Single-cell transcriptomics analysis and multimodal profiling

Abstract

A sample preparation, computational system, sample analysis workflow, and associated methods are presented herein which utilize imaging-based transcriptomics and proteomics in place of conventional single cell sequencing methods to significantly scale-up cell numbers at substantially reduced costs for single cell analysis, while preserving the advantages of molecule detection at a single-cell level. Traditionally, spatial transcriptomics and proteomics have been applied in the context of tissue profiling to map the composition and architecture of complex samples, such as organs or tumors. A sample preparation including one or more monolayers of individual cells is analyzed using image-based transcriptomics and proteomics equipment which can include software to take advantage of new aspects of the sample preparation.

Description

Attorney Docket #: 243734.000229SJ-25 -0018-02SINGLE-CELL TRANSCRIPTOMICS ANALYSIS AND MULTIMODAL PROFILINGCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 723,044, filed on Nov. 20, 2024, and U.S. Provisional Application No. 63 / 737,985, filed on Dec. 23, 2024, the contents of which are herein incorporated by reference in their entirety.FIELD

[0002] This application relates generally to sample preparation for cellular analysis and uses thereof, and more particularly to preparation of a high-density fixed monolayer of single cells or tissue fragments for analysis by and calibration of single-cell spatial molecular imager platforms.BACKGROUND

[0003] Over the last decade, single-cell sequencing, particularly single-cell RNA sequencing (scRNA-seq), has revolutionized our understanding of complex tissues and organs by providing high-resolution maps of their individual cells. The profiling of single cells has offered invaluable insights into cellular composition and states during steady conditions and dynamic processes, such as differentiation or perturbations in diseases. Innovations in microfluidics and combinatorial indexing have enabled the profiling of increasingly large cell numbers, generating atlases of entire organisms, including the fly, mouse and human.

[0004] Despite these advancements, current single-cell transcriptomic methods face significant limitations. The reliance on sequencing and the need to index cells individually in wells or droplets results in high costs that scale linearly with cell numbers. Additional limitations relate to the random sampling of cellular transcripts, resulting in a bias and overrepresentation of highly abundant transcripts at the cost of lowly expressed, often cell lineage-defining genes, such as transcription factors.

[0005] Single-cell sequencing methods also suffer from inherent inefficiencies, with dropletbased microfluidics being affected by cell damage, inefficient encapsulation, and dropletAttorney Docket #: 243734.000229SJ-25 -0018-02 instability, leading to significant sample loss. On the other hand, plate-based combinatorial indexing methods show limited cell capture, cross-contamination and inefficient indexing. Factors such as cell size, fragility and RNA content can further affect the capture efficiency, resulting in underrepresented or entirely missed cell populations. As a consequence, current single-cell datasets may not accurately reflect the cellular composition and sample complexity.

[0006] Conventional scRNA-seq workflows, whether used alone or in combination with multimodal or multiplexing strategies have relatively low throughput, typically handling thousands of cells per experiment. Current methods face difficulties with both ultra-low (hundreds of cells) and ultra-high (millions of cells) experimental scales, the latter related to high costs for library preparation and sequencing and high input material. Scaling single-cell profiling is crucial though for studying rare cell populations and for capturing the full complexity of heterogeneity and cell plasticity in health and disease. In addition, sequencing-based methods destroy cells during molecule capture and library preparation, limiting the ability to combine molecular profiles with cell morphology (e.g., shape and size) or functional attributes (e.g. metabolic activity).

[0007] Single-cell technologies have transformed our ability to detect donor-, cell type-, and cell state- specific variations within mixed clinical samples but challenges like identifying and removing cell doublets, controlling batch effects to accurately detect biologically or clinically relevant patterns and high sequencing costs limit large-scale studies.SUMMARY

[0008] A sample preparation, computational system, sample analysis workflow, and associated methods are presented herein which utilize imaging-based transcriptomics and proteomics in place of conventional single cell sequencing methods to significantly scale-up the number of single cells analyzed at substantially reduced costs, while preserving the advantages of molecule detection at a single-cell level. Advances in spatial transcrip tomic imaging assays now offer large gene panel designs, operating at transcriptome-wide scale, closely mirroring the capabilities of scRNA-seq methods. These molecular imager platforms can also perform proteomics. Traditionally, spatial transcriptomics and proteomics have been applied in the context of tissue profiling to map the composition and architecture of complex samples, such as organs or tumors. In these systems, a tissue mass (e.g. slice) is affixed to a slide which is inserted into a spatial molecular imager tool which performs repeated hybridization with fluorescently labeled probes that bind to specificAttorney Docket #: 243734.000229SJ-25 -0018-02 mRNAs and microscopy imaging to capture the spatial distribution of transcripts. Single cell analysis using spatial molecular imager equipment has been attempted with very limited success due to challenges with cell adhesion, cell distribution, and hydration of cells with reagent.

[0009] To address these, Single-Cell Transcriptomics Analysis and Multimodal Profiling (STAMP), is described herein as a cost effective approach that transforms commercial imaging platforms into scalable and flexible profiling tools for spatial transcriptomics and proteomics readouts in suspension cells. STAMP provides a unified solution that integrates high-throughput analysis, cost efficiency, and multi-sample profiling, expanding the possibilities of single-cell analysis for diverse research and clinical applications, and for advancing our understanding of complex biological systems and diseases.

[0010] STAMP is demonstrated in various examples herein with the main objectives to verify: 1) specificity, 2) reproducibility, 3) scalability, 4) sensitivity, 5) resolution, 6) flexibility, and 7) multimodality. Cancer cell lines were used to confirm specificity and to identify potential artifacts, ensuring that STAMP accurately targeted specific gene sets within individual cells without crosshybridization or background noise. For reproducibility and scalability, millions of peripheral blood mononuclear cells (PBMCs) were analyzed across different platforms and panel sizes, demonstrating STAMP'S consistent performance. Flexibility was assessed using mixtures of cancer cell lines and profiling nuclei, together with various multiplexing experiments. STAMP demonstrated adaptability with diverse sample sizes, multiplexing capabilities, and conditions, achieving high granularity in blood cell immunophenotyping and embryonic stem cell differentiation studies. Its sensitivity was proven in CTC-mimic experiments, detecting rare populations down to 0.00025%.

[0011] A critical advantage of the STAMP technology is its cost-effectiveness, providing a significant reduction in expenses compared to other single-cell imaging modalities. Even when using a non- specialized panel as seen in the hESC BMP4 differentiation experiment, the STAMP sc-imaging approach generates valuable and actionable insights matching the gold standard of transcriptome scRNA-seq technology, which we believe highlights the versatility and potential of this technology.

[0012] Building on proof-of-concept experiments such as the BMP4 hESC perturbations, combining affordable single-cell imaging technologies like STAMP with a refined or expanded probe set, such as one tailored to differentiation or larger panels (5K, 6K, or whole transcriptome),Attorney Docket #: 243734.000229SJ-25 -0018-02 would significantly improve resolution and data depth. This would enable more precise identification of cellular trajectories and lead to more accurate biological interpretations. The ability to customize panels for specific research needs, while keeping costs low, makes STAMP an especially valuable tool for spatial transcriptomics. The integration of STAMP technology with tailored or larger probe sets could be a future-proof solution, offering a balance between accessibility, precision, and scalability. This makes it a strong contender for researchers looking to perform high-quality, cost-effective single-cell spatial transcriptomics on a wide range of biological questions.

[0013] STAMP’S cost-effectiveness makes it accessible to a broader audience, delivering results comparable to scRNA-seq even with non- specialized probe panels. This multimodal integration points to STAMP’S potential as a powerful tool for multi-layered data analysis, where RNA and protein information can be combined to enrich cell-type characterization and functional insights. Its ability to support single-modal (RNA or protein) and multimodal (RNA and protein) profiling bridges gaps between transcriptomics and proteomics, enhancing cell-type characterization and multiomic analysis. As protein panels expand, STAMP will provide deeper insights through comprehensive multi-layered data. By replacing sequencing with advanced imaging, STAMP captures spatially indexed morphological and molecular data simultaneously, supporting visual inspection, reducing false positives, and enabling multimodal profiling of RNA, protein, and histological features. Its scalability supports applications ranging from rare cell population studies to large-scale atlases and CRISPR screens.

[0014] STAMP addresses key limitations in single-cell research by combining scalability, costefficiency, and flexibility. Its accessibility and precision make it an indispensable tool for diverse experimental and clinical needs, setting a new standard for high-throughput single-cell omic analysis. Unlike sequencing-based methods, STAMP employs advanced imaging techniques to spatially index individual cells, capturing morphological and molecular information simultaneously. This dual capability allows researchers to visually inspect cells — valuable in clinical settings where histopathology validation is the standard of care — and reduces false positives by cross-referencing molecular data with visual characteristics. Furthermore, STAMP'S non-destructive nature supports simultaneous analysis of RNA, protein, and histological features in the same cells, enabling comprehensive multimodal profiling for multiscale phenotypic analysis.Attorney Docket #: 243734.000229SJ-25 -0018-02Its versatility allows for a wide range of applications, from studying small, rare cell populations to creating large-scale cell atlases and conducting CRISPR or drug screens.

[0015] By addressing current limitations in single-cell genomics. STAMP offers an integrated solution that combines scalability, cost-effectiveness, multimodality and flexibility in a single approach. This enables researchers to perform experiments on a broad range of sample sizes, from limited biopsy specimens to extensive cellular aliasing, without compromising data quality or increasing costs. STAMP’S ability to balance accessibility, precision, and scalability makes it an invaluable tool for a wide array of experimental needs, from basic research to clinical applications, setting a new benchmark for high-throughput single-cell analysis.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] While the specification concludes with claims, which particularly point out and distinctly claim the subject matter described herein, it is believed the subject matter will be better understood from the following description of certain examples taken in conjunction with the accompanying drawings, in which like reference numerals identify the same elements. The figures depict one or more implementations of the inventive devices, by way of example only, not by way of limitation.

[0017] FIG. 1 is an image of an example sample preparation having a substrate and multiple sample regions each with a respective monolayer of fixed cells and / or nuclei.

[0018] FIG. 2 is a flow diagram including steps of a method of preparing a sample such as the sample preparation illustrated in FIG. 1.

[0019] FIG. 3 is an image of an example sample preparation having a substrate having a first sample region with a known control sample of a monolayer of fixed cells and / or nuclei and a second sample region with a test sample.

[0020] FIG. 4 is a flow diagram including steps of a method of preparing a sample such as the sample preparation illustrated in FIG. 3.

[0021] FIG. 5 is an image of a substrate with a compartmentalization apparatus thereon with openings forming wells with a suspension of fixed cells and / or nuclei as step for preparing a sample.

[0022] FIG. 6 is a flow diagram including steps of a method of preparing a sample including a step corresponding to FIG. 5.Attorney Docket #: 243734.000229SJ-25 -0018-02[0023| FIGs. 7A-7D include images demonstrating size and density of respective monolayers of fixed PBMCs in each sample region in which FIG. 7A includes an image of a sample preparation; FIG. 7B is an image of the sample regions annotated to show the number of cells / nuclei in each respective sample region; FIG. 7C includes zoomed-in images of the sample regions illustrated in FIG. 7B; and FIG. 7D includes counts and analysis of the density of fixed cells and / or nuclei in the sample preparation.

[0024] FIG. 8 is an image of a substrate with a gasket thereon with openings forming wells with a suspension of fixed cells and / or nuclei forming a surface tension dome above the well as step for preparing a sample.

[0025] FIG. 9 is a block diagram of an imaging system configured to receive a sample preparation having discrete sample regions and perform computational analysis of the sample preparation considering the discrete sample regions.

[0026] FIG. 10 is a flow diagram including steps of a computational method which may be stored in memory and executed by a processor on a computational system such as by the imaging system illustrated in FIG. 9.

[0027] FIGs. 11A-F illustrate a STAMP experimental design and data analysis workflow related to Example 1. FIG. 11A includes a schematic of the STAMP workflow. FIG. 11B depicts an immunofluorescence (IF) image of STAMP-C highlighting DAPI staining (blue), CD298 / B2M (red), and pan-cytokeratin (PanCK, green) across the four sub-STAMPs, including an enlarged region of interest (ROI 1), where also post-STAMP hematoxylin and eosin staining is shown. Marker genes specific to each cell line are highlighted across the entire STAMP and in a selected ROI (ROI 2): K1.K3 for LNCaP (yellow), GATA3 for MCF-7 (magenta), and KRT7 for SK-BR-3 (cyan), where each dot represents a transcript, and white lines indicate segmented cell borders (scale bar = 750 pm unless otherwise noted). FIG. 11C includes charts of quality metrics for the mixed sub-STAMP containing equal proportions of MCF-7. LNCaP. and SK-BR-3 cell lines, showing distributions and median values of transcript counts, detected features, and cell areas prior to filtering. FIG. 11D includes a UMAP of the mixed sub-STAMP (as in c), colored by InSituType unsupervised clustering. FIG. HE includes a chart of the composition of the 99 imaged fields of view (FOVs) in the mixed sub-STAMP (as in d), showing the distribution of cell lines. FIG. HF includes a heat map displaying the top 10 marker genes for each cluster identified in D, normalized by feature and representing each cell line.Attorney Docket #: 243734.000229SJ-25 -0018-02[0028| FIGs. 12A-I illustrate STAMP as a robust, scalable and flexible platform for imagingbased single cell transcriptomics of cells in suspension in relation to Example 2. FIG. 12A includes a spatial plot showing the experimental design of STAMP-C with 6 sub-STAMPs to showcase low numbers of tumor cells seeded (sub-STAMPs 100 C, 250 C, 500 C and 1,000 C and sub-STAMPs 20K C and 20K N for cells and nuclei comparison). Each dot is a cell, as detected by imaging, px: pixels. FIG. 12B includes a chart of percentage of cells retrieved from input cells across different sub-STAMPS. Text labels indicate absolute cell counts. FIG. 12C includes charts of proportions of tumor cell lines across sub-STAMPs. Figures 12D through 12E include box plots displaying the number of counts D, features E, and cell area F for each sub-STAMP with respective medians. FIG. 12G includes a chart of Pearson correlation of raw counts averaged by cell number between cells and nuclei. FIG. 12H includes charts of the number of detected (segmented) cells, number of detected transcripts and genes, and cell area for each cell line in each STAMP-X (replicate 1 and 2). FIG. 121 includes a chart of a Pearson correlation of raw counts averaged by cell number between MCF-7 and SK-BR-3 replicates, (g, i) Each dot is a gene. Red line shows the fitted linear regression

[0029] FIGs. 13A-H include information showing that STAMP enables high-level multiplexing on a single slide, facilitating the simultaneous analysis of multiple samples in relation to Example 3. FIG. 13A includes drawings of sample layout of highly multiplexed conditions profiled with the STAMP-C (top) and STAMP-X (bottom) platforms, each with two replicates. FIG. 13B includes charts of Pearson correlations comparing the number of counts, number of features, and cell area across replicates for STAMP-C (top) and STAMP-X (bottom). FIG. 13C includes a chart of gene-wise aggregated counts showing high Pearson correlations across samples and replicates. FIG. 13D includes a diagram showing overlap of genes between the Xenium Prime 5K Human Pan Tissue & Pathways Panel and the CosMx Human 6K Discovery Panel, demonstrating complementary profiling capabilities. FIG. 13E includes principal component analysis (PCA) plots showing unintegrated data (left) and integrated data (right), with PCI vs. PC2 plots colored by technology (top), replicate uniqueness (middle), and sample identity (bottom). FIG. 13F includes charts of Local Inverse Simpson’s Index (LISI) scores calculated by technology (left) and replicate (right), illustrating improved data integration performance. FIG. 13G includes a diagram of AUCell scores for the BIOCARTA_LYMPHOCYTE_PATHWAY across Xenium replicates, demonstrating pathway-specific activity. FIG. 13H includes a scatter plot of AUCell scores forAttorney Docket #: 243734.000229SJ-25 -0018-02HALLMARK_HYPOXIA (x-axis) versus HALLMARK_GLYCOLYSIS (y-axis), where each point represents a sample and point size reflects GOBP_LACTATE_METABOLIC_PROCESS scores, highlighting metabolic pathway activity across conditions.

[0030] FIGs. 14A-F include information related to profiling ultra-high cell numbers with STAMP-X in relation to Example 4. FIG. 14A includes charts of an overview of total cell numbers and proportions for the entire PBMC dataset and for each immune lineage, as defined in FIGs. 19B-D. FIG. 14B includes a dot plot displaying cell type-defining marker genes normalized by feature for clusters obtained through sub-clustering of major immune lineages, as described in FIGs. 19B-D and Methods. FIG. 14C includes UMAP visualizations of T cell and myeloid compartments from the PBMC dataset analyzed using the Xenium Prime 5k Human Pan Tissue and Pathways panel, with cells colored by type. FIG. 14D includes a dot plot showing normalized expression of cell type-specific marker genes for clusters identified in FIG. 14C, with further details in FIG. 19E. Abbreviations: DC, dendritic cells; pDC, plasmacytoid dendritic cells; nonclass. mono, non-classical monocytes; int. mono, intermediate monocytes; infl. mono, inflammatory monocytes; class, mono, classical monocytes; TN, naive T cells; TCM, central memory T cells; TEM, effector memory T cells; TPM, peripheral memory T cells; T eff., effector T cells; T act., activated T cells; T cyto., cytotoxic T cells; T inf. resp., interferon responder T cells. FIG. 14E includes charts showing cell type proportions in PBMCs cultured under control conditions (left), LPS stimulation (middle), or anti-CD3 / CD28 stimulation (right) at 4 h (top) and 24 h (bottom). FIG. 14F includes volcano plots of differentially expressed genes in activated monocytes, comparing LPS versus control at 14 h (left) and 24 h versus 4 h under LPS stimulation (right).

[0031] FIGs. 15A-G include information related to profiling human embryonic stem cells (hESC) differentiation after BMP4 induction with STAMP in relation to Example 5. FIG. 15A includes representative images of Hl (WA01) hESCs treated with BMP4 (50 ng / mL) for 0. 6, 12, 24, 48, 72, 96, and 120 hours, captured using a Zeiss Primovert microscope (scale bars: 200 pm). FIG. 15B is an image of the STAMP slide layout showing the eight BMP4 treatment timepoints along with an H&E-stained image of the slide post-STAMP-C. FIG. 15C includes a schematic illustrating the expected differentiation trajectories of hESCs following BMP4 induction. FIG. 15D includes plots of force-directed layout of cells from all eight timepoints is shown, based on diffusion maps computed using the Palantir algorithm. Cells are colored by annotated cell statesAttorney Docket #: 243734.000229SJ-25 -0018-02(top) and displayed separately for each timepoint (bottom). FIG. 15E includes a chart showing the proportions of annotated cell states at each timepoint, using the same color scheme as panel d. FIG. 15F includes a dot plot highlighting the main marker genes used for cell state annotation. FIG. 15G includes charts showing differentiation trajectories for the amnion, endoderm, and mesoderm branches inferred using Palantir, with expression trends of selected marker genes shown along the pseudotime trajectories.

[0032] FIGs. 16A-J include information related to detection of circulating tumor cells mimics (CTC-mimics) and multimodal using STAMP in relation to Example 6 (FIGs. 16A-16D) and Example 7 (FIGs. 16E-G). FIG. 16A includes an immunofluorescence image of a representative field of view showing 3 CTCs, with DAPI, pancytokeratin (PanCK), the pan-membrane marker CD298 / B2M, and CD45. FIG. 16B includes a heat map of MCF-7 and PBMC gene signatures derived from differential expression analysis of the Flex dataset, subsetted for the CosMx IK panel. FIG. 16C includes a scatter plot showing the relationship between the MCF-7 signature score (as extracted in b) and PanCK mean fluorescence intensity (MFI) in the 10 CTC-mimic spike-in substamp. Each dot represents a cell, with size proportional to cell area and color-coded by the number of counts. FIG. 16D includes an immunofluorescence image of a region of interest showing DAPI (blue), PanCK (yellow), CD298 / B2M (cyan), CD45 (magenta), and cell segmentation outlines (white), highlighting a DAPI+ PanCK-i- CD45- CTC-mimic (#1.2) identified in c). FIG. 16E includes a multimodal visualization of a CTC-mimic identified in STAMP-X (Xenium with the Xenium Prime 5K Human Pan Tissue and Pathways panel), including images of i) H&E and cell-line-specific markers at low and ii) high magnification, iii) DAPI and cell-line-specific markers, iv) Xenium cell segmentation markers and cell-line-specific markers, v) segmented cells colored by cluster ID in Xenium Explorer and cell-line-specific markers, vi) cell boundaries colored by cluster ID showing the CTC-mimic stained with PanCK plus cell-line-specific markers, and vii) cell boundaries colored by cluster ID stained with immune cell markers plus cell-line-specific markers. FIG. 16F includes charts showing protein counts of STAMPs of PBMCs run on CosMX and Phenocycler Fusion, including i) aggregated cell counts and ii) cell area (pm2). FIG. 16G includes plots of average expression of all STAMPs run on protein panels showing high correlation. FIG. 16H include a chart showing cell proportions detected in multimodal profiling of all STAMPs (STAMP-X / X-CP / CP and STAMP-X / X- PCF / PCF), run for both RNA and protein. FIG. 161 includes UMAP visualization of RNAAttorney Docket #: 243734.000229SJ-25 -0018-02STAMP-X-CP showing unsupervised clustering and cell type annotations, demonstrating that all clusters of PBMCs are detected with further sub-clustering of each major immune cell type. FIG. 16J includes UMAP visualization of protein STAMP-X-CP, which illustrates that all clusters of PBMCs are detected, with sub-clusters of each major immune cell type. R and p-value statistics are provided for Pearson correlation analysis.

[0033] FIGs. 17A-J includes information related to STAMP analysis of cells isolated from mouse tissues as related to Example 8. FIG. 17A includes a diagram of tissue STAMP design performed on cells dissociated from mouse tissues: brain, heart, kidney, liver, and lung, as well as a 1 : 1 : 1 : 1 : 1 mixture of cells from these organs. FIG. 17B includes a barplot showing the proportion of high- and low-quality cells and nuclei for each mouse organ. FIG. 17C includes a box plot showing the number (in natural log) of transcripts captured by tissue STAMP and cell isolation. FIG. 17D includes a box plot of number of genes (in natural log) detected in cells from tissue STAMP across different organs. FIG. 17E includes a box plot of the distribution of cell areas captured in tissue STAMP. FIG. 17F includes a scatter plot of average gene expression comparison between cells and nuclei across all tissue STAMP datasets. FIG. 17G includes UMAP visualizations of cell clusters from individual mouse tissues: I brain, (ii) heart, (iii) lung, (iv) liver, (v) kidney, and (vi) a mixed cell population from all five organs. FIG. 17H includes a chart showing proportion of cells detected across organ types in the cell mixture. FIG. 171 includes UMAP visualization of all cell types identified in the mixed population from the five organs. FIG. 17J includes a dot plot highlighting the most highly expressed marker genes for each organ type.

