissociation, enzymatic dissociation, chemical dissociation, or any combination of any thereof. 18. The method of any one of claims 1 to 17, wherein the dissociation comprises: treating the biological sample with a cell dissociation reagent at about 37°C for a period of time; mechanically agitating the sample and reagent mixture; and filtering the agitated mixture to obtain a composition comprising a plurality of single cells. 19. The method of any one of claims 1 to 18, wherein the detection comprises sequencing, polymerase chain reaction (PCR) or next-generation sequencing. 20. The method of any one of claims 1 to 19, further comprising reconstructing a multidimensional image using the positional data. 21. The method of any one of claims 1 to 20, further comprising analyzing the dissociated single cells using one or more analysis methods, wherein the one or more analysis methods is one or more of a transcriptomics analysis, a proteomic analysis, a metabolomic analysis, a lipidomic analysis, or any combination of any thereof.

22. The method of claim 21, wherein the analysis method is next-generation sequencing (NGS). The method of any one of claims 1 to 22, further comprising, after implanting or linking, rotating the first implantation device, second implantation device, or both, at an angle of about 30 degrees to about 90 degrees, in relation to the first positional axis, second positional axis, or both. 24. The method of claim 23, wherein the rotation angle is 45 degrees. 25. The method of any one of claims 1 to 24, wherein the first implantation device, second implantation device, or both, comprise a laser-actuated supercritical injector (LASI) array. 26. The method of any one of claims 1 to 25 wherein the positional markers comprise a plurality of nucleic acids comprising barcode sequences specific for the implantation device from which the nucleic acids originate; and the method further comprises: forming the dissociated cells into different partitions; linking oligonucleotides comprising partition-specific barcodes sequences to cellular DNA, or cDNAs from cellular RNA, and linking the oligonucleotides comprising partition- specific barcodes sequences to the nucleic acids comprising barcode sequences specific for the implantation device. 27. The method of claim 26, wherein the plurality of nucleic acids further comprise a poly A tail sequence and the oligonucleotides comprising partition-specific barcodes sequences comprise a poly T tail sequence and the method comprises performing reverse transcription with the oligonucleotides comprising partition-specific barcodes sequences to form (i) cDNAs from cellular mRNA and (ii) a nucleic acid comprising oligonucleotides comprising partition- specific barcodes sequences linked to the nucleic acids comprising barcode sequences specific for the implantation device.

28. The method of claim 26 or 27, where the partitions are droplets or microwells. 29. The method of any one of claims 27 or 28, further comprising nucleotide sequencing products of the linking and associating the partition-specific barcodes with the positional marker to determine a position of the cell in the biological sample.