[0034] FIGs. 18A-I figures related to Example 1 which shows that clustering analysis of tumor cell lines in STAMP-C recapitulates classical single cell transcriptomics data and can be combined with H&E. FIG. 18A shows H&E staining performed post-STAMP-C highlights single-cell and nuclear morphology while illustrating the experimental design. The spatial distribution of counts is shown in FIG. 18B, features are shown in FIG. 18C. and cell area is show in FIG. 18D for each sub-STAMP. FIG. 18E includes a line plot which shows the total number of counts relative to the distance from the field of view (FOV) border, with horizontal red lines indicating the moving median and a vertical dashed red line marking the threshold applied. FIG. 18F includes a bar plot which quantifies the number of cells excluded from each FOV due to border effects. FIG. 18G includes a UMAP visualization that is color-coded by cluster ID for MCF-7, LNCaP, and SK-BR- 3 sub-STAMPs pooled together. FIG. 18H includes a heat map which illustrates transcriptionalAttorney Docket #: 243734.000229SJ-25 -0018-02 profiles of each cell line as defined by InSituType (1ST). FIG. 181 includes a Spearman correlation plot which compares gene expression between the 10X Flex dataset and STAMP-C data from the same cell suspensions, with individual dots representing genes and red dots highlighting cell line marker genes identified in FIG. 18F.

[0035] FIGs. 19A-H are figures related to Example 4 which shows profiling PBMCs with STAMP-X across platforms and panels. FIG. 19A includes charts of quality metrics of the 1.7M PBMC STAMP-X dataset showing the distribution of counts, features, and cell area before filtering. Red dotted lines and red text indicate the threshold set for filtering. FIG. 19B shows principal component analysis (PCA) color-coded by cluster identity, based on Leuven clustering of the STAMP-X PBMC dataset analyzed with the Immuno-oncology panel. FIG. 19C includes charts showing loadings of the 3 first PCs. FIG. 19D includes box plot displays of the number of counts, features and cell area split by cell lineage. Text labels indicate median values. FIG. 19E shows UMAPs and dotplots of NK and B cell lineages showing population markers. FIG. 19F shows PBMCs perturbations in a STAMP-C layout. FIG. 19G includes charts of quality metrics distributions for FIG. 19F. Red lines show median values, while dotted gray lines show filtering thresholds. FIG. 19H includes a dotplot of annotated PBMCs populations with marker genes. FIG. 19H is a dotplot of myeloid sub-populations showing Toll Like Receptors (TLRs) z-scored mean expression.

[0036] FIGs. 20A-E are figures related to Example 5 showing profiling of human embryonic stem cell (hESC) differentiation upon BMP4 treatment with Single Cell Gene Expression Flex technology. FIG. 20A includes a schematic which illustrates the anticipated cell trajectories following BMP4 treatment of human embryonic stem cells (hESCs). FIG. 20B includes a diagram showing cells from eight time points post-BMP4 treatment which are visualized using a force- directed layout on diffusion maps computed with the Palantir algorithm. The upper panel shows cells colored by their annotated states, while the lower panel separates cells by time point. FIG. 20C includes a bar plot which quantifies the proportion of each annotated cell state at each time point, using the same color scheme as in FIG. 20B. FIG. 20D includes dot plot displays of the key marker genes used to define each cell state. FIG. 20E includes a diagram showing differentiation trajectories for Amnion, Endoderm, and Mesoderm lineages inferred with Palantir (right). Corresponding expression trends of selected marker genes are plotted along the trajectories’ pseudotime (left).Attorney Docket #: 243734.000229SJ-25 -0018-02

[0037] FIG. 21A-C are figures related to Example 5 showing iPSC differentiation. FIG. 21A includes Principal Component Analysis (PCA) plots displaying PCI and PC2, with points color- coded by sub-STAMP identity to highlight clustering patterns. FIG. 21B includes a dot plot which showcases the key marker genes utilized for cell type annotation, indicating their expression levels across identified sub-STAMPs. FIG. 21C includes plots of Spearman gene expression correlation comparing the 10X Flex dataset with the STAMP-C dataset derived from the same cell suspensions. Each dot represents a gene, while red dots highlight marker genes identified in FIG. 21A and FIG. 21B.

[0038] FIGs. 22A-B are figures related to Example 6 showing identification of CTCs-mimic with STAMP-X and STAMP-X-CP. FIG. 22A includes images of a full STAMP-X showing cells clustered in Xenium explorer. Two regions of interest (ROIs) are shown as white frames, from where the zoom-in images are taken. Enlargements show cells color-coded by cluster identity, hematoxylin and eosin staining (H&E), DAPI nuclear segmentation marker in blue, cytoplasmic and membrane segmentation markers ATPlAl / CD45 / e-Cadherin in pink, 18S RNA in yellow, alphaS M A / Vimen tin in green, in that order. EPCAM single transcripts for epithelial / cancer cells are shown as green dots in each image. FIG. 22B includes images of a full STAMP-X-CP showing clustered cells with two ROIs (white frames), from where the zoom-in images are taken, with one SK-BR-3 and one MCF-7 cell. Enlargements show H&E staining (i), the fluorescence image with the segmentation markers (ii; DAPI nuclear segmentation marker in blue, cytoplasmic and membrane segmentation markers ATPlAl / CD45 / e-Cadherin in pink, 18S RNA in yellow, alphaS MA / Vimentin in green), and the protein markers used to identify epithelial / tumor cells (iii; DAPI nuclear staining in blue and PanCK in red) and to identify immune cells (iv; CD45 in red, CD8 in yellow and CD4 in cyan). SK-BR-3-specific transcripts are shown as dots in the first column (D11RS2 in cyan, EPCAM in magenta and ERBB2 in yellow) and MCF-7-specific transcripts are shown in the last column (EEF1A2 in green, EPCAM in magenta and USP32 in purple).

[0039] FIGs. 23A-B are figures related to Example 7 showing multimodal STAMP for RNA (Xenium) and protein (PhenoCycler Fusion). FIG. 23A includes UMAP visualization of RNA STAMP-X-PCF showing unsupervised clustering and cell type annotations, demonstrating that all clusters of PBMCs are detected with further sub-clustering of each major immune cell type. FIG. 23B includes UMAP visualization of protein STAMP-X-PCF illustrates that all clusters of PBMCsAttorney Docket #: 243734.000229SJ-25 -0018-02 are detected, with sub-clusters of each major immune cell type. R and p-value statistics are provided for Pearson correlation analysis.

[0040] FIGs. 24A-F are figures showing STAMP analysis of nuclei isolated from mouse tissues. FIG. 24A includes a scatter plot of tissue STAMP transcript count by number of genes detected across five mouse tissue types for cells (burgundy dot) and nuclei (light blue dots). FIG. 24B is an image of a sample preparation with a tissue STAMP design performed on cells dissociated from mouse tissues: brain, heart, kidney, liver, and lung, as well as a 1:1:1: 1:1 mixture of cells from these organs. FIG. 24C includes UMAP visualizations of nuclei clusters from individual mouse tissues: (i) brain, (ii) heart, (iii) lung, (iv) liver, (v) kidney, and (vi) a mixed nuclei population from all five organs. FIG. 24D includes UMAP visualization of all cell types identified in the mixed population from the five organs. FIG. 24E is a bar chart showing proportion of nuclei detected across organ types in the nuclei mixture. FIG. 24F is a dot plot highlighting the most highly expressed marker genes for each organ type.DETAILED DESCRIPTION

[0041] The following detailed description should be read with reference to the drawings, in which like elements in different drawings are identically numbered. The drawings, which are not necessarily to scale, depict selected embodiments and are not intended to limit the scope of the invention. The detailed description illustrates by way of example, not by way of limitation, the principles of the invention. This description will clearly enable one skilled in the art to make and use the invention, and describes several embodiments, adaptations, variations, alternatives, and uses of the invention, including what is presently believed to be the best mode of carrying out the invention.

[0042] As used herein, the terms “about” or “approximately” for any numerical values or ranges indicate a suitable dimensional tolerance that allows the part or collection of components to function for its intended purpose as described herein. More specifically, “about” or “approximately” may refer to the range of values ±10% of the recited value, e.g. “about 90%” may refer to the range of values from 81% to 99%.

[0043] As used herein, the term “compartmentalizing apparatus” is a generic device or tool designed to create distinct, isolated sections or compartments within a given space or on a surface. The compartmentalizing apparatus may include features such as partitions, walls, or openings thatAttorney Docket #: 243734.000229SJ-25 -0018-02 allow for the organization, separation, or containment of different materials, substances, or components in a controlled and precise manner. The compartmentalizing apparatus can be made of silicone, plastic, PDMS, metal, or glass, depending on factors like durability, chemical resistance, or flexibility.

[0044] As used herein, the term “memory” and “non-transitory computer-readable media” are used interchangeably and are understood to include, but are not limited to. random access memory (RAM), read-only memory (ROM), electronically erasable programmable ROM (EEPROM), flash memory or other memory technology, compact disc ROM (CD-ROM), digital versatile disks (DVD) or other optical storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, or any other tangible, physical medium which can be used to store computer readable information.

[0045] Alternative apparatus and system features and alternative method steps are presented in example embodiments herein. Each given example embodiment presented herein can be modified to include a feature and / or method step presented with a different example embodiment herein where such feature and / or step is compatible with the given example as understood by a person skilled in the pertinent art as well as where explicitly stated herein. Such modifications and variations are intended to be included within the scope of the claims.

[0046] Single-cell RNA sequencing (scRNA-seq) has revolutionized understanding of cellular diversity, but remains constrained by scalability, high costs, and the destruction of cells during analysis. To address the limitations of scRNA-seq methods, examples presented herein include devices, systems, and methods which utilize imaging-based transcriptomics and proteomics readouts to significantly scale-up cell numbers at substantially reduced costs, while retaining the advantages of single-cell feature detection. Recent advancements in spatial transcriptomic imaging assays have significantly expanded gene panel designs, moving towards transcriptome-wide scales with the potential to match and surpass the capabilities of current scRNA-seq methods. Traditionally, spatial transcriptomics and proteomics have been applied to tissue profiling, mapping composition and architecture of complex samples, such as organs or tumors.

[0047] A highly scalable approach for the profiling of single cells is presented here, referred to as STAMP (Single-Cell Transcriptomics Analysis and Multimodal Profiling). By leveraging transcriptomics and proteomics imaging platforms. STAMP may significantly reduce or practically eliminate sequencing costs, enabling single-cell genomics of hundreds to millions ofAttorney Docket #: 243734.000229SJ-25 -0018-02 cells at an unprecedented affordability. Immobilizing (‘stamping’) cells in suspension onto imaging slides, STAMP supports single-modal (RNA or protein) and multimodal (RNA, protein and H&E) profiling, while retaining cellular structure and morphology. Its flexible, ultra-high- throughput formats facilitate the analysis of single or multiple samples in the same experiment, enhancing experimental scalability and adaptability. In some embodiments, cells are grown or cultured directly on a substrate and subsequently fixed in situ. This adherent-culture implementation permits downstream analysis via imaging and / or sequencing technologies. Multiple examples of applications of the STAMP workflow are provided herein, demonstrating STAMP'S versatility across diverse experimental contexts, including the profiling of peripheral blood mononuclear cells (PBMCs), cell lines and stem cells. Cells and nuclei from dissociated tissues from mouse organs are stamped to simulate the generation of cell atlases. Accessibility was further enlarged by analyzing nuclei from archival formalin fixed and paraffin embedded (FFPE) tissue samples. Combining RNA and protein profiling, STAMP was applied for high-throughput immuno-phenotyping of millions of blood cells, providing multimodal insights into cellular heterogeneity. STAMP was used to identify ultra-rare cell populations, simulating clinical applications for detecting circulating tumor cells (CTCs). STAMP was used to capture lineage dynamics during stem cell differentiation and subtle changes in in vitro activated PBMCs, thereby demonstrating its utility for large-scale perturbation studies. These results validate STAMP as a first-of-its-kind single-cell imaging analysis strategy. Data for 10,962,092 high quality cells / nuclei and 6,030,429,954 high quality transcripts are presented. By eliminating sequencing, in some embodiments, STAMP may make high-resolution cellular profiling more accessible, scalable and affordable. Designed to meet the needs of research laboratories, diagnostic cores, and pharmaceutical companies, STAMP holds the potential to revolutionize our capacity to map biological diversity and dynamics, accelerating disease diagnosis and drug discovery.

[0048] Examples presented herein transform three imaging platforms, the Xenium Analyzer (lOx Genomics), the CosMx Spatial Molecular Imager (SMI, Nanostring Technologies / Bruker), and the PhenoCyler Fusion (Akoya Biosciences), into scalable and flexible single-cell profiling tools. Samples processed for the Xenium Analyzer and CosMx Spatial Molecular Imager are respectively referred to herein as STAMP-X and STAMP-C samples. The adaptation of singlemolecule imaging enables the analysis of hundreds to millions of individual cells with unprecedented flexibility and scale. STAMP designs support single-modal (RNA or protein) orAttorney Docket #: 243734.000229SJ-25 -0018-02 multimodal (RNA, protein and H&E) profiling in single- or multi-sample configurations. We demonstrated its utility across diverse sample types and experimental scenarios.

[0049] STAMP'S scalability is demonstrated by profiling a wide range of cell numbers, ranging from fewer than a hundred to millions of cells, showcasing its adaptability to diverse sample sizes. The ability to profile both whole cells and nuclei (the latter prepared from fresh-frozen and FFPE samples) with STAMP was demonstrated, highlighting its suitability in handling samples with different RNA contents and its potential to profile archival tissues. Both single-modal and multimodal profiling was applied in the context of immuno-phenotyping millions of peripheral blood mononuclear cells (PBMCs), successfully capturing subtle compositional and transcriptional changes, for example those induced by in vitro perturbations. STAMP was used to simulate the identification of ultra-rare cell populations, such as circulating tumor cells (CTCs), which are challenging to detect using conventional scRNA-seq methods. To explore the potential of our single-cell imaging approach in studying cellular differentiation and lineage specification, STAMP was used to a differentiation model by treating human embryonic stem cells (hESCs) with bone morphogenetic protein 4 (BMP4). This treatment induced a coordinated, multi-lineage differentiation process, effectively simulating early embryonic gastrulation. STAMP was applied to models derived from induced pluripotent stem cells (iPSCs), a model system for large-scale perturbation and drug screening studies.

[0050] Aspects of the invention are next described in relation to figures which depict one or more implementations of the inventive concept by way of example only, not by way of limitation.

[0051] FIG. 1 is an image of an example sample preparation 10a having a substrate 11 and multiple sample regions each with a respective monolayer of fixed cells and / or nuclei 13. The cells / nuclei in each sample region are accessible for hybridization with a probe configured to bind cellular mRNA while remaining fixed to the substrate. The example sample preparation 10a (as well as other example preparations described herein) are preferably configured such that each monolayer of fixed cells and / or nuclei 13 is available for analysis using sequencing and imagingbased transcriptomics and proteomics equipment and techniques. Several examples of such equipment and techniques are described elsewhere herein and are otherwise known to a person skilled in the pertinent art. The fixed cells and / or nuclei 13 are preferably affixed with sufficient adhesion to the substrate such that the sample preparation can be processed using techniques typically applied to a tissue sample while at least a majority (preferably at least 90%, 95%, or atAttorney Docket #: 243734.000229SJ-25 -0018-02 least 99%) of the fixed cells and / or nuclei 13 remain affixed to the substrate 1 1. In some embodiments, the sample preparation includes a surface coating including poly-D-lysine which adheres the fixed cells and / or nuclei to the surface of the substrate 11. As used herein, “poly-D- lysine” refers to a synthetic peptide comprising the D-enantiomer of the positively charged amino acid lysine. In some embodiments, positively charged peptide coatings include poly-D-lysine and poly-L-lysine, either alone or in combination, optionally with other polymers or peptides suitable for cell adhesion. The fixed cells and / or nuclei 13 in each of the monolayers can include peripheral blood mononuclear cells, dissociated cancer cells, dissociated cells from tissues, differentiated embryonic stem cell cultures, cells from a perturbation assay or any combination thereof including nuclei.

[0052] As illustrated, the sample regions are arranged in a grid. Alternatively, or additionally, the sample regions may be arranged in an alternative configuration to accommodate samples of differing size and shape. As illustrated, the sample preparation includes multiple sample regions. The monolayers are separated from each other so that there is dead space on the substrate between sample regions. Alternatively, the sample preparation may include only one sample region with a monolayer of fixed cells and / or nuclei. For instance, in one embodiment, a majority of the viewing area 12 may be occupied by a single sample region having a high-density monolayer of fixed cells and / or nuclei. Monolayer density is described in greater detail in relation to FIG. 7. In another embodiment, a small area of the viewing area 12 can include a monolayer of fixed cells and / or nuclei of a known control sample as described in greater detail in relation to FIG. 3.

[0053] The substrate can be a standard glass slide, such as a Superfrost or Superfrost Plus slide, coated or uncoated. In other embodiments, the substrate can be technology-specific slides or flowcells including, for example, Xenium slides (lOx Genomics), Visium HD slides (lOx Genomics), coverslip (MERSCOPE), Aviti24 substrates (Element Biosciences), Illumina sequencing flowcells / chips and multicolored fluorophore microscope systems. Any substrate configured to receive and retain monolayers or adherent cultures of cells or nuclei for downstream imaging or sequencing is contemplated. The substrate preferably includes a planar surface upon which the monolayers are affixed.

[0054] FIG. 2 is a flow diagram including steps of a method 100 of preparing a sample such as the sample preparation illustrated in FIG. 1 and other sample preparations described and illustrated herein, alternatives thereto, and variations thereof. The method 100 can be modified to includeAttorney Docket #: 243734.000229SJ-25 -0018-02 compatible steps of other methods described or illustrated here including methods illustrated in figures, methods provided with the examples hereinbelow, methods otherwise described herein, and variations and alternatives thereto as understood by a person skilled in the pertinent art informed by the disclosure herein.

[0055] At block 110, one or more suspensions are prepared of fixed cells and / or nuclei in a water solution. The suspension(s) may each be prepared to have a respective concentration of fixed cells and / or nuclei that is based at least in part on an area of the respective sample regions, a volume of a respective portion of the suspension deposited in the respective sample regions, and a cell size of the fixed cells and / or nuclei. The concentration can be tailored to achieve a desired density of cells / nuclei in the monolayer in a respective sample region in the completed sample preparation. The sample preparation need not have a consistent cell density in each sample region, in which case, multiple suspensions having different concentrations may be prepared in block 110 such that concentration within each suspension is tailored to achieve a target density of cells / nuclei within a respective sample region. Additionally, or alternatively, suspensions may respectively have cells and / or nuclei with an average size that is different from one suspension to the next and the concentration of each suspension may be tailored to achieve a target density within a respective sample region based in part on the average size of the cells / nuclei for that sample region. Monolayer density and suspension concentration are discussed further in relation to FIG. 7.

[0056] In some embodiments, the one or more suspensions of fixed cells and / or nuclei are prepared by fixing and permeabilizing cells and / or nuclei in a solution containing 4% formaldehyde and detergent, thereby obtaining fixed and permeabilized cells and / or nuclei, washing the fixed cells and / or nuclei in nuclease-free water to remove residual salts, resuspending the cells / nuclei in a solution of Triton-XlOO (e.g., 0.01% to 0.2% or 0.1% to 0.2%) in water, counting the fixed cells and / or nuclei, bringing the cell and / or nucleus suspension to the desired working concentration or density, namely cells / nuclei per microliter. In some examples, instead of formaldehyde and detergent, an alcohol solution may be used.

[0057] The suspension of fixed cells and / or nuclei may include peripheral blood mononuclear cells, dissociated cancer cells, differentiated embryonic stem cell cultures, or any combination thereof. The water solution may include between approximately 0.1% and approximately 0.2% Triton-X.Attorney Docket #: 243734.000229SJ-25 -0018-02[0058| The one or more suspensions can otherwise be prepared as described elsewhere herein, or by using alternatives or variations to examples presented herein as understood by a person skilled in the pertinent art.

[0059] At optional block 120, a substrate can be prepared to receive the suspension(s). In some embodiments, the substrate includes a glass slide or cover slip. In some embodiments, the substrate can be coated with an amino acid coating to the surface of the substrate to form a coated surface. For instance, a solution of poly-D-lysine can be applied to the surface of the substrate at approximately 37 °C for a duration ranging from about 1 hour and to about 20 hours. The surface can then be washed with nuclease-free water to remove any residual poly-D-lysine solution, then dried. Drying the solution may involve baking the substrate with the solution thereon at approximately 37 °C for between approximately 1 hour and approximately 20 hours or until evaporation in a thermocycler and air drying the solution until almost fully dry. In some embodiments, the solution of Poly-D-Lysine has a concentration of approximately 1 milligram per milliliter. In one example, the Xenium Thermocycler Adapter plate is used on the 96-well block of the Cl 000 Touch Thermal Cycler for Xenium slides, and incubated overnight at 37 °C. Notably, in some embodiments, the substrate comprises a sequencing platform flowcell, chip, or slide, permitting subsequent library construction and / or sequencing directly or indirectly from the fixed, immobilized, or grown-in-place cells.

[0060] At block 130, the suspension(s) are deposited in the sample regions on the surface of the substrate. As described in greater detail elsewhere herein, the suspension(s) may be deposited into wells that are shaped by a compartmentalizing apparatus on the surface of the substrate. Alternatively, the suspension(s) may be deposited directly onto a planar surface of the substrate, relying on surface tension of the water solution, interaction with the substrate surface (which may include an adhesive coating), and gravity to hold together and maintain the position of the suspension without a well. As another alternative, the suspension(s) may be deposited using a centrifuge by adapting processes and equipment (e.g. cytofunnel) for forming pellets.

[0061] The suspension(s) can be deposited spaced apart from each other such that suspension in each respective sample region does not touch suspension in another sample region. The one or more suspensions can otherwise be deposited in the sample regions as described elsewhere herein, or by using alternatives or variations to examples presented herein as understood by a person skilled in the pertinent art.Attorney Docket #: 243734.000229SJ-25 -0018-02

[0062] At block 140, water is evaporated leaving a respective monolayer of fixed cells and / or nuclei within each respective sample region such that the respective monolayer of a given sample region is separated from the respective monolayer of another sample region. The respective monolayers can resemble the monolayers of fixed cells and / or nuclei 13 depicted herein, alternatives and variations thereof described herein, and alternatives and variations thereof as otherwise understood to a person skilled in the pertinent art.

[0063] In some embodiments, the water can be evaporated by placing a loaded substrate into a thermocycler and cooling in an initial ambient temperature of between about 4 °C and about 15 °C, preferably 4 °C. In one example, the loaded substrate is initially cooled in an ambient temperature of about 4 °C for between about 10 minutes and 30 minutes. The pre-cooling step reduces movement of the cells / nuclei within the suspension so that they are evenly distributed. The initial cooling time and temperature may be adjusted based on the volume of the suspension in each sample region such that smaller suspensions may require less pre-cooling. After the initial cooling of the loaded substrate, the temperature of the thermocycler can be increased from the initial ambient temperature to an intermediate ambient temperature between about 20 °C to about 30 °C (e.g., about 20 °C). After remaining at the intermediate temperature for between about 2 minutes and about 10 minutes (e.g., about 5 minutes), the temperature of the thermocycler can be increased from the intermediate ambient temperature to a drying temperature of between about 35 °C and about 50 °C. The time and temperature at the intermediate temperature may be adjusted to reduce shock to the sample due to the temperature change from the initial pre-cooling temperature to the drying temperature. The drying temperature can be held at least until the water of the water solution of the suspension of fixed cells and / or nuclei is essentially entirely evaporated. Times and / or temperatures may vary based on the loaded volume of suspension in each sample region and the overall thermal mass of the loaded substrate. Water in smaller volume suspensions may change temperature and evaporates more quickly than water in higher volume suspensions. In particular, the time held at the drying temperature may be determined based on the loaded volume of the suspension in a sample region. The sample preparation can be held at approximately 22 °C until use and then thermally cycled according to the protocols of a particular imaging system.

[0064] FIG. 3 is an image of another example sample preparation 10b having a substrate 11 having a first sample region with a known control sample 13a of a monolayer of fixed cells and / or nuclei and a second sample mounting region 18 with a test sample 17. Both samples 13a, 17 areAttorney Docket #: 243734.000229SJ-25 -0018-02 positioned in a viewing area 12 on a surface of the substrate 11. The sample preparation 10b as illustrated is ready for analysis using imaging-based transcriptomics and / or proteomics equipment and techniques as described elsewhere herein and otherwise understood by a person skilled in the pertinent art. Data collected from the known control sample can be used to calibrate data collected from the test sample. Using currently existing sequencing and imaging-based transcriptomics and proteomics equipment and techniques, drawing meaningful conclusions based on comparison of test sample data on different sample preparations can be challenging due to lack of a baseline control sample. Variations in sample preparation, equipment hardware, equipment configuration, and test equipment setup can result in differences in sample data between similar samples. As such, it can be difficult to determine whether differences in data from one sample to the next are due to actual differences between the samples or variations due to other factors using current equipment and techniques. The illustrated example sample preparation 10b addresses this issue by providing a known control sample 13a which can be used to calibrate data from the test sample 17. The known control sample 13a can include one or more known cell / nucleus types. In some embodiments, cells and / or nuclei of a known type are included in the known control sample 13a at a known density. In some examples, multiple sample preparations may be prepared, each having a known control sample 13a with known types, optionally at known concentrations. Each of the sample preparations can include a sample mounting region 18 configured to receive a test sample. Test samples can be mounted to the substrate 11 as needed. The known control sample 13a can be tailored to indicate targets of interest relevant to the test sample. The data from each respective known control sample 13a can be compared from one run to the next, or to a stored reference file, and this comparison can be used to compensate for variations between test samples due to external factors.

[0065] The known types may include peripheral blood mononuclear cells, dissociated cancer cells, differentiated embryonic stem cell cultures, cells from perturbation experiments or any combination thereof.

[0066] The test sample 17 may include a tissue sample, similar to as currently processed by existing imaging-based transcriptomics and / or proteomics equipment, one or more monolayers of fixed cells and / or nuclei, or a combination thereof. In some embodiments, the sample mounting region 18 may be sub-divided into multiple test sample regions.Attorney Docket #: 243734.000229SJ-25 -0018-02[0067| FIG. 4 is a flow diagram including steps of a method 200 of preparing a sample such as the sample preparation 10b illustrated in FIG. 3. The method 200 can be modified to include compatible steps of other methods described or illustrated here including methods illustrated in figures, methods provided with the examples hereinbelow, methods otherwise described herein, and variations and alternatives thereto as understood by a person skilled in the pertinent art informed by the disclosure herein.

[0068] At block 210, a suspension is prepared having fixed cells and / or nuclei including a known control sample in a water solution using different fixation methods. The suspension may be prepared to have a concentration that is based at least in part on an area of the respective sample regions, a volume of a respective portion of the suspension deposited in the respective sample regions, and an average size of the fixed cells and / or nuclei. The suspension may be prepared to achieve a target density of known control sample in the monolayer of fixed cells and / or nuclei. In some embodiments, the known control sample may include different known cell / nucleus types, and the suspension may be prepared to have respective concentrations of each of the known cell / nucleus types to achieve a target density for each of the known types in sample region of the known control sample.

[0069] In some embodiments, the suspension of fixed cells and / or nuclei of known type are prepared by fixing and permeabilizing cells and / or nuclei in a solution of formaldehyde and detergent, washing the fixed cells and / or nuclei in nuclease-free water to remove residual salts, counting the fixed cells and / or nuclei, and bringing the suspension to the desired working concentration or density. The water solution may include between approximately 0.1% and approximately 0.2% Triton-X.

[0070] The suspension can otherwise be prepared as described elsewhere herein, or by using alternatives or variations to examples presented herein as understood by a person skilled in the pertinent art.

[0071] The method 200 can optionally include preparing the substrate such as described in block 120 of method 100 in FIG. 2, described elsewhere herein, or a variation or alternative thereto as understood by a person skilled in the pertinent art informed by the disclosure herein. In some embodiments a portion of the substrate including the first sample region is prepared such as described in block 120 of method 100 in FIG. 2 while a portion of the substrate including at leastAttorney Docket #: 243734.000229SJ-25 -0018-02 a portion of the test sample mounting region 18 is not. The test sample mounting region 18 of the substrate may be prepared as appropriate for the test sample 17.

[0072] At block 220, the suspension is deposited in a first sample region on a surface of a substrate. Preferably the first sample region is disposed within the viewing area 12 such that a majority of the viewing area 12 remains available for the test sample mounting region 18. As described in greater detail elsewhere herein, the suspension may be deposited into a well that is shaped by a compartmentalization apparatus, such as a gasket, on the surface of the substrate. The one or more suspensions can otherwise be deposited in the sample regions as described elsewhere herein, or by using alternatives or variations to examples presented herein as understood by a person skilled in the pertinent art.

[0073] At block 230, water can be evaporated from the suspension leaving a monolayer of fixed cells and / or nuclei of the known control sample within the first sample region. The water can be evaporated as described in greater detail elsewhere herein, for example as described in relation to block 140 of method 100 in FIG. 2.

[0074] At optional block 240, a test sample is affixed to a second sample region on the substrate. For instance, a test sample similar to sample 17 illustrated in FIG. 3 may be affixed to the substrate. The test sample 17 may include tissue or other material similar to typical sample preparations for analysis using imaging-based transcriptomics and proteomics equipment and techniques. Alternatively, the test sample 17 may include fixed cells and / or nuclei deposited as a monolayer. In some embodiments, multiple test samples may be affixed to the substrate. The test samples may include a mixture of various sample types. For instance, the second sample region 18 illustrated in FIG. 3 may be subdivided into respective sample regions each having a different test sample. In some embodiments, one or more of the monolayers of fixed cells and / or nuclei in a sample preparation having multiple sample regions each having a respective monolayer of fixed cells and / or nuclei can include known control sample cells / nuclei and function as a known control sample.

[0075] As an alternative to affixing a test sample to the substrate, the sample preparation with the known control sample can be used to evaluate repeatability of sample data collection for the purpose of calibrating equipment, improving sample preparation consistency, or otherwise addressing outside factors that may result in data variation between tested samples.Attorney Docket #: 243734.000229SJ-25 -0018-02[0076| At optional block 250, the known control sample can be compared to the test sample, or the known control sample can otherwise be used to calibrate data collected from the test sample.

[0077] FIG. 5 is an image of a substrate 11 with a compartmentalizing apparatus 14 (gasket) thereon with openings forming wells 15, 15a with a suspension of fixed cells and / or nuclei 16 as step for preparing a sample. The bottom of each well defines a respective sample region on the surface of the substrate 11. Sidewalls of each well are formed by a surface of the compartmentalizing apparatus within a respective opening. The compartmentalizing apparatus 14 is shown having openings that are a rounded square shape. A rounded square is a square-like shape with straight, or nearly straight, sides and smoothly curved comers, created by replacing the sharp right angles of a square with uniform arcs or other smooth fillets. It looks square in overall proportion but transitions gently at the comers rather than meeting at hard 90° angles. Alternatively, at least one opening of the compartmentalizing apparatus 14 can have a circular shape. The openings are arranged in a grid. The compartmentalizing apparatus 14 may be a single piece or may include separate parts that are positioned next to each other as illustrated. In some embodiments, the overall size of the compartmentalizing apparatus 14 is sized according to a viewing area 12 of a spatial molecular imager, which can include any of the molecular imagers discussed herein and / or a fluorescent microscope or a sequencer, and predetermined arrangement of sample regions within the viewing area. The compartmentalizing apparatus 14 may otherwise be configured as described elsewhere here.

[0078] The compartmentalizing apparatus 14 includes a large well 15a that has a larger corresponding sample region at the surface of the substrate 11 on the bottom of the well 15a and also has higher sidewalls than the remainder of the wells 15. The volume of each well 15, 15a is a function of the area of the bottom of the well (sample region) and the height of the well sidewalls. Given the same base area, increasing the sidewall height allows for a larger overall volume of each well 15, 15a. In some embodiments, the compartmentalizing apparatus has a height of approximately 0.5 mm (millimeter) to approximately 10 mm, or more preferably of approximately 1 mm to approximately 3 mm. In some embodiments, the height may be between 0.5 mm to approximately 1 mm. In some embodiments, one or more of the wells has a respective volume of between approximately 5 microliters and approximately 500 microliters. At least one well may have a volume of approximately 10 microliters.Attorney Docket #: 243734.000229SJ-25 -0018-02[0079| The compartmentalizing apparatus 14 may include one or multiple openings to form one or multiple wells. The opening(s) may be sized to form a wide range of sample region areas essentially as large as practical for imaging equipment, e.g.. 700 mm2or greater, and as small as practical for suspension deposition and sample handling, e.g., possibly 0.1 or 0.2 mm2. In some embodiments, each of the openings of the compartmentalizing apparatus 14 is sized such that the respective sample region of the bottom of each of the one or more wells has an area of between approximately 3 mm2and approximately 700 mm2. At least one opening may be sized such that the respective sample region has an area of between approximately 3 mm2and approximately 15 mm2. At least one opening may be sized such that the respective sample region has an area of between approximately 3 mm2and approximately 15 mm2. In some embodiments, the compartmentalization apparatus 14 has an opening between approximately 0.2 and approximately 3 mnr.

[0080] The loaded substrate 11 is shown on a thermocycler 20 which controls temperature during evaporation of water from the suspension 16. The compartmentalizing apparatus 14 is configured to be removed from the substrate 11 after the water is evaporated, thereby leaving a monolayer of fixed cells and / or nuclei in each sample region. The compartmentalizing apparatus 14 is preferably formed of a flexible material that can be peeled away from the substrate 11 after the water is evaporated. For instance, the compartmentalizing apparatus 14 may include silicone, a flexible polymeric material, or other suitable material as understood by a person skilled in the pertinent art. The illustrated compartmentalizing apparatus 14 includes a silicone gasket.

[0081] The suspension 16 in each well can have a concentration calculated to achieve a target density of the monolayer of fixed cells and / or nuclei formed at the bottom of the well 15, 15a after the water is evaporated from the suspension 16. The illustrated loaded substrate has a large well 15a and smaller wells 15 having differing volume. A different concentration in the suspension may be required in the large well 15a as compared to the smaller wells 15 to achieve the same target cell density in each well 15, 15a. The target concentration may be calculated by calculating a first target concentration based at least in part on the volume of the large well 15a and the area of the respective sample region of the larger well 15a and calculating a second target cell concentration based at least in part on the volume of a smaller well 15 and the area of the respective sample region of the smaller well 15. The average size of the cells / nuclei in each suspension may also be used to determine the target concentration.Attorney Docket #: 243734.000229SJ-25 -0018-02

[0082] FIG. 6 is a flow diagram including steps of a method of preparing a sample including a step corresponding to FIG. 5. The method 300 can be modified to include compatible steps of other methods described or illustrated here including methods illustrated in figures, methods provided with the examples hereinbelow, methods otherwise described herein, and variations and alternatives thereto as understood by a person skilled in the pertinent art informed by the disclosure herein.

[0083] At block 310, a compartmentalizing apparatus having one or more openings can be applied to a surface of a substrate to form a well at each opening such that the bottom of each well includes a respective sample region and the sidewall of each well includes a surface of a respective opening. For instance, a gasket similar to as illustrated in FIG. 5 can be applied to a substrate 11. Alternatively, a compartmentalizing apparatus having openings shaped and positioned to correspond to the respective monolayers of fixed cells and / or nuclei of the sample preparations shown elsewhere herein (e.g. FIGs. 1, 3, 7A-D, 11A, 12A, 13A, 13G, 15B, 17A, 18A-D, 24B), variations thereof, and alternatives thereto as understood by a person skilled in the pertinent art informed by the disclosure herein can be applied to the substrate surface. Alternatively, a tape gasket 14a similar to as illustrated in FIG. 8, variations thereof, and alternatives thereto as understood by a person skilled in the pertinent art informed by the disclosure herein can be applied to the substrate surface. In one embodiment, the compartmentalizing apparatus includes a single opening forming one well. The single opening may be small and near a perimeter of the viewing area such as shown in FIG. 3. Alternatively, the single opening may occupy a majority of the viewing area 12. In some embodiments, the compartmentalizing apparatus includes multiple openings arranged in a grid.

[0084] Prior to applying the compartmentalizing apparatus, the method 300 can optionally include preparing the substrate such as described in block 120 of method 100 in FIG. 2, described elsewhere herein, or a variation or alternative thereto as understood by a person skilled in the pertinent art informed by the disclosure herein. In one embodiment, an amino acid coating can be applied to the surface of the substrate to form a coated surface, and the gasket can be applied to the coated surface.

[0085] At block 320, a suspension of fixed cells and / or nuclei in a water solution can be prepared to have a concentration that is based at least in part on an area of the respective sample region, volume of a respective well, and average size of the fixed cells and / or nuclei. One or moreAttorney Docket #: 243734.000229SJ-25 -0018-02 suspensions can be prepared as described in relation to block 1 10 of FIG. 2 and / or as described in relation to block 210 of FIG. 4.

[0086] A target concentration for the suspension can be calculated such that a volume of the suspension that is equal to a volume of the respective well includes a number of fixed cells and / or nuclei predicted to cover a target percentage of total area of the respective sample region with a monolayer of the fixed cells and / or nuclei. Meaning, when the volume of the well is filled to a desired amount with the suspension, the well holds all fixed cells and / or nuclei in that suspension which will ultimately form the monolayer of fixed cells and / or nuclei at the target percentage of the total area of the sample region. The number of fixed cells and / or nuclei needed to cover the total area of the sample region at the target percentage can be calculated a function of the total area of the sample region and cell size. The target cell concentration can therefore be calculated as a function of the calculated number of fixed cells and / or nuclei and the volume of the well. Average size of the fixed cells and / or nuclei can be used to determine the target cell concentration. Water can be added to the fixed cells and / or nuclei such that the cell concentration of the suspension of fixed cells and / or nuclei is approximately equal to the target cell concentration.

[0087] In some embodiments, the target percentage is between approximately 50% and approximately 100%. The target percentage may be between 70% and 100%. In some embodiments, the target cell concentration is between approximately 1,000 cells per microliter and approximately 3,000 cells per microliter. The target cell concentration may be approximately 2,500 cells per microliter.

[0088] In some embodiments, multiple suspensions having differing average cell / nucleus size may be prepared. A first suspension of fixed cells and / or nuclei having cells of a first average size may be prepared, and a second suspension of fixed cells and / or nuclei having cells of a second average size different from the first average size may be prepared. A first target cell concentration may be calculated for a first suspension based at least in part on the first average size, a second target cell concentration for a second suspension may be calculated based at least in part on the second average size.

[0089] At block 330, a quantity of the suspension can be loaded into each well based at least in part on the concentration and the area of the respective sample region. The quantity of suspension loaded into each well of the one or more wells includes a respective number of fixed cells / nuclei determined to cover a target percentage of the respective sample region of the bottom of each wellAttorney Docket #: 243734.000229SJ-25 -0018-02 of the one or more wells. In some embodiments, the volume of each respective well is entirely filled. Alternatively, the well may be partially filled. In some embodiments, the quantity of suspension of fixed cells and / or nuclei loaded into each well of the one or more wells is approximately equal to the volume of the respective well. In some embodiments, a surface tension dome of the suspension that extends above the sidewall can be formed.

[0090] In some embodiments, at least one well can be loaded with a known control sample.

[0091] At block 340, water can be evaporated from the well(s) leaving a respective monolayer of fixed cells and / or nuclei on each respective sample region. In some embodiments, the loaded substrate, including the compartmentalizing apparatus and suspension, can be placed into a thermocycler. The thermocycler can provide ambient temperatures as described in relation to block 140 of method 100 in FIG. 2. Additionally, or alternatively, the water can be evaporated as described in greater detail elsewhere herein, alternatives thereto, and variations thereof as understood by a person skilled in the pertinent art informed by the disclosure herein.

[0092] At optional block 350, the gasket can be removed from the substrate. In some embodiments, the gasket is peeled away from the substrate. Removing the gasket from the substrate results in a sample preparation having the substrate and one or more sample regions each having a respective monolayer of the fixed cells and / or nuclei.

[0093] At optional block 360, the sample can be hydrated in a flow cell assembly such that fluid flows over each of the sample regions.

[0094] FIG. 7 includes images demonstrating size and density of respective monolayers of fixed PBMCs in each sample region in which FIG. 7A includes an image of a sample preparation; FIG. 7B is an image of the sample regions annotated to show the number of cells in each respective sample region; FIG. 7C includes zoomed-in images of the sample regions illustrated in FIG. 7B; and FIG. 7D includes count and analysis of the density of fixed cells and / or nuclei in the sample preparation. Each sample region has a different cell density. Sample regions with higher cell density appear more opaque in FIG. 7A and darker in color in FIGs. 7B-D. Cells within each sample region are uniformly distributed over a majority of a respective area of each sample region.

[0095] The number of cells that can fit in a given area varies based on the average size of the fixed cells / nuclei. There is a maximum of fixed cells / nuclei one could fit side to side for 100% coverage of a given area based on the average size. The number of fixed cells / nuclei per sample region can be tuned based on the desired percentage of the sample region to be covered. ForAttorney Docket #: 243734.000229SJ-25 -0018-02 example, given a PBMC cell average size of 9 pm, in a 22.45 mm by 10.45 mm viewing area entirely occupied by a single sample region about approximately 4.5 million PBMCs can form a monolayer with 100% coverage and approximately 3.6 million PBMCs would provide approximately 80% coverage. In other words, a cell concentration of about 20,000 PBMCs per square millimeter provides approximately 100% coverage. The relationship between cell concentration and percent coverage of a sample region is based on average size of fixed cells / nuclei in the monolayer. Compartmentalizing the viewing area introduces smaller “dead” areas between each sample region.

[0096] FIGs. 7A-7D show a sample preparation with sample regions having differing concentrations of PBMCs. Each sample region is a rounded square shape with a side length of 3.5 mm. Each sample region has an area of approximately 10 to 12 mm2. The viewing area of the sample preparation includes twelve (12) sample regions. As shown, approximately 225,000 cells form a monolayer of fixed cells and / or nuclei at about 100% coverage in a sample region. Cells begin to overlap at higher densities and there is no longer a monolayer of cells. A density of approximately 5% coverage was also demonstrated.

[0097] Each sample region can be analyzed using approximately 30 fields of view (FOV), which include a total of approximately 120,000 fixed cells and / or nuclei with 100% coverage. All twelve sample regions can be imaged with approximately 360 FOV. Imaging time can be saved by excluding FOV that include dead zones between sample regions. The slide includes approximately 1.4 million fixed cells. Using two slides with approximately 1.4 million fixed cells apiece, 2.8 million total cells can be imaged in 24 distinct sample regions.

[0098] In some embodiments, a monolayer between approximately 1.000 PBMCs per square millimeter and approximately 20,000 PBMCs per square millimeter within each sample region. A respective monolayer of PBMCs of at least one sample region can include approximately 18,000 and 20,000 cells per square millimeter. In some embodiments, the sample region(s) include a total sum of between approximately 1,000,000 and approximately 4,000,000 total cells. Similar cell densities may be achieved for samples having an average cell / nucleus size around 9 pm. Achievable cell densities for samples having larger or smaller average cell / nucleus size can be estimated based on their proportion to the PBMC cell size.

[0099] FIG. 8 is an image of a substrate with a gasket 14a thereon with openings forming wells with a suspension of fixed cells and / or nuclei forming a surface tension dome above the well asAttorney Docket #: 243734.000229SJ-25 -0018-02 step for preparing a sample. The gasket 14a is thin and flexible to provide ease of applying and removing the gasket 14a. The volume of the wells is less than the suspension 16 loaded into each individual well, and the surface tension of the suspension is relied upon to confine the portions of the suspension above each respective sample region.

[0100] Materials to assemble a sample preparation including a monolayer of fixed cells / nuclei can be provided in a kit. The kit can include a substrate, fixative and permeabilization buffer, RNase inhibitor, resuspension reagent, a coating configured to provide an adhesive coating to the substrate, and a compartmentalization apparatus having openings shaped to cover a predetermined area of the surface of the substrate. The various components of the kit can each be configured as described in greater detail elsewhere herein.

[0101] FIG. 9 is a block diagram of an imaging system 50 configured to receive a sample preparation having discrete sample regions and perform computational analysis of the sample preparation considering the discrete sample regions. The imaging system 50 can be configured in part similar to current transcriptomic and protein imaging platforms, such as the Xenium Analyzer, the CosMx SMI, the MERSCOPE, and the PhenoCyler Fusion. The imaging system 50 includes a sample receptacle and processing system 55 sized, shaped, and otherwise mechanically configured to receive a sample preparation, such as those described herein, variations thereof, and alternatives thereto as understood by a person skilled in the pertinent art. The sample receptacle and processing system 55 is further configured to facilitate steps for image analysis such as applying and removing gene set probes, oligo-conjugated antibodies, or other such products similar to as currently used with current transcriptomic and protein imaging platforms for tissue sample analysis. The imaging system 50 includes an automated control system 51 including memory 52, one or more processors 53, and a data store module store 54.

[0102] The automated control system 51 is configured to automatically execute processes associated with the imaging system 50 such as data acquisition. The memory 52, processor 53. and data store module 54 may be included in one pieces of hardware such as an imaging platform, or may be distributed across multiple devices. The memory 52 is a non-transitory computer-readable medium (which may be distributed) with instructions thereon, that when executed by the processor 53 (which may include one or more processors) to cause the imaging system 50 to perform automated tasks to gather data from the sample preparation. The data from the sample preparationAttorney Docket #: 243734.000229SJ-25 -0018-02 may be stored in the data store module 54 and / or compared against data stored in the data store module 54 (which may be distributed).

[0103] The automated control system 51 may be configured similar to current transcriptomic and protein imaging platforms, alternatives thereof, or variations thereto as understood by a person skilled in the pertinent art informed by the disclosure herein and include modified instructions in the memory 52 to facilitate processing of a sample preparation having multiple sample regions.

[0104] FIG. 10 is a flow diagram including steps of a computational method which may be stored in memory and executed by a processor on a computational system such as by the imaging system 50 illustrated in FIG. 9.

[0105] At block 410, the imaging system 50 receives an identification of a multi-sample substrate preparation. For instance, the imaging system 50 may be configured to receive sample preparations that have viewing areas partitioned in various configurations as illustrated and described in greater detail elsewhere herein. In some embodiments, the sample preparation may include a monolayer of fixed cells and / or nuclei in at least one of the sample regions. In some embodiments, the imaging system 50 may include a user interface, and the memory 52 may include instructions thereon which cause the processor 53 to provide, to a user via the user interface, the identification as one of a plurality of predetermined sample identifications; and receive, from the user interface, the identification as a selection of one of the plurality of predetermined sample identifications. For instance, the user interface may provide a drop-down menu of possible sample preparation layouts which are associated with the plurality of predetermined sample identifications, and the user may make a selection from the drop-down menu so that the processor receives the identification of the multi-sample preparation based on the user selection.

[0106] At block 420, the viewing area is partitioned into predetermined sample regions based at least in part on the identification. For instance, the identification may indicate that the sample preparation has a grid layout of sample regions such as illustrated in FIG. 1. and the entire viewing area 12 may be partitioned based on the layout of the respective monolayers of fixed cells and / or nuclei 13 (corresponding to the sample regions). Similarly, the entire viewing area 12 can be partitioned for other example sample preparations having multiple sample regions such as those described herein, variations thereof, and alternatives thereto as understood by a person skilled in the pertinent art informed by the disclosure herein.Attorney Docket #: 243734.000229SJ-25 -0018-02[0107| Once the viewing area is partitioned, there are several optional steps that the imaging system 50 may perform based on the identification and partitioning.

[0108] At optional block 430, the viewing area can be imaged such that at least a portion of the predetermined sample regions are imaged while at least a portion of the viewing area outside of the predetermined sample regions is excluded. For instance, FIG. 7D illustrates a 3.5 mm by 3.5 mm sample region which in which 30 FOV are drawn over a central portion of the sample region. In one embodiment, each of the twelve sample regions can be imaged with 30 respective FOV for each sample region, and the remainder of the FOV within the viewing area can be excluded from imaging. By excluding portions of the viewing area outside of the predetermined sample regions imaging time can be significantly reduced, which can increase sample throughput. In this embodiment, portions of the sample regions at the perimeter of the sample region are excluded from imaging. Alternatively, these regions may be included in imaging. In some embodiments, the entirety of each predetermined sample region is imaged. In some embodiments, at least a majority of the viewing area inside the predetermined sample regions is imaged while excluding at least a majority of the viewing area outside of the predetermined sample regions.

[0109] At optional block 440, data can be distinguished from the predetermined sample regions as individual, separable data sets. In some embodiments, data from each of the plurality of predetermined sample regions can be saved as separate data files. In some embodiments, each of the separable data sets include a plurality of gene counts per cell datasets such that each of the gene counts per cell datasets are each associated with a respective predetermined sample region of the plurality of predetermined sample regions.

[0110] At optional block 450. a control sample region may be identified from among the plurality of predetermined sample regions. Data from the control sample region may be used to perform several useful functions. In one embodiment, the imaging system 50 is configured to use data from the control sample to calibrate data from some or all of the remainder of plurality of sample regions. In some embodiments, data from the control sample is compared to other control sample datasets of a similar known control sample type. For instance, data from the control sample may be compared to data stored in data store module 54. The comparison may provide an indication of external factors which may influence data acquisition from test sample regions in the sample preparation.Attorney Docket #: 243734.000229SJ-25 -0018-02

[0111] Aspects of the aforementioned methods, systems, and sample preparations were used to perform a variety of analysis as outlined in the examples and methods presented below.EXAMPLE 1: SEQUENCING FREE SINGLE-CELL GENOMICS THROUGH IMAGING

[0112] To address current limitations in single-cell genomics, STAMP is presented herein as a flexible and scalable approach for cost-efficient, massive-parallel single-cell profiling. STAMP enables the profiling of single cells on slides integrated with state-of-the-art transcriptomic and protein imaging platforms, such as the Xenium Analyzer, the CosMx SMI, the Merscope, and the Phenocyler Fusion. The STAMP workflow begins with the fixation and permeabilization of cells in suspension, followed by anchoring ("stamping") cells onto instrument-compatible glass slides to form monolayers. While certain protocols describe stamping suspensions onto slides, in other embodiments STAMP is applied to adherent cultures grown on slides or chambered substrates and then fixed in situ. The same downstream imaging, multiplex probing, and analysis apply to both suspension- stamped monolayers and adherent cultures. Flexibility is achieved through the adaptable format of stamping areas, allowing versatile multi-sample profiling. After stamping, the cells are hybridized with gene set probes or oligo-conjugated antibodies followed by cyclic decoding through imaging, which can accommodate any gene or protein panel size and design (FIG. 11 A). This versatility allows STAMP to be used for targeted or data-driven, hypothesis-free strategies to explore the complexity of hundreds to millions of cells in a single experiment.

[0113] To evaluate STAMP'S suitability and specificity for imaging-based transcriptomic s, three cancer cell lines (LNCaP, MCF-7, and SK-BR-3) were profiled using the CosMx SMI platform (STAMP-C) with the 1000-plex Human Universal Cell Characterization RNA Panel. The multi-sample slide array contained four sub-STAMPs, each containing -35,000 cells: three with individual cancer cell lines and one with a 1:1:1 pooled mixture (FIG. 18A and FIG. 18B). For imaging, contiguous fields of view (FOVs) were selected to comprehensively scan each sub- STAMP. Cell staining information was obtained for DAPI, pancytokeratin (PCK) and panmembrane markers (B2M / CD298), used for cell segmentation (see Methods; FIG. 11B). A uniform distribution of transcripts, genes and cell areas across each sub-STAMP were initially confirmed pointing to homogeneous cell stamping (FIGs. 18C-D). Next, the pooled mixture was interrogated to distinguish different cell types in a spatially mixed environment. On average,Attorney Docket #: 243734.000229SJ-25 -0018-0211 ,103 cells per cell line were obtained, with a median of 3,637 transcripts and 413 genes per cell (FIG. 11C). Stringent quality control steps included removing segmentation artifacts, filtering out low transcript and gene counts (<2.5 median absolute deviations, MAD), and excluding high counts and large cell areas (>2.5 MAD). Cells near the FOV borders (<30 pixels) were also excluded due to reduced counts and features (FIG. HD). Overall, 4.63% of cells were removed from the analysis (FIG. HE).

[0114] Using the InSituType (1ST) algorithm for unsupervised clustering, three distinct clusters with unique transcriptional profiles (FIG. 11F,G) were identified. The pooled mixture consistently contained an average of 33.33% (range: 32.6 - 33.4%) of each cell line per FOV (FIG. 11H). To validate the clustering results of the pooled mixture, 1ST was applied to the sub- STAMPs of the individual cell lines, which also separated into three distinct clusters with transcriptional profiles matching those found in the mixture (FIGs. 18E,F). The gene signatures of both pooled and individual cell lines were correlated with data from a scRNA-seq reference dataset generated using the Single Cell Gene Expression Flex assay (lOx Genomics) on the same suspension of fixed cancer cells (FIG. 18G).EXAMPLE 2: SENSITIVE CAPTURE OF LOW-INPUT CELL NUMBERS

[0115] To explore the scalability to ultra-low cell densities, cancer cell line mixtures (1:1:1 ratio of MCF-7, SK-BR-3, and LNCaP) were stamped into four sub-STAMPs with varying cell counts of approximately 100, 250, 500. and 1,000 cells (FIG. 12A). The performance of STAMP at low cell numbers was assessed, which are challenging to capture with current droplet microfluidics or combinatorial indexing scRNA-seq methods. Additionally, 20,000 cells and -20,000 nuclei of the cell line mixture were separately profiled in two sub-STAMPs, enabling a direct side-by-side comparison of high-RNA (whole cell) and low-RNA (nuclear) content samples within a multiplexed experimental setup. Again, contiguous FOVs were selected, ensuring comprehensive coverage of the low-density samples.

[0116] The mean recovery rate of high-quality cells was 85.33% (range: 65 - 97 %) from the input material (FIG. 12B), with an average mix of 32.5% for each cell line per sub-STAMP (range: 18.6 - 56.9%, FIG. 12C). The median gene counts per cell were consistent across sub- STAMPs representing higher loadings, with reduced genes detected in the lowest sub-STAMP (100 cells; FIG. 12D). The number of detected features per cell followed similar trends,Attorney Docket #: 243734.000229SJ-25 -0018-02 indicating suboptimal performance when profiling extremely low cell densities (FIG. 12E). Cell areas remained stable in less densely seeded sub-STAMPs, but halved in those with higher confluence, likely due to reduced cell expansion during segmentation (Fig.l2F). Expectedly, nuclei had fewer counts and features than intact cells, yet gene expression levels were highly correlated across cells and nuclei (FIG. 12D,E,G).

[0117] Next, the reproducibility of this approach was tested by splitting MCF-7 and SK-BR-3 cell suspensions into two aliquots, imaged in two slides as technical replicates (scanned in the same Xenium run using the Xenium Prime 5k Human Pan Tissue and Pathways panel, STAMP- X). While recovered cell numbers slightly varied, both replicates showed a highly consistent number of transcripts and genes detected, as well as equal cell areas (FIG. 12H). Accordingly, gene expression levels correlated highly across replicates (FIG. 121).EXAMPLE 3: SAMPLE MULTIPLEXING ACROSS PLATFORMS

[0118] To evaluate the multiplexing capabilities of STAMP, 27 distinct samples, including primary cells, cell lines and nuclei samples extracted from prostate cancer FFPE tissues were prepared. We included a diverse range of cell types, such as PBMCs, cancer cell lines, cancer- associated fibroblasts (CAFs), stem cells, and mixed cell populations. The samples were sub- STAMPed onto two replicate slides for each assay, STAMP-C and STAMP-X, with 21 sub- STAMPs shared between the assays. This setup ensured consistency and reproducibility, while enabling a comprehensive evaluation of the assays' multiplexing capabilities (FIG. 13A). The largest gene panels available for each platform were selected to match the cell type diversity represented by the sub-STAMPs. Specifically, the Xenium Prime 5k Human Pan Tissue and Pathways Panel and the CosMx Human 6K Discovery Panel were selected.

[0119] Gene and transcript number per cell, as well as cell areas, were highly correlated across replicates, once again underscoring the robustness of the assay (FIG. 13B). Furthermore, experimental conditions at both the upper and lower ends of the gene, molecule count, and area distributions were consistent across platforms. Aggregating genes across all samples and replicates revealed high correlations, not only within replicates, but also across platforms in 18 out of 21 conditions (FIG. 13C). The suboptimal correlation observed for CHP-134, SK-N-DZ, and KELLY cell lines may be explained by the low overlap between the two panels (29,6%, FIG. 13D), with only one intersecting marker gene (ALK). High correlations were also detected across biologicallyAttorney Docket #: 243734.000229SJ-25 -0018-02 similar samples. For instance, iPSC 32F, hESC, and MD3iPSC clustered together, as did LNCaP and V16D (a castration-resistant prostate cancer cell line derived from LNCaP cells). Similar trends were evident for individual cancer cell lines LNCaP. SK-BR-3. and MCF-7 compared to their pooled mixtures (MX1 and MX2). Given the high correlation of STAMP across platforms, we performed PCA on all datasets together. The batch effect introduced by the panels and platforms, captured by PCI, was successfully removed using Harmony integration (FIG. 13E). This integration also improved the Local Inverse Simpson’s Index (LISI score), reflecting enhanced consistency across both platform (technical) and replicate batches (FIG. 13F). Next, we analyzed curated pathways from the MSigDB Database on the sub-STAMPs using AUCell. As an example, we identified a specific enrichment in the Biocarta Lymphocyte Pathway on the stamped PBMCs, which was consistent across both STAMP-X replicates (FIG. 13G). Hallmark Glycolysis and Hypoxia pathways and GOBP Lactate metabolic processes were similarly enriched in the cancer cell lines, but showed low scores in bone marrow progenitor cells and prostate cancer FFPE nuclei sub-STAMPs (FIG. 13H). These findings align with the enhanced lactate production under hypoxic conditions due to increased glycolytic rates, commonly observed as a metabolic adaptation in cancer cells and differentiated cell lines in in vitro cultures.EXAMPLE 4: IMMUNO-PHENOTYPING OF MILLIONS OF CIRCULATING BLOOD CELLS

[0120] The STAMP protocol was expanded to stamp millions of cells per experiment. An ultra high-density STAMP containing ~1.7 million PBMCs was generated, on which the Xenium Immuno-Oncology panel (380 target genes) was applied. A median of 83 transcripts per cell (range: 27 - 259) and 49 genes per cell (range: 24 - 103) were imaged. After quality control to remove segmentation errors and cells with extreme counts and features (FIG. 19A), 88,53% of high-quality cells were retained. Subsequently, conventional single-cell analyses were applied including dimensionality reduction and principal component analysis (PCA, see Methods). Principal components (PCs) 1-3 effectively resolved the three main immune lineages, namely Myeloid, T and B cells, based on cell lineage marker genes (FIG. 19B,C). As expected, the myeloid compartment displayed a higher number of genes and transcripts and larger cell areas (FIG. 19D). Further clustering of each immune lineage identified all major PBMC cell types and states at expected frequencies, defined by cell-state markers (FIGs. 14A,B). In total, 13 immune clusters representing the main PBMC populations were identified.Attorney Docket #: 243734.000229SJ-25 -0018-02[0121| To test STAMP’S power for high-resolution immunophenotyping, we stamped an additional 750,000 cells using a larger probe panel (Xenium Prime 5K Human Pan Tissue and Pathways panel). This analysis generated a high-resolution map of 31 immune cell states in circulation, providing the foundation for large-scale atlasing projects across dimensions such as time (e.g., age) or genetics (e.g., ethnic background; FIG. 14C,D and FIG. 19E). Notably eight CD4+ T cell subsets were identified, ranging from naive to effector and memory populations, including Thl, Th2 and Th 17 functional cell types that represent the three arms of adaptive immune responses. Similarly, eight subclusters within the CD8+ T cell pool were annotated, ranging from naive to central / effector memory populations and different types of effector populations, including interferon-responding CD8 T cells. Within the circulating NK cell pool, we distinguished CD56dlmCD16brlghtfrom CD56brlghtCD16dlmNK cells, which exhibit divergent antibody-dependent cellular cytotoxicity and migratory properties, along with a small proportion of proliferating EOMES- and CD34-expressing cells, likely representing NK progenitors. Ig- related genes and other markers relevant for B cell phenotyping were missing in the panel, which limited a deeper annotation of B cell differentiation states. However, the myeloid compartment revealed well-defined monocyte subsets (classical, intermediate and non-classical monocytes) and enabled detailed characterization of DCs. We next evaluated the suitability of STAMP for multiplexed perturbation studies, such as the responses to classic immune activators (anti- CD3 / CD28 for adaptive and LPS for innate immunity). Therefore, PBMCs were cultured with either media alone (control), LPS, or CD3 / CD28 beads, harvested at 4 and 24 h (FIG. 19F). Using the CosMx 1000-plex Human Universal Cell Characterization RNA Panel, a median of 320 transcripts (range: 33 - 1630) and 183 genes (range: 33 - 55) per cell were detected (FIG. 19G). After filtering out 18% of low-quality cells, unsupervised clustering with the InSituType (1ST) algorithm identified 12 distinct immune cell clusters (FIG. 19G,H). In line with the results obtained with the Xenium Immuno-Oncology panel, we captured immune cells from B, T and myeloid lineages at expected proportions (4%, 68%, 28%, respectively). Anti-CD3 / CD28 stimulation progressively decreased naive CD4+ T cells and increased effector and exhausted CD4+ T cells at the 4-hour and 24-hour time points (FIG. 14E). At 24h, naive CD8+ T cells also decreased markedly, while activated CD8+ T cells increased in numbers. The T cell activation also altered myeloid cell proportions, with classical monocytes and plasmacytoid DCs (pDCs) increasing at 4h, while inflammatory monocytes decreased. At 24h conventional DCs expanded,Attorney Docket #: 243734.000229SJ-25 -0018-02 and activated DCs declined relative to controls. LPS stimulation caused subtler compositional changes, consistent with previous reports. LPS is known to activate monocytes through the cell surface Toll-like receptor 4 (TLR4) complex, and, accordingly, activated monocytes showed the highest TLR4 expression levels among PBMC myeloid cell subtypes (FIG. 191). Overall, 97 genes were differentially expressed in activated monocytes (62 up- and 35 down-regulated; FDR < 0.05) upon LPS treatment, among them pro-inflammatory cytokine and chemokine genes (e.g., 1L1B, 1L6, CCL3, CCL5, CXCL8) and other inflammation-associated transcripts (e.g., PTGS2, STAT4, CSF3', FIG. 14F). By 24h, the upregulation of DUSP1, B2M, and IL10 in activated monocytes suggested a transition from early inflammatory responses to regulatory feedback mechanisms, limit inflammation.EXAMPLE 5: PROFILING CELL STATE DYNAMICS DURING STEM CELL DIFFERENTIATION

[0122] Next the resolution to which cell states emerging from differentiating hESCs in response to BMP4 treatment could be identified, leveraging the multiplexing capabilities of STAMP. Adding BMP4 to cultured hESC colonies triggers spatially-resolved signaling cascades, recapitulating those observed in the epiblast during gastrulation. This process results in concentric rings of embryonic germ lineages and the extra-embryonic cell type, amnion. To profile the trajectories of these lineages as they arise, we combined eight BMP4 treatment timepoints (Oh to 120h) in a single STAMP-C experiment using the CosMx Human Universal Cell Characterization RNA panel (FIGs. 15A,B)- We then applied pseudotime trajectory analysis with Palantir to model the differentiation pathways of hESCs in response to BMP4 treatment (FIG. 15C-F). The initial trajectory bifurcation in this system, determined by the presence or absence of BMP pathway activity (FIG. 15C), is influenced by cell position within the colony. Due to the basolateral localization of BMP receptors, cells in the center of large colonies are occluded from BMP4 in the media. However, as this sample contained relatively few large colonies, the majority of BMP4-treated hESCs transitioned from a pluripotent state to an intermediate BMP-induced state as early as 6h, persisting until 12-24h after BMP4 exposure. This transition was marked by the rapid upregulation of BMP4 pathway target genes, such as GATA3, followed by a downregulation of pluripotency marker SOX2.

[0123] By 48h, a mesendoderm-like state emerged, characterized by the expression of EOMES and KDR, alongside an early amnion-like state strongly expressing GATA3. After 72h, theAttorney Docket #: 243734.000229SJ-25 -0018-02 mesendoderm-like cells gave rise to early mesoderm-like cells (SNAI2, PDGFRA). endodermlike (CXCR4. APOA1), and primordial germ cell-like (PGCLC; NANOG, CXCR4) cells. By 72h, early amnion-like cells differentiated into late amnion-like cells expressing TGFBI, while the mesoderm branch progressed into a late mesoderm-like state, characterized by upregulation of DUSP6 and F0XF1. Using Palantir, we also computed the gene expression dynamics of key marker genes along the amnion, mesoderm, and endoderm branches, demonstrating the progressive downregulation of pluripotency genes and the upregulation of lineage-defining markers (FIG. 15g). Equivalent analyses performed on cells from the same samples, processed with the Single Cell Gene Expression Flex assay (lOx Genomics), revealed similar trajectories (FIG. 20). Both trajectories and marker gene dynamics are consistent with previous scRNA-seq studies of hESC-based gastrulation models and a gastrulating human embryo.

[0124] Induced Pluripotent Stem Cells (iPSCs) are pluripotent cells generated from differentiated donor cells, e.g. skin fibroblasts, through cellular reprogramming. Subsequently, these can be differentiated into various cell types and represent an invaluable tool to model human development and genetic diseases. Hence, iPSCs have been used to drive discovery in applications, such as disease modeling, drug screening and toxicity testing, regenerative medicine, immune research, and studying lineage specification. Given these extensive application areas, applying STAMP to iPSCs would enlarge the molecular profiling toolbox and significantly broaden its utility across multiple research fields. To explore such potential, we differentiated iPSCs into (neuro)ectoderm and mesodermal lineages, and performed scRNA-seq using the Gene Expression Flex assay (lOx Genomics) alongside with STAMP-C using the CosMx 1000-plex Human Universal Cell Characterization RNA Panel. Dimensionality reduction and PCA showed the first two PCs to resolve undifferentiated parental cells, ectoderm, and mesoderm in both assays (FIG. 21A). Compared to the parental iPSCs, the ectoderm sample exhibited increased expression of neuroectodermal markers (SOX2. NRG1) and decreased expression of pluripotency markers (POU5F1, FGF2-, FIG. 21B). Similarly, the mesoderm cells showed an upregulated expression of mesoderm-associated markers (PDGFRA, FOXF1, WNT5A). Gene signatures of each cell culture analyzed in STAMP-C were highly correlated with the scRNA-seq reference dataset generated on the same suspension of fixed iPSCs (FIG. 21C).Attorney Docket #: 243734.000229SJ-25 -0018-02EXAMPLE 6: SENSITIVE DETECTION OF RARE CELL TYPES...

[0125] Given the scalable design of STAMP, this next example is aimed to simulate clinically relevant scenarios, particularly the identification of circulating tumor cells (CTCs). Tumor cells circulating in the blood are valuable for quantifying tumor burden, identifying tumor heterogeneity, and detecting actionable alterations. To simulate CTC detection from blood samples and approximate the sensitivity of STAMP in this experimental setting, MCF-7 cancer cells were spiked at dilutions of 1:100,000 and 1:50,000 into PBMC samples, stamped onto the same slide (total 1.1 million cells). Next, cell mixtures were imaged with the CosMx Human Universal Cell Characterization RNA panel. Here, the CosMx cell segmentation markers provided the ground-truth, allowing for the visual detection of the CTC-mimics, which were randomly distributed across each sub-STAMP (PCK staining, FIG. 16A). To automate CTC- mimic detection, gene expression signatures were generated using scRNA-seq performed on fixed cells and each imaged single cell was scored based on their MCF-7 or PBMC profiles (FIG. 16B). Information of the PCK mean fluorescence intensity, the number of transcript counts and the cell areas were combined, to jointly identify CTC-mimic with high accuracy (FIG. 16C). Thereby, in sub-STAMPs with 10 and 20 target spike-in cells, 7 and 28 CTC-mimics were identified, respectively. Here, CTC-mimic accounted for 0.001% of the overall sample size, demonstrating the capacity to detect extremely rare cellular types with clinical application potential. Of note, the immuno-fluorescence images allowed us to manually validate the identified CTC-mimic based on their coordinates registered with the STAMP experiments (FIG. 16D). The detection of these rare events was independent of the platform, panel, or segmentation method, as demonstrated by the identification of CTC-mimics using the Xenium Analyzer with the Immuno-Oncology panel (FIG. 22A) and the Xenium Prime 5K Human Pan Tissue and Pathways panel (FIG. 16E and FIG. 22B).

[0126] Here, using the Xenium Explorer to align RNA, protein, and H&E images, for visual integration. This analysis successfully identified and confirmed the presence of two cancer cells among the 852,677 total cells in the sample (FIG. 22B), demonstrating the sensitivity of STAMP in detecting rare cells.Attorney Docket #: 243734.000229SJ-25 -0018-02EXAMPLE 7: COMBINED RNA AND PROTEIN MULTIMODAL PROFILING

[0127] Building on the scalable imaging-based approach for single-cell transcriptomics, in this next example, RNA and protein readouts were integrated and combined into the STAMP framework for multimodal cell profiling. Two high-density STAMPS (-750,000 to 900,000 PBMCs) were prepared to generate and integrate RNA and protein imaging data. First STAMP-X was performed using the Xenium Prime 5K Human Pan Tissue and Pathways panel for high- resolution RNA profiling. The same slides were reused for subsequent protein profiling on two separate platforms: the CosMx SMI (64-protein panel; STAMP-X-CP) and the Akoya Phenocycler Fusion (42-protein panel, STAMP-X-PCF). Parallel single-modal protein STAMP experiments were conducted on the CosMx SMI (STAMP-CP) and the Phenocycler Fusion (STAMP-PCF) to assess the impact of prior STAMP-X transcriptomic processing on the protein profiling outputs.

[0128] All protein assays yielded high-quality protein profiles with substantial protein counts per cell, demonstrating the robustness of protein detection within the STAMP framework (FIG. 16F). For STAMP-CP, 582,087 cells were detected with a mean intensity of 1430.7 and a mean cell area of 77.25 pm2. The STAMP-X-CP sample detected 548,800 cells with a mean intensity of 2022 and a mean cell area of 73.62 pm2For the Phenocycler, the STAMP-PCF sample contained 615,566 cells with a mean intensity of 350.8 and a mean cell area of 70.57 pm2, whereas the STAMP-X-PCF sample included 630,691 cells with a higher mean intensity of 923.1 and a mean cell area of 65.17 pm2. To further evaluate the quality of multimodal profiling, average protein expression was compared for each protein across STAMP-CP / X-CP and STAMP-PCF / X-PCF samples, i.e. with and without prior RNA profiling. Here, protein detection in both platforms correlated significantly (STAMP-CP / X-CP: R = 0.93, p < 0.01; STAMP-PCF / X-PCF: R = 0.82, p < 0.01; FIG. 16G). It is important to note though that significant differences were observed for specific proteins between the single- and multimodal workflows. For the Phenocycler Fusion platform, Beta- Actin levels were notably higher in STAMP-PCF (1269.09) compared to STAMP- X-PCF (99.54), while CD68 showed the opposite trend (81.41 in STAMP-PCF versus 689.86 in STAMP-X-PCF). In the CosMx samples, differences were observed for CCR7 (62.63 in STAMP- CP versus 486.3 in STAMP-X-CP) and Tim-3 levels (217.15 in STAMP-CP versus 436.64 in STAMP-X-CP).

[0129] To validate that the RNA and protein data from single- and multimodal workflows could be integrated for immuno-phenotyping across modalities, we conducted cluster analysis of theAttorney Docket #: 243734.000229SJ-25 -0018-02PBMCs from multimodal STAMP-X-CP (FIGs. 16H-J) and STAMP-X-PCF (FIG. 23), identifying the major cell types across modalities (FIG. 16H-J, FIG. 23A-B). Sub-clustering the transcriptome data of the major immune cell types revealed 26 subpopulations of T, B and natural killer (NK) and myeloid cells (FIG. 161). Protein profiling identified the same major cell types, but faced difficulties to describe more subtle cell states (FIG. 16J). Thus, while multi-modal profiling proved feasible with STAMP, protein profiling was less effective at resolving PBMC subclusters due to the smaller protein panel (42 targets) compared to the RNA panel (-5,000 targets), an inherent limitation for current protein analysis. However, protein profiling can provide complementary information about cell identities and function, as well as druggable targets and, thus, provides a valuable extra layer of information in such experimental designs.EXAMPLE 8: EVALUATING STAMP FOR DISSOCIATED TISSUE PROFILING

[0130] Despite the transformative potential of spatial transcriptomics, single-cell analysis of dissociated cells remains the gold-standard for characterizing tissues, offering unparalleled resolution for the understanding of cellular heterogeneity and the identification of rare populations. As spatial technologies continue to mature, single-cell and spatial analysis are expected to evolve into complementary tools. STAMP represents a prime example of this co-evolution, bridging the strengths of both approaches. To assess the versatility and applicability of STAMP for dissociated tissues, tissue analyses was performed of five major mouse organs: kidney, liver, brain, heart, and lung (FIG. 17A). These organs were chosen for their diverse cellular compositions to provide an evaluation across various tissue architectures.

[0131] For each organ, both cell and nuclei suspensions were prepared and processed. Nuclei were included because their preparations provide a less biased cell type coverage, avoid transcriptional artifacts associated with cell isolation, and for their compatibility with archived frozen specimens. Cells and nuclei suspensions were immobilized onto Xenium-compatible glass slides for STAMP-X analysis and profiled using the Xenium Prime 5K Mouse Pan Tissue & Pathways panel. Additionally, a portion of the fixed cells and nuclei was profiled using the Gene Expression Flex assay (1 Ox Genomics) to compare quality and performance between STAMP and established scRNA-seq protocols. In total, 601,417 cells were profiled in two STAMP-X experiments (215,578 cells and 385,839 nuclei).Attorney Docket #: 243734.000229SJ-25 -0018-02

[0132] The STAMP data from cells and nuclei showed comparable frequencies of high quality cells and the numbers of detected transcripts and genes (FIGs. 17B-E). A strong correlation was observed between cells and nuclei across all organs (R = 0.73, p < 0.01: FIG. 17F), with the liver being a notable exception for all metrics. Isolating single cells from liver tissue presents significant challenges, primarily due to the organ's complex architecture, the delicate nature of hepatocytes, and the limitations of current dissociation methods performed at a temperature at which liver enzymes are highly active. Brain single-cell and single-nuclei preparations were due to the distinct biases to capture highly connected cell types, such as neurons. Here, applying STAMP on brain nuclei outperformed single-cell preparations yielding a total of 6,862 cells and 45,487 nuclei.

[0133] Using the mouse reference cell atlas, a total of 44 different cell types across all organs were identified (FIG. 17G). The lung dataset contained the highest number of cell types (n=15), including various types of pneumocytes (FIG. 17G, iii.). In the sub-STAMP with a 1:1: 1:1:1 mixture of cells from all organs, cell identities were computationally separated by applying gene signatures derived from each respective tissue (FIG. 171). This approach yielded 26,705 high- quality cells, with 3.3% (n=880) identified as brain, 27.6% (n=7,391) as heart, 24.25% (n=6,477) as kidney, 16.66% (n=4,451) as liver, and 28.1% (n=7,506) as lung cells in line with the above- mentioned biases to isolate intact cells from brain and liver tissues. To further confirm the cell’s origin, a one-vs-all differential expression analysis was conducted for each organ and the organspecificity of the top differentially expressed genes was validated (FIG. 17J). For example. Hspg2, the top biomarker for heart cells, is a gene that encodes for perlecan, a heparan sulfate proteoglycan integral for muscle homeostasis in mice. These results collectively support STAMP as a versatile and powerful method for generating high-quality single-cell and single-nuclei data across a wide range of tissue types, making it a valuable alternative to more traditional single-cell transcriptomics approaches.DISCUSSION OF EXAMPLES

[0134] STAMP is demonstrated as a cost-effective approach that transforms commercial imaging platforms into scalable and flexible profiling tools for transcriptomics and proteomics readouts of cells in suspension. STAMP provides a unified solution that integrates high-throughput analysis and multi-sample profiling, expanding the possibilities of single-cell analysis for diverse research and clinical applications. STAMP was evaluated to verify: 1) specificity, 2)Attorney Docket #: 243734.000229SJ-25 -0018-02 reproducibility, 3) scalability, 4) sensitivity, 5) resolution and 6) flexibility. Cancer cell lines were used to confirm specificity and to ensure the accurate targeting of gene sets within individual cells, without cross-hybridization or background noise. For reproducibility and scalability, the analysis of millions of PBMCs was tested across replicates, platforms and gene panel sizes, demonstrating STAMP'S consistent performance. Flexibility was assessed through the profiling of cells and nuclei from fresh and preserved samples, together with various experiments multiplexing diverse cell and sample types. STAMP demonstrated adaptability to diverse sample sizes, cell types and experimental conditions. Its sensitivity was proven in CTC-mimic experiments, detecting rare populations down to 0.00025%.[0135| One advantage of the STAMP technology is its cost-effectiveness, providing a significant reduction in expenses compared to single cell sequencing approaches. Even when using nonspecialized gene panels, as shown in the hESC (BMP4 treated) differentiation experiment, the STAMP approach generates insights that match scRNA-seq datasets. Refining or expanding probe sets is expected to further improve resolution for an even more precise mapping of cellular trajectories, as the basis for large-scale gene and drug perturbation studies. The ability to customize gene panels also offers a path towards further cost reduction, while keeping precision and scalability high. The multimodal integration highlights STAMP’S potential as a powerful tool for multi-layered data analysis, where RNA and protein information can be combined to improve phenotype and functional mapping. As protein panels expand. STAMP is expected to provide deeper insights through more comprehensive multi-layered data. By replacing sequencing with imaging, STAMP also captures spatially-indexed morphology data, supporting visual inspection to reduce false positive results and enabling multimodal integration of RNA, protein, and histological features.

[0136] By addressing current limitations in single-cell genomics, STAMP offers an integrated solution that combines scalability, cost-effectiveness, and flexibility in a single approach. This enables researchers to perform experiments on a broad range of sample sizes, from limited biopsy specimens to extensive cellular aliasing, without compromising data quality or increasing costs. STAMP’S ability to balance precision and scalability makes it an invaluable tool for a wide array of experimental needs, from basic research to clinical applications, setting a new benchmark for high-throughput single-cell analysis.Attorney Docket #: 243734.000229SJ-25 -0018-02METHODS: MAINTENANCE OF HUMAN CELL LINES

[0137] All cell lines used in this study were cultured and maintained under the conditions outlined in Supplementary Table 1. Cell counting and viability measurements, both before and after storage, were performed using the Luna-FX7 Automated Cell Counter (Logos Biosystems) with the AO / P Viability Kit (F23011). Cell viability after thawing was consistently >85%. On the day of fixation, cells were thawed in a 37 °C water bath and washed twice with lx PBS (without Ca27Mg2+) supplemented with 0.05% BSA (Miltenyi Biotec, 130-091-376). For the tongue cancer- associated fibroblasts, sample collection was conducted with informed patient consent and approved by the Central Adelaide Local Health Network Human Research Ethics Committee (approval number: 17981).METHODS: TISSUE COLLECTION AND PREPARATION

[0138] Heart, brain, liver, kidney, and lung tissues from scavenged C57B1 / 6 female mice were obtained from Dr. Hon Yeung Chan, Robertson Lab, The University of Adelaide. The mice were humanely killed by cervical dislocation under the approval of the Adelaide Animal Ethics Committee (approval number: M-2019-087). The scavenged organs were harvested within 10 minutes and immediately immersed in cold lx PBS to maintain cellular integrity. Cells and nuclei were prepared as follows. To create single-cell suspensions, tissues were mechanically dissociated into smaller fragments and subjected to enzymatic digestion using the gentleMACS Octo Dissociator with Heaters (Miltenyi Biotec, 130-096-427). The enzyme used for digestion was Liberase TH (Roche, 792347) at a concentration of 0.5 mg / mL for 30 min at 37 °C (program 37C_Multi_G). Following digestion, cell suspensions were filtered through a 40 pm strainer to obtain a pure single-cell suspension. Cell viability was assessed using the Luna FX7 to ensure suitability for single-cell analysis. Nuclei suspensions were prepared following the SaltyEz protocol as described at dx.doi.org / 10.17504 / pfotocols.io.bx64prgw. For the FFPE prostate samples (31944 IN and 31568 F), ethical approval for tissue collection and experimentation was obtained from the Royal Adelaide Hospital (approval #04101 If) and the University of Adelaide Human Research Ethics Committees (approval number: H-2018-222).Attorney Docket #: 243734.000229SJ-25 -0018-02METHODS: DIFFERENTIATION OF HUMAN EMBRYONIC STEM CELL (HESC)

[0139] Human embryonic stem cells (hESCs, WA01, WiCell) were cultured in vessels coated with growth factor-reduced Matrigel (Coming, 354230) and maintained in mTeSR-i- growth media (STEMCELL Technologies, 85850). Cultures were passaged by removing the medium, washing with DPBS (Sigma-Aldrich, D8537), and treating with Gentle Cell Dissociation Reagent (STEMCELL Technologies, 100-0485,) at sufficient volume to cover cells for 3 min at 37 °C. The dissociation reagent was aspirated, growth media was added, and cells were lifted by scraping with a cell lifter to produce small cell clumps. One-sixth volume of lifted cells was added to a new culture vessel and topped up with mTeSR-i- growth media. All cultures were grown at 37 °C, 5% CO?. For differentiation, hESCs were seeded as colonies into 6-well trays by passaging as described. The following day, hESCs were treated with 50 ng / mL human recombinant BMP4 (STEMCELL Technologies, 78211) in mTeSR-i- media for 0, 6, 12, 24, 48, 72, 96, or 120 h, with daily media replacement. To harvest, media was removed, cells were washed with DPBS, and Gentle Cell Dissociation Reagent was added to cover the cells. Cultures were incubated at 37 °C until dissociated, then growth media was added. Dissociated cells were pelleted by centrifugation for 5 min at 200 ref, and the media was aspirated. Cell pellets were resuspended in 1 mL mFreSR (STEMCELL Technologies, 05855) and transferred to a cryotube, then frozen in a CoolCell Container (Corning) at -80 °C overnight. Cells were transferred to -150 °C storage the next day. Cell counting and viability measurements before and after storage were carried out using the Luna- FX7 Automated Cell Counter. Viability of cells after thawing was >85%.METHODS: DIFFERENTIATION OF INDUCED PLURIPOTENT STEM CELLS (IPSCS) AND IMMUNOFLUORESCENCE

[0140] Induced Pluripotent Stem Cells (iPSCs, 32F) were generated via Sendai virus reprogramming as previously describedl9 and maintained in E8 media (ThermoFisher Scientific, A2656101) on MatrigeLcoated plates (Corning, 354277). For mesodermal differentiation, iPSCs were seeded at 90% confluency in E8 medium. On day 1, cells were switched to RPMI (ThermoFisher Scientific, 12633012) supplemented with 0.5% B27 without insulin (ThermoFisher Scientific, A1895601) and 40 ng / mL BMP4 (PeproTech, 120-05ET-500UG). On day 2, the medium was replaced with the same medium plus 6 pM CHIR99021 (STEMCELL Technologies, 72052). On day 3, 5 pM IWR1 (STEMCELL Technologies, 72562) was added, removed the nextAttorney Docket #: 243734.000229SJ-25 -0018-02 day, and replaced with RPMI + 0.5% B27 without insulin. For ectodermal differentiation, iPSCs were plated in E8 medium until 80% confluency. On day 1, the medium was replaced with neural induction medium (NIM) composed of DMEM / F12 (ThermoFisher Scientific, 11320033) + lx NEAA (ThermoFisher Scientific, MEM Non-Essential Amino Acids, 11140050) + 0.5% B27 (ThermoFisher Scientific, 17504044), 5 uM SB431542 (STEMCELL Technologies, 100-1051), and 0.1 uM LDN193189 (Sigma Aldrich, SML0559) for 5 days, after which cells were harvested. For endodermal differentiation, cells at 50% confluency were switched to RPMI supplemented with 0.5% B27 without insulin, 100 ng / mL Activin A (PeproTech, 120-14E), 10 ng / mL BMP4, and 20 ng / mL FGF2 (PeproTech, 100-18B). On day 3, cells were switched to RPMI supplemented with 0.5% B27 without insulin and 100 ng / mL Activin A until harvest on day 5. Cells were harvested using Accutase (ThermoFisher Scientific, Al 110501), centrifuged for 2 min at 400 ref, and washed twice with PBS (without Ca2+ / Mg2+) + 0.05% BSA. Viability at harvesting was >90% as measured using the Luna-FX7 Automated Cell Counter. All cultures were grown at 37 °C, 5% CO2. Cells were processed for STAMP on the same day of collection. Expression of canonical marker genes for all embryonic lineages was assessed as part of sample quality control. Based on the absence of marker gene expression (SOX17, HNF4A, GATA6, PRDM1) it was concluded that iPSCs in the Endoderm sample had failed to sufficiently differentiate towards endoderm. This sample was therefore excluded from further analysis. For immunofluorescence analysis, cells of all lineages were seeded on MatrigeLcoated chamber slides (ThermoFisher Scientific, Nunc Lab- Ttek, 171080), fixed with 4% PFA for 15 min at room temperature (RT), and permeabilized with 0.2% Triton X-100 (ThermoFisher Scientific, A16046.AE) in PBS for 10 min. Cells were blocked with PBS (without Ca2+ / Mg2+) + 1% BSA and 0.2% Triton X-100 for 30 min at RT, followed by incubation with the primary antibody in blocking buffer overnight at 4 °C. After washing three times with blocking buffer, cells were incubated with secondary antibodies at RT for 1 h in the dark, washed three times with blocking buffer, and stained with lx DAPI (ThermoFisher Scientific, R37606). Confocal images were acquired using an Olympus FV3000 Confocal Microscope. Analysis was performed using Image! software (U.S. National Institutes of Health, Bethesda, Maryland, USA). The primary antibodies used were: GATA4 (G-4, sc-25310), Nestin (SAB4200347), Pax6 (AMAB91372), SOX2 (14-9811-82), Oct3 / 4 (C-10, sc-5279), and TRA-1- 60 (MAI-023Attorney Docket #: 243734.000229SJ-25 -0018-02METHODS: SLIDE PREPARATION AND ‘STAMPING’ PROCEDURE

[0141] Superfrost Plus Micro Slides (VWR, 48311-703) for STAMP-C / CP / PCF and Xenium slides (lOx Genomics, PN-3000941) for STAMP-X / X-CP / X-PCF / PCF, were placed on Xenium slide holders were coated with 1 mL of Poly-D-Lysine for 1 h and overnight at 37 °C. respectively, in a thermocycler using the Xenium Thermocycler Adapter plate positioned atop the 96-well block of a C1000 Touch Thermal Cycler (BioRad) with the lid closed and set at 37 °C. After coating, the slides were washed with 1 mL of Nuclease Free Water (ThermoFisher Scientific, 10977023) 3 times and air dried. Custom single or multiplex (multi-sample) arrays of various areas / volumes were created using either 10X Genomics’ gaskets (PN- 370017) from the Chromium Single Cell Reagent Kits and using a hole punch or punch pliers (Total Tools, 9070220SB), micro-Slide 8- well (ibidi, 80841) and 12- well (ibidi, 81201) cell culture chambers or a silicone gasket for ProPlate® microarray system (Grace Bio-Labs, 246875) by placing them on the coated slides within the scanning area of the CosMx, Xenium or Phenocycler. For the STAMPing procedure (i.e. attaching cells on slides for STAMP), up to 5 million single cells / nuclei in suspension, with >80% viability, were first fixed with 4% Formaldehyde (Sigma Aldrich, 252549-500ML) + lx Concentrated Cell Fixation and Permeabilization Buffer (lOx Genomics, PN- 2000517), as per the Fixation of Cells & Nuclei for Chromium Fixed RNA Profiling protocol (CG000478, RevD). After 2 h incubation at RT, cells were pelleted at RT for 5 min at 850 ref and resuspended in 1 mL of lx Quenching Buffer (lOx Genomics, PN-2000516), washed 2 times with nuclease-free water and finally resuspended in water + 0.01% Triton-X and counted in duplicates or triplicates using the Luna-FX7 Automated Cell Counter. To maximize the use of the area in each stamp (or substamp, when in multi-sample settings) for high and ultra-high cell profiling we used the average cell size data provided by the Luna-FX7 Automated Cell Counter and estimates generated using ChatGPT 4o to calculate the number of cells that would fully (or partially) cover a desired area of the given custom array. Before STAMPing, cell concentrations were adjusted so that the minimum volume of cell suspension added to the wells would evenly cover the bottom surface for the desired number of cells to profile and fully dry within 1 h of incubation at 42 °C. This volume was dependent on the well area and was approximated in advance for each well size. Cells were then loaded into the wells, and the slide was placed in a thermocycler using the Xenium Thermocycler Adapter plate, running the following program: 4 °C for 30 min, 25 °C for 5 min, 42 °C for up to 1 h (or until the volume dried), followed by 42 °C for 2 h, and then held at 22 °C. After STAMPing, custom wellsAttorney Docket #: 243734.000229SJ-25 -0018-02 were carefully removed, and the slides were either processed immediately or placed in a mailer with desiccant at RT for STAMP-X / X-CP / X-PCF or at 4 °C for STAMP-C / CP / PCF until further processing. The same procedure was carried out for STAMP profiling using PBMCs from a healthy donor (STEMCELL Technologies, 200-0470), human cell lines (MCF-7, SK-BR-3, LNCaP, EndoC-betaHl, hTERT-HMEl, TF-1, U-373 MG, UMSCC-1, V16D, CHP-134, HEK293T. SK-N-DZ. SK-N-SH, SHSY-5Y and KELLY either individually or mix 1:1:1 MCF-7:SK-BR-3:LNCaP or MCF-7:SK-BR-3:LnCaP:KELLY), iPSCs, iTSCs, Human dermal fibroblasts, hESC, Tongue Cancer CAFs, BM CD34+ (STEMCELL Technologies. 70002), nuclei isolated from FFPE prostate tumour samples 31944 IN and 31568 F, cells and nuclei isolated from mouse heart, lung, brain, liver and kidney and CTC-mimics. For CTC-mimics, MCF-7 and SK- BR-3 cell lines were counted in triplicates and spiked into 1 million PBMCs either as a single line (MCF-7) or as a mix (1:1 MCF-7:SK-BR-3). Spike-in ratios varied from less than 10 to more than 10 but fewer than 50 cancer cells per approximately 1 million PBMCs.METHODS: ACTIVATION OF PBMCS

[0142] Cryopreserved -80°C Human Peripheral Blood Leukopak (Stem Cell) were thawed in a 37°C water bath and transferred with a bored tip to a 15 ml Falcon containing 14 ml of 37°C prewarmed RPMI (L-Glutamine) media supplemented with 10% FBS (Thermo Fisher Scientific) and 100 U / ml Penicillin / Streptomycin (Gibco). PBMCs were centrifuged at 300x g for 7 min at RT, supernatant was removed, and pellet resuspended in 1 ml of IX PBS (Thermo Fisher Scientific) supplemented with 1% BSA (Miltenyi Biotec) and 10 pL of DNase I (PN LS002007, Worthington-Biochem). After incubation at RT for 10 min with periodic shaking, cells were filtered with a 40 pm strainer (PN 43-10040-40, Cell Strainer) into a new 15 ml falcon on ice and filter was washed by adding 9 ml of cold IX PBS. PBMCs were spun down by centrifuging at 300x g for 7 min at 4°C and resuspended in IX PBS with 0,05% BSA for assessment of cell numbers and viability with the TC20™ Automated Cell Counter (Bio Rad), obtaining a viability >85%. PBMCs were then cultured in a 6 well plate at 4 million cells / ml in complete RPMI media at 37°C + 5% CO2 under three different culture conditions: (1) Dynabeads™ Human T-Activator CD3 / CD28 for T Cell Expansion and Activation (PN 11132D, Thermo Fisher Scientific) following manufacturer’s protocol, (2) lipopolysaccharide (LPS) at 100 ng / ml (InvivoGen); andAttorney Docket #: 243734.000229SJ-25 -0018-02(3) complete medium only as control. After 4 and 24 h in culture, cells in each condition were harvested, washed with filtered IX PBS with 0,05% BSA and STAMPed as previously described.METHODS: COSMX RNA SLIDE PREPARATION (STAMP-C)

[0143] STAMPed slides were placed on a Xenium Thermocycler Adapter plate atop the 96-well block of a thermocycler with the lid open and incubated at 60 °C for 2 h, then equilibrated to RT for 3-5 min. The slides were subsequently processed according to the guidelines provided in the CosMx SMI Slide Preparation for FFPE RNA Assays manual (NanoString, MAN-10184-02 or MAN- 10184-03 for 6k), starting from page 38 or 36 respectively. Slides were immersed directly in pre-heated lx Target Retrieval Solution (NanoString, CosMx FFPE Slide Preparation RNA Kit) in a pressure cooker at 100 °C for 8 min (as per MAN-10184-02 and MAN-10184-03). The slides were immediately transferred to water for 15 s, then washed in 100% ethanol for 3 min, and airdried at RT for 30 min to 1 h. Incubation frames were attached to each slide, and a pre-warmed digestion buffer containing 1.5 pg / mL Proteinase K (NanoString, CosMx FFPE Slide Preparation RNA Kit) and lx PBS (ThermoFisher Scientific, AM9625) was applied. Slides were incubated in a hybridization chamber at 40 °C for 15 min. The slides were then rinsed twice in water, and fiducials were applied at 0.001%, followed by a 5 min incubation at RT, shielded from light. The slides underwent a lx PBS wash for 1 min, followed by fixation in 10% NBF for 1 min, and then two washes in NBF stop buffer (Tris Base, Sigma S6639-1L, and Glycine, Sigma, G7126) for 5 min each, and a 5 min wash in lx PBS. A 100 mM NHS-acetate solution (ThermoFisher Scientific, 26777) was applied to the tissue for 15 min at RT, followed by two washes in 2X SSC (ThermoFisher Scientific, AM9763) for 5 min each. The CosMx Human Universal Cell Characterization RNA Panel targeting 950 human genes, and a 50-target add-on panel set (NanoString, CMX-H-USCP-1KP-R), or the CosMx 6k Discovery Panel (Nanostring, 121500041), were denatured at 95 °C for 2 min, cooled on ice for 1 min, and then added to a probe mix containing RNase inhibitor, Buffer R, and nuclease-free water. This mix was applied to the slide and incubated for 18 h in the hybridization chamber at 37 °C. After incubation, the slides were washed twice in a final concentration of 50% deionized formamide (ThermoFisher Scientific, AM9342) and 2x SSC mix for 25 min each, followed by two washes in 2x SSC for 2 min each. DAPI nuclear stain stock was diluted to 1:40 with blocking buffer (Nanostring, CosMx FFPE Slide Preparation RNA Kit) and applied to the slides for 15 min at RT, protected from light. The slidesAttorney Docket #: 243734.000229SJ-25 -0018-02 were then washed in 1 x PBS for 5 min and stained for 1 h with a cocktail of CD298, B2M, PanCK, and CD45 antibodies (Nanostring, 121500020, 121500021). The slides were washed three times in lx PBS for 5 min each and then stored in 2X SSC. The pre-bleaching profile followed configuration A, while the cell segmentation profile adhered to configuration C (MAN- 10161-03- 2 or MAN-10161-05, Nanostring).METHODS: COSMX PROTEIN SLIDE PREPARATION (STAMP-X-CP / CP)

[0144] STAMPed slides (for both single and multimodal STAMP) were processed according to the guidelines provided in the CosMx SMI Slide Preparation for FFPE Protein manual (MAN- 10185-01-1, NanoString) from page 31. Briefly, slides were baked at 65 °C for 2 h, equilibrated to RT for 3-5 min, then rehydrated in lx PBS for 5 min. Slides were immersed in pre-heated lx Target Retrieval Solution (Nanostring, CosMx Protein Slide Preparation FFPE Kit) in a pressure cooker at 100 °C for 8 min and then allowed to equilibrate to RT in the same solution for 60 min. Subsequently, the slides were washed three times in lx PBS for 5 min each, and incubation frames were attached. Slides were then covered with Buffer W and incubated at RT for 1 h in the dark. The 64-plex human immuno-oncology protein antibody panel (Nanostring, CMX-H-IOP-64P-P) was combined with CD298, B2M, PanCK and CD45 segmentation markers in Buffer W. The primary antibody mix was incubated at 4 °C for 18 h, followed by three washes with lx TBS-T buffer for 10 min each and a wash with lx PBS for 2 min. Fiducials, prepared at the recommended concentration of 0.00005%, were applied to the slides at RT for 5 min, protected from light. Slides were then washed once with lx PBS for 5 min and fixed with 4% PFA for 15 min, followed by three washes in lx PBS for 5 min each. Sections were stained with a 1:40 diluted nuclei stain for 10 min, washed twice with lx PBS for 5 min, and incubated with 100 mM NHS acetate for 15 min, before a final wash in lx PBS for 5 min. The selected pre-bleaching profile was Configuration A, and the cell segmentation profile was Configuration C.METHODS: COSMX SMI SETUP AND DATA ACQUISITION (STAMP-X-CP / CP / C)

[0145] The setup and scan acquisition for the CosMx SMI instrument were performed according to the CosMx SMI Instrument User Manual (MAN-10161-03-2 or MAN-10161-05 for 6k, Software Version 1.3.0.209, NanoString). A new acquisition process was initiated through the CosMx SMI Control Center web interface. Before insertion into the CosMx Flow Cell AssemblyAttorney Docket #: 243734.000229SJ-25 -0018-02Tool, the slides were carefully dried in the areas surrounding the imaging region. The assembly process involved lowering the tailgate, placing the slide, and securing it by raising the tailgate. After removing the adhesive backing, a new flow cell coverslip was precisely aligned above the slide's imaging area. The Assembly Tool’s lid was then closed securely to attach the coverslip to the slide, creating a functional CosMx flow cell. Following assembly, 2x SSC (for RNA assay) or lx PBS (for protein assay) was gently introduced through one of the flow cell ports to hydrate the samples. For multi-sample STAMPs, prior to placement of the flow cell, each STAMP was hydrated with 4 pl of 2x SSC (for RNA assay) or lx PBS (for protein assay). The flow cell coverslip was slowly lowered from left to right allowing the liquid to spread evenly across the stamps. A PIO tip was used to help guide the coverslip down. Once positioned, the tip was removed and the assembly tool closed to secure the flow cell. This method prevented the formation of “rivers” inside the flow cell chamber and ensured full sample hydration.

[0146] Flow cell configuration data, including the flow cell's barcode, slide ID number, and the maximum tissue thickness of 7 pm, were entered into the Control Center interface (MAN-10161- 03-2 or MAN-10161-05, Nanostring). All slides were scanned using Configuration A for the prebleaching profile and Configuration C for the cell segmentation profile. Additional details regarding the probe panel, cell segmentation, and supplemental markers were also entered into the flow cell configuration data for each section. Assembled flow cells / slides, along with Buffer Bottles 1-4, were loaded into the instrument. Before positioning Bottle 4, catalase and pyranose oxidase enzymes were added directly before loading, or added the previous day for 6k runs. For RNA runs, RNase inhibitor was added to a designated well in the CosMx imaging tray, which was placed in the instrument after equilibration to RT. The Control Center configuration was verified, followed by a pre -run check and a tissue find scan for each slide conducted by the instrument. After completing the tissue find scans, rectangular scan areas were placed around each tissue section for the preview scan. The preview scan images enabled the selection of regions of fields of view (FOVs), ensuring thorough coverage of the STAMP areas on each slide. FOV selections for each slide were confirmed before starting the cycling process. CosMx scan data was automatically uploaded to NanoString’s cloud-based AtoMx Spatial Analysis Platform during the run, as detailed in the CosMx Data Analysis Manual (MAN-10162-03, Software Version 1.3.2, NanoString). Upon completion of the run and full upload of scan data to AtoMx, a study wasAttorney Docket #: 243734.000229SJ-25 -0018-02 created for each CosMx scan. Within AtoMx, pipelines were executed for each study, and data was exported in various formats, including TileDB arrays and Seurat objects, for in-house analysis.METHODS: XENIUM SLIDE PREPARATION (STAMP-X)

[0147] STAMPed Xenium slides were placed on a Xenium Thermocycler Adapter plate atop the 96-well block of a thermocycler with the lid open and incubated at 60 °C for 2 h for Xenium vl or 30 min (cells) or 1 h (dissociated tissues) for Xenium Prime. Slides were then equilibrated to RT for 7 min, assembled into Xenium cassettes, and hydrated with lx PBS for Xenium vl or PBS-T or IX PBS for Xenium Prime. The slides were processed following the Xenium In Situ for FFPE Deparaffinization and Decrosslinking protocol from step 1.4.a (page 42) (CG000580 Rev D or Rev E, 10X Genomics). Briefly, the slides were reverse crosslinked using a decrosslinking buffer containing tissue enhancer, urea, and perm enzyme B at 80 °C for 30 min, followed by three washes with PBS-T. For Xenium vl , slides were then immediately processed according to the Xenium In Situ Gene Expression Cell Segmentation User Guide (10X Genomics, CG000749 Rev A) for the remaining slide preparation steps. Briefly, the pre-designed gene expression probe set, Xenium Human Immuno-oncology Panel (10X Genomics, PN- 1000654), targeting 380 human genes, was denatured at 95 °C for 2 min, crash-cooled on ice for 1 min, and then equilibrated to RT before being added to the probe hybridization buffer and TE buffer (Fisher Scientific, BP24731) to make the probe hybridization mix. The slides were incubated with the hybridization mix at 50 °C for 20 h. Slides were washed three times with PBS-T for 1 min each and then incubated with a posthybridization wash buffer for 30 min at 37 °C. Slides were washed three times with PBS-T for 1 min each and incubated with the ligation mix for 2 h at 37 °C. Three 1 min PBS-T washes were followed by a 2 h incubation at 30 °C with amplification mix and enzyme (10X Genomics, PN- 2000392, 2000399). Slides were then washed three times with TE buffer for 1 min each, 70% ethanol for 2 min, twice with 100% ethanol, and once with 70% ethanol for 2 min each before rehydration with PBS-T. Slides were incubated at RT for 1 h with lx Xenium Block and Stain Buffer (10X Genomics, PN-2001083). For segmentation staining, slides were incubated for 20 h at 4 °C with the Xenium Multi-Tissue Stain Mix (10X Genomics, PN-2000991), which contains a cocktail of antibodies labeling the membranes (anti-ATPlAl / CD45 / E-cadherin), antibodies labeling the cell interior (anti-alphaSMA / Vimentin), and a universal interior label against Ribosomal RNA (18S rRNA) (10X Genomics, PN-2000991). Staining was enhanced by theAttorney Docket #: 243734.000229SJ-25 -0018-02 addition of Xenium Staining Enhancer reagent (10X Genomics, PN-2000992), followed by treatment with Xenium Autofluorescence Mix (10X Genomics, PN-2000753) to diminish unwanted autofluorescence and enhance the signal-to-noise ratio. Subsequently, Xenium Nuclei Staining Buffer (10X Genomics, PN-2000762) was used to facilitate the identification of tissues or regions of interest during the instrument’s overview scan. For Xenium Prime, slides were immediately processed following the Xenium Prime In Situ Gene Expression with Optional Cell Segmentation User Guide (CG000760 Rev A, 10X Genomics) for the remaining slide preparation steps. Briefly, following decrosslinking steps in CG000580, Xenium 5K Human or Mouse PTP Panel Priming Oligos (10X Genomics, PN-2001224 or 2001226) were denatured at 95 °C for 2 min, crash-cooled on ice for 1 min, and then equilibrated to RT before being added to Priming Hybridization Mix with TE buffer and Priming Hybridization Buffer (10X Genomics, PN- 001228). A Xenium Cassette Insert was placed onto the Xenium Cassette v2, and the slides were incubated with the priming hybridization mix at 50 °C for 1.5 h, then washed twice with PBS-T and incubated with Post-Priming Wash Buffer (10X Genomics, PN-2001229) at 50 °C for 30 min. Following three 1 min PBS-T washes, slides were incubated with an RNAse mix containing 2X RNAse buffer, RNase enzyme (10X Genomics, PN-2000411, 3000953), and water at 37 °C for 20 min. Slides were then washed three times with 0.5X SSC-T and incubated with a polishing reaction mix containing Polishing Buffer, Polishing Enzyme (10X Genomics, PN-2001231, 2001230), and water at 37 °C for 1 h. The pre-designed gene expression probe set, Xenium Prime 5K Human or Mouse Pan Tissue & Pathways Panels (10X Genomics, PN-1000724, 1000725), was denatured at 95 °C for 2 min, crash-cooled on ice for 1 min, and then equilibrated to RT before being added to the probe hybridization buffer and TE buffer to make the probe hybridization mix. Following three 1 min PBS-T washes, Xenium Cassette Inserts were placed onto the Xenium slide, and the slides were incubated with the hybridization mix at 50 °C for 20 h. Slides were washed twice with PBS- T for 1 min each, then incubated with a post-hybridization wash buffer (10X Genomics, PN- 2000395) for 15 min at 35 °C. Slides were washed three times with PBS-T for 1 min each and incubated with ligation mix containing ligation enzymes A, B, and ligation buffer (10X Genomics, PN-2000397, 2000398, 2001233) for 30 min at 42 °C. Following ligation, slides were washed three times with PBS-T for 1 min each and incubated with Amplification Enhancement Master Mix containing Amplification Enhancer Buffer and Amplification Enhancer (10X Genomics, PN- 2001234, 2001235) at 4 °C for 2 h. Amplification Enhancer Wash Buffer (10X Genomics, PN-Attorney Docket #: 243734.000229SJ-25 -0018-022001236) was added, and slides were incubated with amplification mix (10X Genomics, PN- 2000392) at 30 °C for 1.5 h, followed by three 1 min washes with TE buffer. Slides were processed for segmentation staining as outlined above for Xenium vl.METHODS: XENIUM ANALYZER SETUP AND DATA ACQUISITION[0148| STAMPed Xenium slides, assembled within Xenium cassettes, were imaged using the Xenium Analyzer in accordance with the guidelines specified in the Xenium Analyzer User Guide (CG000584 Rev F, 10X Genomics). The Xenium Decoding Consumables Kit (10X Genomics, PN-1000487) was used for instrument loading. Briefly, Xenium slide ID numbers and information on pre-designed gene expression probes were input into the Analyzer, and the necessary consumables and reagents were loaded into the instrument. Reagent modules B, and C for Xenium vl, were thawed at 4 °C overnight and equilibrated to RT for 30 min before loading into the instrument, while reagent module A was stored at 4 °C until loaded. Instrument wash buffer (100% Milli-Q water), sample wash buffer A (lx PBS, 0.05% Tween-20 (ThermoFisher Scientific, 28320). sample wash buffer B (100% Milli-Q water), and probe removal buffer (50% DMSO (Sigma Aldrich, D8418) 50 mM KC1 (ThermoFisher Scientific, AM9640G), 0.1% Tween-20) were prepared and loaded into the instrument along with buffer caps, a pipette tip rack, an extraction tip, and the objective wetting consumable. After loading, the samples were scanned to generate images of the fluorescently labeled nuclei in each section, which were used for Field of View (FOV) selection prior to run initiation. Each STAMP area was selected as a separate region and labeled accordingly. Upon completion of the run, the instrument was cleared of consumables, and the Xenium slides were carefully removed. Fresh PBS-T was applied to each slide / cassette, which were then covered and stored in the dark at 4 °C for up to 3 days until post-run H&E staining. The data were acquired using the Xenium Explorer software suite (v3.1.0, 10X Genomics), which provides a set of applications for analyzing and visualizing in situ gene expression data produced by the Xenium Analyzer.METHODS: FROM XENIUM RNA TO COSMX PROTEIN (STAMP-X-CP) AND PHENOCYCLER-FUSION (STAMP-X-PCF)

[0149] Following the completion of the Xenium Prime 5k Human RNA run, slides were removed from the instrument and stored in the Xenium cassette with 50% glycerol in lx PBS at 4 °C. After 6 days of storage, one of the slides (STAMP-X-CP) was washed three times in lx PBS for 1 minAttorney Docket #: 243734.000229SJ-25 -0018-02 each, followed by two additional washes in lx PBS for 5 min each. The Xenium cassette was then removed, and the slide was placed into a wash jar containing lx PBS for 5 min, as described in the CosMx SMI Manual Slide Preparation for Protein Assays (MAN-10185-01-1, NanoString). The slide was then immersed in antigen retrieval solution and incubated for 8 min at 100 °C, according to the CosMx Protein Slide Preparation and CosMx SMI Setup and Data Acquisition protocols. The other slide (STAMP-X-PCF) was covered with 50% glycerol in lx PBS, coverslipped and sealed for shipment to The Jackson Laboratory (CT, USA) for PhenoClycler Fusion profiling (see below).METHODS: PHENOCYCLER-FUSION SLIDE PREPARATION (STAMP-X-PCF / PCF).

[0150] The STAMP-PCF slide was baked at 60 °C for 30 min. During the final 5 min of baking, the STAMP-X-PCF slide was removed from 50% glycerol / PBS storage and placed in lx PBS. After baking, both slides were washed twice in lx PBS for 5 min each. The slides were then immersed in lx antigen retrieval buffer (pH 9.0) (AR9, Akoya Biosciences), and antigen retrieval was performed at 95 °C for 8 min using the TintoRetriever (BioSB). Following antigen retrieval, the slides were cooled in the retrieval buffer to RT and washed in nuclease-free water for 5 min. Slides were then processed according to the PhenoCycler-Fusion User Guide (PD-000011 REV M, Akoya Biosciences), starting from step 4 on page 49. Briefly, the slides were washed in Hydration Buffer, equilibrated in Staining Buffer, and incubated overnight at 4 °C with a 40- marker antibody cocktail (Supplementary Table 2) prepared in Blocking Buffer. The slides were subsequently washed in Staining Buffer, gently fixed with Post-Staining Fixing Solution, washed in lx PBS, and incubated in ice-cold methanol for 5 min. The slides were then washed in lx PBS, fixed with Final Fixative Solution for 20 min, washed three times in lx PBS, and immersed in Storage Buffer prior to the PhenoCycler Fusion run.METHODS: PHENOCYCLER SETUP AND DATA ACQUISITION (STAMP-X-PCF / PCF)

[0151] The experimental protocol was set up using the PhenoCycler Experiment Designer (Version 2.1.0, Akoya Biosciences). A reporter plate containing fluorescently labeled barcode reporters, as per the experimental design, was prepared following instructions on page 73 of the PhenoCycler-Fusion User Guide (PD-000011 REV M, Akoya Biosciences). Slides were prepared for the PhenoCycler-Fusion instrument according to the steps outlined in the Phenoimager-FusionAttorney Docket #: 243734.000229SJ-25 -0018-02User Guide (PD-000001 Rev N, Akoya Biosciences). Briefly, the slide was moved from Storage Buffer to lx PBS and incubated for 10 min. After incubation, a Flow Cell (Akoya Biosciences) was attached to the sample slide using the Flow Cell Assembly Device (Akoya Biosciences). The slide with the attached Flow Cell (Sample Flow Cell) was then placed in lx PhenoCycler buffer for 10 min. PhenoCycler Fusion software (Version 2.2.0) was used to set up the imaging run on the PhenoCycler-Fusion, following the steps on page 57 of the Phenoimager-Fusion User Guide (PD-000001 Rev N, Akoya Biosciences).

[0152] Reagents were prepared and loaded into the appropriate reagent reservoirs on the instrument, and the pre-prepared reporter plate was loaded into the PhenoCycler. A new PhenoCycler run was initiated using the experimental protocol design. A blank flow cell was loaded into the Flow Cell Slide Carrier, and all software prompts during the pre-flight routine were followed. The Sample Flow Cell was then loaded into the carrier, and a leak check was performed. Scan regions were selected following automated sample finding, and imaging was started. Upon completion, the Sample Flow Cell was placed in Storage Buffer at 4 °C. The generated QPTIFF data file was used for downstream image analysis.METHODS: H&E STAINING AND IMAGING

[0153] For post-Xenium (STAMP-X) Hematoxylin and Eosin (H&E) staining, slides were quenched in 10 mM sodium hydrosulfite (Sigma Aldrich, 157953-5G) at RT for 10 min, rinsed three times in water, and then immediately processed through the following sequence: once in water for 2 min, once in Mayer's Hematoxylin (Sigma Aldrich, MHS16) for 20 min, three times in water for 1 min each, once in bluing solution (Dako, CS702) for 1 min, once in water for 1 min, once in 70% ethanol for 3 min, once in 95% ethanol for 3 min, once in Eosin Y Solution, Alcoholic (Leica, 3801615) for 2 min, twice in 95% ethanol for 30 seconds each, twice in 100% ethanol for 30 seconds each, and twice in xylene for 3 min each, as described in the Demonstrated Protocol Xenium HE Staining (CG000613 Rev B, 10X Genomics). The slides were dried for 15 min and then cover slipped using 1.5 mm thick cover glass and Cytoseal Mountant XYL (ProSciTech, 1A013-XYL-118) or toluene-free mounting media (Dako, CS705). For post-CosMx (STAMP-X- CP / CP / C) H&E staining, the glass flow cell coverslip was first removed by adhering clear sticky tape to the top of the flow cell, scoring around the inside of the adhesive edges of the flow cell, then peeling off the sticky tape, which left the adhesive edges attached to the slide and exposedAttorney Docket #: 243734.000229SJ-25 -0018-02 the tissue for staining. Slides were then washed by dipping into water several times to remove any glass shards. The Demonstrated Protocol Xenium HE Staining (CG000613 RevB, 10X Genomics) was then followed starting from step 1.4, as described for post-Xenium H&E staining. Stained sections were covered using custom-cut coverslips fitted inside the flow cell adhesive edges using a glass scribe. Once the mounting media had dried, slides were scanned using a NanoZoomer 2.0HT (Hamamatsu) with a 40X objective or a Zeiss AxioObserver 7 with a 20x objective (Zeiss). For post-PhenoCycler (STAMP-X-PCF / PCF) H&E staining, the Sample Flow Cells were removed from the Storage Buffer and placed in a coplin jar containing xylene for 24 h. The Flow Cells were then carefully removed and disposed of properly. The slides were transferred to 100% ethanol for 2 min, dipping 10-15 times to ensure full coverage of the tissue. This step was repeated using 95% ethanol, followed by DI water. The slides were then placed in Mayer’s Hematoxylin for 4 min, followed by a rinse in DI water for 1 min, Bluing Reagent for 1 min, another DI water rinse for 1 min, and stained in Alcoholic Eosin for 2 min. Subsequent washes included 95% ethanol for 1 min, 100% ethanol for 1 min, fresh 100% ethanol again for 1 min, xylene I for 1 min, and finally, the slides were held in fresh xylene. Inside a fume hood, each slide was removed one at a time, the back was dried, and the slide was tilted onto a paper towel to remove excess xylene without allowing the tissues to dry. DPX or another xylene-based mountant was applied over the tissue area using a disposable Pasteur pipette while the remaining slides were kept in xylene to prevent over-drying. The glass coverslip was swiftly cleaned, any particles were removed, and the long edge was placed onto the slide. The slide was tipped towards the user, allowing the mountant to contact the coverslip, and was gently pressed down until the mountant spread evenly across the tissue. Excess mountant was carefully blotted using a paper towel, avoiding contact with the top of the coverslip. Any bubbles over the stained tissue were gently pressed out. The mountant was allowed to cure in the hood for at least 20 min before imaging. The slides with coverslips were then imaged using the NanoZoomer-SQ Digital slide scanner (Hamamatsu) with a 40x objective.METHODS: CHROMIUM FIXED RNA PROFILING OF CANCER CELL LINES AND HESC AND SEQUENCING

[0154] MCF-7, LNCaP, SK-BR-3, iPSC, and hESC cell lines were processed using the Fixation of Cells & Nuclei for Chromium Fixed RNA Profiling protocol (CG000478, RevD, 10X Genomics). After quenching, the cells were counted in replicates using the Luna-FX7 Automated Cell Counter, and 0.5 to 1 million cells were subjected to either the Chromium Fixed RNA KitAttorney Docket #: 243734.000229SJ-25 -0018-02(PN-1000474, 10X Genomics) for cancer cell lines or the Chromium Fixed RNA Kit (PN- 1000475, 10X Genomics) for iPSC lineages and hESC BMP4 treatments. Singleplex or multiplex gene expression libraries were prepared according to the user guides CG000691 (RevB) and CG000527 (RevF), respectively. The libraries were quality controlled using a 5200 Fragment Analyzer System (Agilent, HS NGS Fragment Kit, DNF-474-1000) and sequenced on a NovaSeq 6000 / X instrument following 10X Genomics’ user guide recommendations.DATA ANALYSIS: FIXED RNA PROFILING (FLEX)

[0155] The Flex datasets were aligned to probe set reference from 10X Genomics using cellRanger v8.0 with the filter-probes argument set to false. Filtered feature-barcode matrices were loaded into R (v4.4.1) as a SingleCellExperiment object using the readlOxCounts function from the DropletUtils package (v. 1.24.0).DATA ANALYSIS: QUALITY CONTROL...

[0156] Quality control metrics were computed for each cell using the addPerCellQC function from the scater package (v 1.32.0). Outliers in the number of counts and detected features were identified and removed using the isOutlier function from scater with parameters type = "lower", log - TRUE, and nmads - 2 for counts and nmads - 2.5 for detected features, respectively. Cells with a high percentage of mitochondrial gene expression were filtered out by applying isOutlier with type = "higher", log = FALSE, and nmads = 5.DATA ANALYSIS: PRE-PROCESSING

[0157] The count data were log-normalized using the logNormCounts function from scater. Feature selection was performed by modeling the mean-variance relationship with the modelGeneVar function from the scran package (vl.32.0). Highly variable genes were selected using getTopHVGs with an FDR threshold of 0.7. Principal Component Analysis (PCA) was conducted using thefixedPCA function from scran, specifying the subset. row argument to include the selected highly variable genes. The first 25 PCs were selected for downstream analyses based on the variance explained. UMAP dimensionality reduction was then applied using the runUMAP function from scater on these components.Attorney Docket #: 243734.000229SJ-25 -0018-02DATA ANALYSIS: CLUSTERING

[0158] Cells were clustered using the clusterCells function from scran, specifying dimred = "PCA" and BLUSPARAM = NNGraphParam(k = 50, cluster.fun = "louvain").DATA ANALYSIS: DOUBLET IDENTIFICATION

[0159] Potential doublets were identified using the scDblFinder function from the scDblFinder package (vl.16.0), incorporating cluster information via the clusters = colLabels(sce) argument, and subsequently removed by looking at the scDblFinder.score metrics together with canonical markers exclusive of specific populations.COSMX RNA DATASETS: CELL SEGMENTATION IN ATOMX

[0160] Default cell segmentation yielded suboptimal results (e.g. over- segmentation and / or over-expanded cell boundaries) as assessed by visual inspection of the immunofluorescence images (e.g. DAPI, panCK, CD45 and CD298 / B2M). Thus, we re-run cell segmentation for all STAMP-C experiments within the AtoMx platform using Configuration C (Cell Pellet Array) and setting the Basic Parameters as follows: CellDilation at 2 pm, CellDiameter at 30 pm for cell lines and 10 pm for PBMCs and NuclearDiameter at 10.8 pm for cell lines and at 5 pm for PBMCs. We additionally adjusted the Advanced Parameters for STAMP-C shown in FIG. 1 and Suppl. FIG. 1 as follows: BackgroundPercentile at 0.4, LogBlurSigma at 2, LoGThreshold at 5, Nuclei and CytoplasmModels at CP, NucleiProbability at -2, CellProbability at -3. CellFlowThreshold at 0.1 and MinCellSize at 4.3. Flat-files exported from AtoMx were imported into R (v4.4.1), generating SingleCellExperiment objects using the SingleCellExperimens package (vl.26.0).COSMX RNA DATASETS: QUALITY METRICS

[0161] For each cell, we considered the cell area, number of counts and number of detected genes (the latter computed using the addPerCellQC function from the scaler package (vl .32.0)). Lower and Higher outlier cells in any of these distributions were identified and removed using the isOutlier function from scater with the parameter log=TRUE. The nmads parameter, which specifies the number of median absolute deviations for outlier detection, was determined independently for each dataset based on visual inspection of the distributions. Additionally, cells located within 30 pixels of any field of view border were filtered out.Attorney Docket #: 243734.000229SJ-25 -0018-02COSMX RNA DATASETS: CLUSTERING

[0162] Cells were clustered using the unsupervised approach provided by the InSituType (1ST) package (vl.0.0), with the n_clust parameter varying according to the annotation step. Background noise was calculated using the mean negative probe counts. The fastCohorting function was used to perform cohorting on mean immune fluorescence values (CD298 / B2M, CD45, PanCK, DAPI, CD68_CK8_18), cell area, and aspect ratio. Cells with a posterior assignment probability below 0.8 were excluded from further analysis.COSMX RNA DATASETS: MARKER GENES

[0163] 1ST generates cluster profiles by aggregating counts for each gene within each cluster, and correcting for background (mean negative probe count). To compute marker genes, we normalized these cluster- level counts using the normalizeCounts() function from the scater package, and calculated the log2 fold change between each cluster and the average across remaining clusters (adding a small constant of le-6 to both).COSMX RNA DATASETS: UMAP

[0164] Prior to dimensionality reduction, the count matrices were normalized by total counts and loglp-transformed. Principal Component Analysis (PCA) was performed using the prcompjrlba function from the irlba package (v2.3.5.1), a subset of PCs chosen based on the elbow plot, and Uniform Manifold Approximation and Projection (UMAP) was applied using the umap function from the uwot packagehttps: / / www.zotero.org / google-docs / ?kdpZ3G (vO.2.2).COSMX RNA DATASETS: SIGNATURE SCORING

[0165] The circulating tumor cell mimics signature was derived by subsetting a lOx Genomics Flex dataset containing peripheral blood mononuclear cells (PBMCs) and MCF-7 cells, retaining only genes present in the CosMx IK panel. We then applied the scoreMarkers function from the scran package (vl.32.0) to identify marker genes. The top 100 markers were then selected by highest absolute log2FC and the signature was subsequently scored in the PBMCs-CTCs CosMx dataset using the A UCell_run function from the A UCell package (v 1.26.0).Attorney Docket #: 243734.000229SJ-25 -0018-02COSMX RNA DATASETS: DIFFERENTIATION TRAJECTORY

[0166] After the preprocessing of the hESC samples, we used more strict thresholds to remove low quality cells. More specifically, for the STAMP-C samples we removed cells with fewer and more than 300 and 600 genes and 1500 and 6000 counts, respectively. For the Flex samples we removed cells with fewer and more than 2000 and 1000 genes and 5000 and 60000 counts, respectively. Then, for each time point, we normalized and scaled the data using the NormalizeDataQ and ScaleDataQ functions of Seurat (version 5.0.1) followed by a principal component analysis and clustering using 30 principal components (PCs) and a clustering resolution of 0.1. In the STAMP-C samples, as we expected the distinct cell states to be uniformly distributed across the FOVs, we decided to remove cell clusters that were present in less than half of the FOVs. We then inferred a differentiation trajectory using the Palantir python package (version 1.0.0). For each STAMP-C and Flex data, we first created a subset of -40,000 cells and then computed an augmented affinity matrix using the 10 main PCs. Briefly, Palantir constructs this by augmenting the kNN graph affinity matrix with mutually nearest neighbors between successive time points. This matrix forms the basis to generate a force directed layout for visualization and as input for computing the diffusion operator which can be used for trajectory detection. We generated the force directed layout using 200 iterations. We then clustered the cells using the determine_cell_clusters() function of Palantir using the default parameters and 30 PCs. This generated 16 and 25 cell clusters for the STAMP-C and Flex data respectively, that were aggregated and annotated into nine cell types using canonical gene markers. To generate the Amnion, Endoderm and Mesoderm trajectories we determined the cell of origin and the terminal cells for each trajectory and ran the run_palantir() function with 500 waypoints. Gene expression trends of selected marker genes along the trajectories’ pseudotime were generated with the run_magic_imputation() and compute_gene_trends() functions.XENIUM DATASETS: QUALITY METRICS

[0167] Low-quality cells were filtered out by applying data-driven adaptive thresholds to the distributions of counts, genes, and cell area, following the same approach as with the CosMx datasets.Attorney Docket #: 243734.000229SJ-25 -0018-02XENIUM DATASETS: PRE-PROCESSING

[0168] SingleCellExperiment objects of the Xenium datasets were log-normalized using theogNormCounts function from the scuttle package (vl.14.0). For the Immune-Oncology datasets, no feature selection was performed. PCA was conducted using the fixedPCA function from scran with the parameter BSPARAM=IrlbaParam ). The proportion of variance explained by each principal component guided the selection of components for downstream analyses. UMAPs were then generated using the runUMAP function from the scater package.XENIUM DATASETS: CLUSTERING

[0169] A shared nearest neighbors (SNN) graph was constructed using the buildSNNGraph function from scran, specifying type= "jaccard", k = 50, and BNPARAM=AnnoyParam( ). Louvain clustering was performed on this graph using the cluster_louvain function from the igraph package with resolutions 0.5 or 1 depending on the annotation step. Following the initial round of annotation to identify major lineages, a second round of pre-processing and clustering was conducted within these lineages to achieve more detailed annotation.XENIUM DATASETS: FEATURE SELECTION

[0170] Following an initial annotation round — conducted by applying the aforementioned steps to the entire panel — and only in the 5K panel datasets, we performed feature selection to identify highly variable genes (HVGs) within PBMC lineage subsets. The mean-variance relationship was modeled using the modelGeneVar function from scran, and HVGs were selected using the getTopHVGs function setting the fdr. threshold - 0.9. PCA was then rerun as previously described, incorporating the subset, row parameter to specify the selected HVGs.STAMP PROTEIN DATASETS: COSMX PROTEIN

[0171] Cell segmentation and initial pre-processing were performed using AtoMx. Flat files were downloaded, and background fluorescence was subtracted from each segmented cell to control for non-specific fluorescence signals. Cells with aggregated signals less than 20 or greater than 10,000, as well as those with an area smaller than 40 pm2, were excluded from further analysis. Seurat was used for dimensionality reduction and clustering, selecting all 42 proteins for analysis. Data was normalized by applying centered log ratio transformation (CLR) acrossAttorney Docket #: 243734.000229SJ-25 -0018-02 features. Principal component analysis was performed with nPCs set to 40, and after examining the elbow plot, the top 15 PCs were chosen for constructing the neighborhood graph (sNN, k.param = 20, distance = “euclidean”). Louvain clustering was then conducted with a resolution of 0.8. Cell annotation was achieved by inspecting the expression of each protein in the panel across the identified clusters, resulting in the classification of cells into eight main categories. For quality control of cells profiled in CosMx Protein after Xenium and those profiled in CosMx Protein alone, aggregated expression was calculated for each protein. Using the R packages ggpubr and ggplot2, a scatter plot was generated to compare the average expression of each protein across the different experiments. A linear regression line was added to represent the best-fitting line through the data points, along with Pearson correlation statistics to assess the relationship between the datasets.STAMP PROTEIN DATASETS: AKO YA PHENOCYCLER DATASETS

[0172] Cell segmentation was performed using StarDist in QuPath (v0.5.0) with a custom Groovy script. The following parameters were used for the StarDist function: threshold = 0.5, channel = 0, normalizePercentiles = (1, 99), pixelSize = 0.5, cellExpansion = 5.0, and cellConstrainScale = 1.5. Fluorescence measurements were exported in text format. An expression matrix was constructed, retaining only the mean intensity measurements per channel for each segmented cell. Seurat (v5.1) (REF) was used for quality control and downstream analysis. Cells with a mean aggregated intensity below 200 or an area smaller than 20 pm2were excluded. Data normalization was performed using the centered-log-ratio option of the NormalizeData function in Seurat. All 40 proteins were included in the calculation of 40 principal components, and after reviewing the elbow plot, the top 15 PCs were selected for calculating the neighborhood graph. Louvain clustering was then applied with a resolution of 0.5 for unsupervised clustering.CLAUSES

[0173] The following clauses list non-limiting embodiments of the disclosure:

[0174] Clause 1. A method of preparing a sample, the method comprising: preparing one or more suspensions of fixed cells, nuclei, or combination thereof in a water solution; depositing portions of the one or more suspensions in a plurality of sample regions on a surface of a substrate; evaporating water from the portions of the suspension leaving a respective monolayer of the fixed cells, nuclei, or combination thereof within each sample region of the plurality of sample regionsAttorney Docket #: 243734.000229SJ-25 -0018-02 such that the respective monolayer of a given sample region is separated from the respective monolayer of each of a remainder of the sample regions of the plurality of sample regions.

[0175] Clause 2. The method of clause 1, wherein preparing the one or more suspensions comprises preparing the suspension to have a concentration of fixed cells, nuclei, or combination thereof based at least in part on an area of the respective sample regions, a volume of a respective portion of the suspension deposited in the respective sample regions, and an average size of the fixed cells and / or nuclei.

[0176] Clause 3. The method of clause 1 or 2, comprising: applying a compartmentalizing apparatus comprising a plurality of openings to the surface of the substrate to form a plurality of wells each comprising a bottom and a sidewall such that the bottom comprises a respective sample region of the plurality of sample regions on the surface of the substrate, and the sidewall comprising a surface of a gasket within a respective opening of the plurality of openings, and wherein depositing portions of the suspension in the plurality of sample regions comprises depositing the portions of the suspension into the plurality of wells.

[0177] Clause 4. The method of clause 3, wherein depositing portions of the suspension in the plurality of sample regions comprises loading a quantity of the suspension of fixed cells and / or nuclei into each well of the plurality of wells based at least in part on the cell concentration and the area of the respective sample region.

[0178] Clause 5. The method of any one of clauses 1-4, wherein at least one sample region of the plurality of sample regions comprises a known control sample.

[0179] Clause 6. A method of preparing a sample, the method comprising: preparing a suspension of fixed cells, nuclei, or combination thereof comprising a known control sample in a water solution; depositing the suspension within a first sample region on the substrate; and evaporating water from the suspension leaving a monolayer of the known control sample within the first sample region.

[0180] Clause 7. The method of clause 6, comprising: affixing a test sample to a second sample region on the substrate; and comparing the known control sample to the test sample.

[0181] Clause 8. The method of clause 7, wherein the test sample comprises a tissue sample.

[0182] Clause 9. The method of clause 7, wherein the test sample comprises another respective monolayer of fixed cells and / or nuclei.Attorney Docket #: 243734.000229SJ-25 -0018-02[0183| Clause 10. The method of any one of clauses 6-9, comprising: applying a compartmentalizing apparatus comprising a first opening to the surface of the substrate to form a well comprising a bottom and a sidewall such that the bottom comprises the first sample region on the surface of the substrate and the sidewalls comprises a surface of the gasket within the first opening, wherein depositing the suspension within the first sample region on the substrate comprises loading the suspension into the well.

[0184] Clause 11. A method of preparing a sample, the method comprising: applying a compartmentalizing apparatus comprising one or more openings to a surface of a substrate to form one or more wells each comprising a bottom and a sidewall such that the bottom comprises a respective sample region on the surface of the substrate and the sidewall comprises a surface of the one or more openings to a surface of a substrate to form one or more wells each comprising a bottom and a sidewall such that the bottom comprises a respective sample region on the surface of the substrate and the sidewall comprises a surface of the gasket within a respective opening of the one or more openings within a respective opening of the one or more openings; preparing a suspension of fixed cells, nuclei, or combination thereof in a water solution having a concentration based at least in part on an area of the respective sample region, a volume of a respective well, and an average size of the fixed cells, nuclei, or combination thereof; loading a quantity of the suspension into each well of the one or more wells based at least in part on the concentration and the area of the respective sample region; and evaporating water from the one or more wells leaving a respective monolayer of the fixed cells, nuclei, or combination thereof on the respective sample region of each of the one or more wells.

[0185] Clause 12. The method of any one of clauses 3, 10, or 11 comprising: removing the compartmentalizing apparatus from the substrate.

[0186] Clause 13. The method of any one of clauses 3, 10, 11, or 12, comprising: applying an amino acid coating to the surface of the substrate to form a coated surface, wherein applying the compartmentalizing apparatus to the surface of the substrate comprises applying the compartmentalizing apparatus directly to the coated surface.

[0187] Clause 14. The method of any one of clauses 1-13, comprising: applying a solution of poly-D-lysine to the surface of the substrate at approximately 37 °C for a duration ranging from approximately 1 hour and approximately 20 hours or until evaporation; washing the surface withAttorney Docket #: 243734.000229SJ-25 -0018-02 nuclease-free water to remove any residual poly-D-lysine solution; and drying washed surface prior to applying the suspension to the surface of the substrate.

[0188] Clause 15. The method of clause 14, wherein the solution of Poly-D-Lysine comprises a starting concentration of approximately 0.1 milligram per milliliter and above

[0189] Clause 16. The method of clause 14 or 15, wherein drying the solution on the surface at approximately 37 °C for between approximately 1 hour and approximately 20 hours comprises or until evaporation: baking the substrate with the solution thereon at approximately 37 °C for between approximately 1 hour and approximately 20 hours or until evaporation in a thermocycler; and air drying the solution until almost fully dry.

[0190] Clause 17. The method of any one of clauses 3-5 and 10-16, wherein the compartmentalizing apparatus comprises a plurality of openings such that applying the compartmentalizing apparatus to the surface of the substrate forms a plurality of wells.

[0191] Clause 18. The method of clause 17, wherein the plurality of openings is arranged in a grid.

[0192] Clause 19. The method of any one of clauses 3-5, wherein the compartmentalizing apparatus comprises a single opening configured to utilize any fraction of a viewing area of the surface of the substrate.

[0193] Clause 20. The method of any one of clauses 3-5 and 10-19, wherein the compartmentalizing apparatus comprises silicone, plastic, metal, glass, or combination thereof.

[0194] Clause 21. The method of any one of clauses 3-5 and 10-20, wherein each of the openings of the compartmentalizing apparatus is sized such that the respective sample region of the bottom of each of the one or more wells comprises an area greater than 700 square millimeters.

[0195] Clause 22. The method of any one of clauses 3-5 and 10-20, wherein each of the openings of the compartmentalizing apparatus is sized such that the respective sample region of the bottom of each of the one or more wells comprises an area of between approximately 3 square millimeters and approximately 700 square millimeters.

[0196] Clause 23. The method of clause 22, wherein at least one opening of the one or more openings is sized such that the respective sample region comprises an area of between approximately 3 square millimeters and approximately 15 square millimeters.

[0197] Clause 24. The method of any one of clauses 3-5 and 10-20. wherein at least one of the openings of the compartmentalizing apparatus is sized such that the respective sample region ofAttorney Docket #: 243734.000229SJ-25 -0018-02 the bottom of each of the one or more wells comprises an area between approximately 0.2 square millimeters and about 3 square millimeters.

[0198] Clause 25. The method of any one of clauses 3-5 and 10-24, wherein at least one opening of the compartmentalizing apparatus has a circular shape.

[0199] Clause 26. The method of any one of clauses 3-5 and 10-25, wherein at least one opening of the compartmentalizing apparatus has a rounded square or square shape.

[0200] Clause 27. The method of any one of clauses 3-5 and 10-26, wherein the compartmentalizing apparatus comprises a height of approximately 1 millimeters to approximately 10 millimeters.

[0201] Clause 28. The method of clause 27, wherein the compartmentalizing apparatus comprises a height of approximately 1 millimeters to approximately 3 millimeters.

[0202] Clause 29. The method of any one of clauses 3-5 and 10-26, wherein the compartmentalizing apparatus comprises a height of approximately 0.5 millimeters to approximately 1 millimeters.

[0203] Clause 30. The method of any one of clauses 3-5 and 10-29, wherein the compartmentalizing apparatus is sized according to a viewing area of a spatial molecular imager and a predetermined arrangement of sample regions within the viewing area.

[0204] Clause 31. The method of any one of clauses 1-30, wherein the substrate comprises a glass slide or coverslip.

[0205] Clause 32. The method of any one of clauses 1-26, wherein preparing the suspension comprises: fixing and permeabilizing cells or nuclei in a solution containing one or more of formaldehyde and detergent, or alcohol solutions, thereby , thereby obtaining fixed and permeabilized cells and / or nuclei; washing the fixed cells and / or nuclei in nuclease-free water to remove residual salts and resuspending in a solution of Triton-XlOO in water; counting the fixed cells and / or nuclei: bringing the cell and / or nucleus suspension to a desired working concentration or density at a target cell / nuclei per microliter.

[0206] Clause 33. The method of clause 32, wherein counting the fixed cells and / or nuclei comprises determining the fixed cells and / or nuclei have the desired working concentration or density.Attorney Docket #: 243734.000229SJ-25 -0018-02[0207| Clause 34. The method of any one of clauses 1-33, wherein the suspension comprises peripheral blood mononuclear cells, dissociated cancer cells, differentiated embryonic stem cell cultures, or any combination thereof.

[0208] Clause 35. The method of any one of clauses 1-34, wherein the water solution comprises between approximately 0.01% and approximately 0.2% Triton-X.

[0209] Clause 36. The method of any one of clauses 3-5 and 10-35, further comprising: calculating a target concentration such that a volume of the suspension at the target concentration equal to the volume of the respective well includes a number of fixed cells and / or nuclei predicted to cover a target percentage of total area of the respective sample region with a monolayer of the fixed cells and / or nuclei, wherein the suspension comprises the target concentration.

[0210] Clause 37. The method of clause 36, wherein the target percentage is between approximately 50% and 100%.

[0211] Clause 38. The method of clause 37, wherein the target percentage is between 70% and 100%.

[0212] Clause 39. The method of any one of clauses 36-38, wherein the target cell concentration is between approximately 1,000 cells per microliter and approximately 3,000 cells per microliter.

[0213] Clause 40. The method of clause 39, wherein the target cell concentration is approximately 2,500 cells per microliter.

[0214] Clause 41. The method of any one of clauses 36-40, wherein the one or more openings of the compartmentalizing apparatus comprises a first opening comprising a first area and a second opening comprising a second area different than the first area such that applying the compartmentalizing apparatus to the surface of the substrate forms a first well and a second well and such that the area of the respective sample region of the first well is different than the area of the respective sample region of the second well and the volume of the first well is different than the volume of the second well, and wherein calculating the target concentration comprises calculating a first target concentration based at least in part on the volume of the first well and the area of the respective sample region of the first well and calculating a second target concentration based at least in part on the volume of the second well and the area of the respective sample region of the second well.

[0215] Clause 42. The method of any one of clauses 36-41, wherein preparing the suspension comprises preparing a first suspension of fixed cells, nuclei, or combination thereof comprising aAttorney Docket #: 243734.000229SJ-25 -0018-02 first average size and preparing a second suspension of fixed cells, nuclei, or combination thereof of a second average size different from the first average size, wherein calculating the target concentration comprises calculating a first target concentration based at least in part on the first average size and calculating a second target concentration based at least in part on the second average size.

[0216] Clause 43. The method of any one of clauses 3-5 and 10-42, wherein the volume of the respective well is between approximately 5 microliters and approximately 500 microliters.

[0217] Clause 44. The method of clause 43, wherein the volume of the respective well is approximately 10 microliters.

[0218] Clause 45. The method of any one of clauses 3-5 and 10-44, wherein the quantity of suspension loaded into each well of the one or more wells includes a respective number of the fixed cells and / or nuclei determined to cover a target percentage of the respective sample region of the bottom of each well of the one or more wells.

[0219] Clause 46. The method of any one of clauses 3-5 and 10-44, wherein the quantity of suspension loaded into each well of the one or more wells is approximately equal to the volume of the respective well.

[0220] Clause 47. The method of any one of clauses 3-5 and 10-46, wherein loading the quantity of the suspension into each well of the one or more wells comprises entirely filling the volume of each of the respective wells.

[0221] Clause 48. The method of clause 47, wherein loading the quantity of the suspension into each well of the one or more wells comprises forming a surface tension dome of the suspension of individual fixed cells and / or nuclei such that the surface tension dome extends above the sidewall.

[0222] Clause 49. The method of any one of clauses 3-5 and 6-48, loading a quantity of the suspension of individual fixed cells and / or nuclei into each well of the one or more wells comprises loading at least one well of the one or more wells with a known control sample.

[0223] Clause 50. The method of any one of clauses 1-49, wherein evaporating water from the one or more wells comprises: placing a loaded substrate into a thermocycler and cooling in an initial ambient temperature of between about 4 °C and about 15 °C.

[0224] Clause 51. The method of clause 50, wherein the initial ambient temperature is about 4 °C.Attorney Docket #: 243734.000229SJ-25 -0018-02[0225| Clause 52. The method of clause 50 or 51 , wherein evaporating water from the one or more wells comprises: placing a loaded substrate into a thermocycler and initially cooling in the initial ambient temperature for about 30 minutes.

[0226] Clause 53. The method of any one of clauses 50-52, wherein evaporating water from the one or more wells comprising: increasing temperature of the thermocycler from the initial ambient temperature to an intermediate ambient temperature between about 20 °C to about 30 °C for between about 2 minutes and about 10 minutes; and increasing temperature of the thermocycler from the intermediate ambient temperature to a drying temperature of between about 35 °C and about 50 °C and holding at least until water of the suspension is essentially entirely evaporated.

[0227] Clause 54. The method of clause 53, wherein evaporating water from the one or more wells comprises: placing a loaded substrate into a thermocycler and initially cooling in the initial ambient temperature for about 30 minutes, wherein the initial ambient temperature is about 4 °C, and wherein the loaded substrate comprises the substrate, gasket, and suspension in each well of the one or more wells; increasing temperature of the thermocycler from the initial ambient temperature to the intermediate ambient temperature for about 5 minutes, wherein the intermediate ambient temperature is about 25 °C; and increasing temperature of the thermocycler from the intermediate ambient temperature to the drying temperature and holding for approximately 2 hours after water of the suspension is essentially entirely evaporated, wherein the drying temperature is about 42 °C.

[0228] Clause 55. The method of any one of clauses 11-54, wherein removing the compartmentalizing apparatus from the substrate comprises peeling the compartmentalizing apparatus away from the substrate.

[0229] Clause 56. The method of any one of clauses 11-49, wherein removing the compartmentalizing apparatus from the substrate results in the sample comprising the substrate and one or more sample regions each comprising a respective monolayer of the fixed cells, nuclei, or combination thereof.

[0230] Clause 57. The method of clause 56, further comprising: hydrating the sample in a flow cell assembly such that fluid flows over each of the one or more sample regions.

[0231] Clause 58. A kit comprising: a substrate; fixative and permeabilization buffer; RNAse inhibitor; resuspension reagent; a coating configured to provide an adhesive coating to theAttorney Docket #: 243734.000229SJ-25 -0018-02 substrate; and a compartmentalizing apparatus comprising a plurality of openings and shaped to cover a predetermined area of a surface of the substrate.

[0232] Clause 59. The kit of clause 58, wherein the coating comprises poly-D-lysine.

[0233] Clause 60. The kit of clause 58 or 59, wherein the compartmentalizing apparatus comprises silicone, plastic, metal, glass, or combination thereof.

[0234] Clause 61. The kit of any one of clauses 58-60. wherein the compartmentalizing apparatus comprises one or more openings, and wherein the compartmentalizing apparatus is configured to be applied to a surface of a substrate to form one or more wells each comprising a bottom and a sidewall such that the bottom comprises a respective sample region on the surface of the substrate and the sidewall comprises a surface of the gasket within a respective opening of the one or more openings.

[0235] Clause 62. The kit of clause 61, wherein each of the openings comprises an area greater than 700 square millimeters.

[0236] Clause 63. The kit of clause 61. wherein each of the openings comprises an area between approximately 3 square millimeters and approximately 700 square millimeters.

[0237] Clause 64. The kit of clause 63, wherein at least one of the openings comprises an area of between approximately 3 square millimeters and approximately 15 square millimeters.

[0238] Clause 65. The kit of clause 61, wherein at least one of the openings comprises an area between approximately 0.2 square millimeters and about 3 square millimeters.

[0239] Clause 66. The kit of any one of clauses 61-65, wherein at least one of the openings has a circular shape.

[0240] Clause 67. The kit of any one of clauses 61-66, wherein at least one of the openings has a rounded square shape.

[0241] Clause 68. The kit of any one of clauses 58-67, wherein the compartmentalizing apparatus comprises a height of approximately 1 millimeters to approximately 10 millimeters.

[0242] Clause 69. The kit of clause 68, wherein the compartmentalizing apparatus comprises a height of approximately 1 millimeters to approximately 3 millimeters.

[0243] Clause 70. The kit of any one of clauses 58-67, wherein the compartmentalizing apparatus comprises a height of approximately 0.5 millimeters to approximately 1 millimeters.Attorney Docket #: 243734.000229SJ-25 -0018-02[0244| Clause 71. The kit of any one of clauses 58-70, wherein the compartmentalizing apparatus is sized according to a viewing area of a spatial molecular imager and a predetermined arrangement of sample regions within the viewing area.

[0245] Clause 72. The kit of any one of clauses 58-71, wherein the substrate comprises a glass slide or coverslip.

[0246] Clause 73. A sample preparation comprising: a substrate; and a plurality of sample regions disposed at predetermined locations on the substrate, each sample region of the plurality of sample regions comprising a respective monolayer of fixed cells, nuclei, or combination thereof.

[0247] Clause 74. The sample preparation of clause 73, wherein the respective monolayer comprises between approximately 1,000 fixed cells and / or nuclei per square millimeter and approximately 20,000 fixed cells and / or nuclei per square millimeter within each sample region of the plurality of sample regions.

[0248] Clause 75. A sample preparation comprising: a substrate; and at least one sample region on the substrate, each sample region of the at least one sample regions comprising a respective monolayer of fixed cells, nuclei, or combination thereof, wherein the respective monolayer comprises between approximately 1,000 fixed cells and / or nuclei per square millimeter and approximately 20,000 fixed cells and / or nuclei per square millimeter within each sample region.

[0249] Clause 76. The sample preparation of any one of clauses 73-75, wherein at sample region of the at least one sample region comprises a known control sample.

[0250] Clause 77. The sample preparation of clause 76, further comprising a test sample mounting region configured to receive a test sample for comparison against the known control sample.

[0251] Clause 78. A sample preparation comprising: a substrate; and a first sample region comprising a known control sample comprising a respective monolayer of fixed cells, nuclei, or combination thereof on the substrate; and a test sample mounting region configured to receive a test sample on the substrate for comparison against the known control sample.

[0252] Clause 79. The sample preparation of any one of clauses 75-78, comprising: a tissue sample affixed to the substrate on the test sample mounting region.

[0253] Clause 80. The sample preparation of any one of clauses 73-79, wherein the substrate comprises a coated surface comprising a poly-D-lysine coating.Attorney Docket #: 243734.000229SJ-25 -0018-02[0254| Clause 81. The sample preparation of any one of clauses 73-80, wherein a majority of the fixed cells and / or nuclei in each sample region are accessible for hybridization with a probe configured to bind cellular mRNA while remaining fixed to the substrate.

[0255] Clause 82. The sample preparation of any one of clauses 73-81, wherein a respective monolayer of at least one sample region comprises between approximately 10,000 and approximately 20,000 fixed cells and / or nuclei per square millimeter.

[0256] Clause 83. The sample preparation of clause 82, wherein the respective monolayer comprises between approximately 18,000 and approximately 20,000 fixed cells and / or nuclei per square millimeter.

[0257] Clause 84. The sample preparation of any one of clauses 73-83, wherein the sample region(s) comprise a total sum of between approximately 1,000,000 and approximately 4,000,000 total fixed cells and / or nuclei.

[0258] Clause 85. The sample preparation of any one of clauses 73-84, wherein at least one sample region comprises an area greater than 700 square millimeters.

[0259] Clause 86. The sample preparation of any one of clauses 73-84, wherein each sample region comprises an area of between approximately 3 square millimeters and approximately 700 square millimeters.

[0260] Clause 87. The sample preparation of clause 86, wherein at least one sample region comprises an area of between approximately 3 square millimeters and approximately 15 square millimeters.

[0261] Clause 88. The sample preparation of any one of clauses 73-87, wherein the sample regions are arranged in a grid.

[0262] Clause 89. The sample preparation of any one of clauses 73-88, wherein at least one sample has a circular shape.

[0263] Clause 90. The sample preparation of any one of clauses 73-89, wherein at least one sample has a rounded square or square shape.

[0264] Clause 91. The sample preparation of any one of clauses 73-90, wherein the fixed cells and / or nuclei within each sample region are uniformly distributed over a majority of a respective area of each sample region.Attorney Docket #: 243734.000229SJ-25 -0018-02[0265| Clause 92. The sample preparation of any one of clauses 73-91 , wherein the majority of the fixed cells and / or nuclei in each sample region are configured to remain affixed to the substrate during transcriptomics and / or proteomics imaging.

[0266] Clause 93. The sample preparation of any one of clauses 73-92, wherein the respective monolayer of at least one sample region comprises peripheral blood mononuclear cells, dissociated cancer cells, dissociated cells from tissues, differentiated embryonic stem cell cultures, cells from a perturbation assay or any combination thereof.

[0267] Clause 94. Non-transitory computer readable medium with instructions thereon that, when executed by one or more processors cause the one or more processors to: receive an identification of a multi-sample substrate preparation; partition a viewing area into a plurality of predetermined sample regions based at least in part on the identification; and image at least a portion of the viewing area inside the plurality of predetermined sample regions while excluding at least a portion of the viewing area outside of the predetermined sample regions.

[0268] Clause 95. The non-transitory computer readable medium of clause 94, wherein the instructions, when executed by the one or more processors cause the one or more processors to: distinguish data from each of the plurality of predetermined sample regions as individual, separable data sets.

[0269] Clause 96. The non-transitory computer readable medium of clause 95, wherein the instructions, when executed by the one or more processors cause the one or more processors to: save data from each of the plurality of predetermined sample regions as separate data files.

[0270] Clause 97. The non-transitory computer readable medium of clause 95 or 96, wherein the separable data sets comprise a plurality of gene counts per cell / nuclei datasets each associated with a respective predetermined sample region of the plurality of predetermined sample regions.

[0271] Clause 98. The non-transitory computer readable medium of any one of clauses 94-97, wherein the instructions, when executed by the one or more processors cause the one or more processors to: identify a control sample region among the plurality of predetermined sample regions.

[0272] Clause 99. The non-transitory computer readable medium of clause 98, wherein the instructions, when executed by the one or more processors cause the one or more processors to: calibrate, based at least in part on a dataset associated with the control sample region, respectiveAttorney Docket #: 243734.000229SJ-25 -0018-02 dataset(s) associated with a portion of the plurality of predetermined sample regions excluding the control sample region.

[0273] Clause 100. The non-transitory computer readable medium of any one of clauses 94-99, wherein the instructions, when executed by the one or more processors cause the one or more processors to: provide, to a user via a user interface, the identification as one of a plurality of predetermined sample identifications; and receive, from the user interface, the identification as a selection of one of the plurality of predetermined sample identifications.

[0274] Clause 101. The non-transitory computer readable medium of any one of clauses 94-100, wherein the instructions, when executed by the one or more processors cause the one or more processors to: image the entirety of each of the predetermined sample regions.

[0275] Clause 102. The non-transitory computer readable medium of clause 94-101, wherein the instructions, when executed by the one or more processors cause the one or more processors to: image at least a majority of the viewing area inside the predetermined sample regions while excluding at least a majority of the viewing area outside of the predetermined sample regions.

[0276] Clause 103. A method of preparing a sample, the method comprising: growing or culturing at least one of cells or nuclei on a surface of a substrate; fixing the at least one of cells or nuclei on the substrate to form a monolayer of fixed cells or nuclei within one or more sample regions; and analyzing the fixed cells or nuclei using one of (i) imaging-based transcriptomics or (ii) proteomics or sequencing technologies.

[0277] Clause 104. The method of clause 103 further comprising applying a compartmentalizing apparatus comprising a plurality of openings to a surface of the substrate to form a plurality of wells each comprising a bottom and a sidewall such that the bottom comprises a respective sample region of the one or more sample regions on the surface of the substrate, and the sidewall comprising a surface of the gasket within a respective opening of the plurality of openings.

[0278] Clause 105. A method of analyzing samples comprising: depositing a plurality of distinct samples onto a single slide comprising a plurality of spatial regions, a first spatial region comprising a first sample, and a second spatial region comprising a second sample comprising a different type of cell or nuclei than the first sample; applying a multiplex panel of target-binding reagents to the first sample and the second sample; and performing multi-cycle imaging on the single slide.Attorney Docket #: 243734.000229SJ-25 -0018-02[0279| Clause 106. The method of clause 105 further comprising associating each of the first sample and the second sample with a unique identifier comprising at least one of a spatial region map or a nucleic-acid sample barcode readable during cyclic imaging.

[0280] Clause 107. The method of clause 106 further comprising demultiplexing the per-cell feature measurements to assign each cell identified to its originating first sample or second sample using the unique identifier.

[0281] Clause 108. The method of any of clauses 105 to 107, wherein the type of cell or nuclei of the first sample and the second sample comprises at least one of peripheral blood mononuclear cells, dissociated cancer cells, dissociated cells from tissues, differentiated embryonic stem cell cultures, cells from a perturbation assay or any combination or nuclei thereof.

[0282] Clause 109. The method of any of clauses 105 to 108, wherein performing multi-cycle imaging generates per-cell feature measurements.

[0283] Clause 110. The method of any of clauses 105 to 109, wherein depositing the plurality of distinct samples comprises depositing the first sample and the second sample into a compartmentalizing apparatus comprising a plurality of wells, each well comprising a bottom and a sidewall such that the bottom comprises a respective spatial region of the plurality of spatial regions, and the sidewall comprising a surface of a gasket.

[0284] Clause 111. The method of clause 110 wherein depositing the plurality of distinct samples comprises: preparing a suspension of fixed cells, nuclei, or combination thereof; depositing the suspension within the first spatial region or the second spatial region; and evaporating water from the suspension leaving a monolayer of the first sample or the second sample.

[0285] Clause 112. The method any of clauses 105 to 111, wherein performing multi-cycle imaging comprises using at least two different imaging platforms.

[0286] Clause 113. The method of clause 112, wherein the at least two different imaging platforms are configured to support one of RNA profiling or protein profiling.

[0287] Clause 114. The method of clause 112, wherein the at least two different imaging platforms are configured to support RNA profiling and protein profiling.

[0288] Having shown and described exemplary embodiments of the subject matter contained herein, further adaptations of the methods and systems described herein may be accomplished by appropriate modifications without departing from the scope of the claims. In addition, whereAttorney Docket #: 243734.000229SJ-25 -0018-02 methods and steps described above indicate certain events occurring in certain order, it is intended that certain steps do not have to be performed in the order described, but in any order, as long as the steps allow the embodiments to function for their intended purposes. Further, steps from various methods and processes associated with various example embodiments can be combined when compatible as understood by a person skilled in the pertinent art informed by the disclosure herein. Therefore, to the extent there are variations of the invention, which are within the spirit of the disclosure or equivalent to the inventions found in the claims, it is the intent that this patent will cover those variations as well. Some such modifications should be apparent to those skilled in the art. For instance, the examples, embodiments, geometries, materials, dimensions, ratios, steps, and the like discussed above are illustrative. Accordingly, the claims should not be limited to the specific details of structure and operation set forth in the written description and drawings.

Claims

Attorney Docket #: 243734.000229SJ-25 -0018-02CLAIMSWhat is claimed is:

1. A method of preparing a sample, the method comprising: preparing one or more suspensions of fixed cells, nuclei, or combination thereof in a water solution; depositing portions of the one or more suspensions in a plurality of sample regions on a surface of a substrate; and evaporating water from the portions of the suspension leaving a respective monolayer of the fixed cells, nuclei, or combination thereof within each sample region of the plurality of sample regions such that the respective monolayer of a given sample region is separated from the respective monolayer of each of a remainder of the sample regions of the plurality of sample regions.

2. The method of claim 1, wherein preparing the one or more suspensions comprises preparing the suspension to have a concentration of fixed cells, nuclei, or combination thereof based at least in part on an area of the respective sample regions, a volume of a respective portion of the suspension deposited in the respective sample regions, and an average size of the fixed cells and / or nuclei.

3. The method of claim 1 or 2, comprising: applying a compartmentalizing apparatus comprising a plurality of openings to the surface of the substrate to form a plurality of wells each comprising a bottom and a sidewall such that the bottom comprises a respective sample region of the plurality of sample regions on the surface of the substrate, and the sidewall comprising a surface of a gasket within a respective opening of the plurality of openings, and wherein depositing portions of the suspension in the plurality of sample regions comprises depositing the portions of the suspension into the plurality of wells.

4. The method of claim 3, wherein depositing portions of the suspension in the plurality of sample regions comprises loading a quantity of the suspension of fixed cells and / or nuclei into each well of the plurality of wells based at least in part on the cell concentration and the area of the respective sample region.Attorney Docket #: 243734.000229SJ-25 -0018-025. The method of any one of claims 1-4, wherein at least one sample region of the plurality of sample regions comprises a known control sample.

6. A method of preparing a sample, the method comprising: preparing a suspension of fixed cells, nuclei, or combination thereof comprising a known control sample in a water solution; depositing the suspension within a first sample region on the substrate; and evaporating water from the suspension leaving a monolayer of the known control sample within the first sample region.

7. The method of claim 6, comprising: affixing a test sample to a second sample region on the substrate; and comparing the known control sample to the test sample.

8. The method of claim 7, wherein the test sample comprises a tissue sample.

9. The method of claim 7, wherein the test sample comprises another respective monolayer of fixed cells and / or nuclei.

10. The method of any one of claims 6-9, comprising: applying a compartmentalizing apparatus comprising a first opening to the surface of the substrate to form a well comprising a bottom and a sidewall such that the bottom comprises the first sample region on the surface of the substrate and the sidewalls comprises a surface of the gasket within the first opening, wherein depositing the suspension within the first sample region on the substrate comprises loading the suspension into the well.

11. A method of preparing a sample, the method comprising: applying a compartmentalizing apparatus comprising one or more openings to a surface of a substrate to form one or more wells each comprising a bottom and a sidewall such that the bottom comprises a respective sample region on the surface of the substrate and the sidewall comprises a surface of the one or more openings to a surface of a substrate to form one or more wells each comprising a bottom and a sidewall such that the bottom comprises a respective sample region on the surface of the substrate and the sidewall comprises a surface of the gasket within a respective opening of the one or more openings within a respective opening of the one or more openings;Attorney Docket #: 243734.000229SJ-25 -0018-02 preparing a suspension of fixed cells, nuclei, or combination thereof in a water solution having a concentration based at least in part on an area of the respective sample region, a volume of a respective well, and an average size of the fixed cells, nuclei, or combination thereof; loading a quantity of the suspension into each well of the one or more wells based at least in part on the concentration and the area of the respective sample region; and evaporating water from the one or more wells leaving a respective monolayer of the fixed cells, nuclei, or combination thereof on the respective sample region of each of the one or more wells.

12. The method of any one of claims 3, 10, or 11 comprising: removing the compartmentalizing apparatus from the substrate.

13. The method of any one of claims 3, 10, 11, or 12, comprising: applying an amino acid coating to the surface of the substrate to form a coated surface, wherein applying the compartmentalizing apparatus to the surface of the substrate comprises applying the compartmentalizing apparatus directly to the coated surface.

14. The method of any one of claims 1-13, comprising: applying a solution of poly-D-lysine to the surface of the substrate at approximately 37 °C for a duration ranging from approximately 1 hour and approximately 20 hours or until evaporation; washing the surface with nuclease-free water to remove any residual poly-D-lysine solution; and drying washed surface prior to applying the suspension to the surface of the substrate.

15. The method of claim 14, wherein the solution of Poly-D-Lysine comprises a starting concentration of approximately 1 milligram per milliliter.

16. The method of claim 14 or 15, wherein drying the solution on the surface at approximately 37 °C for between approximately 1 hour and approximately 20 hours comprises or until evaporation: baking the substrate with the solution thereon at approximately 37 °C for between approximately 1 hour and approximately 20 hours or until evaporation in a thermocycler; and air drying the solution until almost fully dry.Attorney Docket #: 243734.000229SJ-25 -0018-0217. The method of any one of claims 3-5 and 10-16, wherein the compartmentalizing apparatus comprises a plurality of openings such that applying the compartmentalizing apparatus to the surface of the substrate forms a plurality of wells.

18. The method of claim 17, wherein the plurality of openings is arranged in a grid.

19. The method of any one of claims 3-5, wherein the compartmentalizing apparatus comprises a single opening configured to utilize any fraction of a viewing area of the surface of the substrate.

20. The method of any one of claims 3-5 and 10-19, wherein the compartmentalizing apparatus comprises silicone, plastic, metal, glass, or combination thereof.

21. The method of any one of claims 3-5 and 10-20, wherein each of the openings of the compartmentalizing apparatus is sized such that the respective sample region of the bottom of each of the one or more wells comprises an area greater than 700 square millimeters.

22. The method of any one of claims 3-5 and 10-20, wherein each of the openings of the compartmentalizing apparatus is sized such that the respective sample region of the bottom of each of the one or more wells comprises an area of between approximately 3 square millimeters and approximately 700 square millimeters.

23. The method of claim 22, wherein at least one opening of the one or more openings is sized such that the respective sample region comprises an area of between approximately 3 square millimeters and approximately 15 square millimeters.

24. The method of any one of claims 3-5 and 10-20, wherein at least one of the openings of the compartmentalizing apparatus is sized such that the respective sample region of the bottom of each of the one or more wells comprises an area between approximately 0.2 square millimeters and about 3 square millimeters.

25. The method of any one of claims 3-5 and 10-24, wherein at least one opening of the compartmentalizing apparatus has a circular shape.

26. The method of any one of claims 3-5 and 10-25, wherein at least one opening of the compartmentalizing apparatus has a rounded square or square shape.

27. The method of any one of claims 3-5 and 10-26, wherein the compartmentalizing apparatus comprises a height of approximately 1 millimeters to approximately 10 millimeters.

28. The method of claim 27, wherein the compartmentalizing apparatus comprises a height of approximately 1 millimeters to approximately 3 millimeters.Attorney Docket #: 243734.000229SJ-25 -0018-0229. The method of any one of claims 3-5 and 10-26, wherein the compartmentalizing apparatus comprises a height of approximately 0.5 millimeters to approximately 1 millimeters.

30. The method of any one of claims 3-5 and 10-29, wherein the compartmentalizing apparatus is sized according to a viewing area of a spatial molecular imager and a predetermined arrangement of sample regions within the viewing area.

31. The method of any one of claims 1-30. wherein the substrate comprises a glass slide or coverslip.

32. The method of any one of claims 1-26, wherein preparing the suspension comprises: fixing and permeabilizing cells or nuclei in a solution containing one or more of formaldehyde and detergent, or alcohol solutions, thereby obtaining fixed and permeabilized cells and / or nuclei; washing the fixed cells and / or nuclei in nuclease-free water to remove residual salts and resuspending in a solution of Triton-XlOO in water; counting the fixed cells and / or nuclei; and bringing the cell and / or nucleus suspension to a desired working concentration or density at a target cell / nuclei per microliter.

33. The method of claim 32, wherein counting the fixed cells and / or nuclei comprises determining the fixed cells and / or nuclei have the desired working concentration or density.

34. The method of any one of claims 1-33, wherein the suspension comprises peripheral blood mononuclear cells, dissociated cancer cells, differentiated embryonic stem cell cultures, or any combination thereof.

35. The method of any one of claims 1-34, wherein the water solution comprises between approximately 0.01% and approximately 0.2% Triton-X.

36. The method of any one of claims 3-5 and 10-35, further comprising: calculating a target concentration such that a volume of the suspension at the target concentration equal to the volume of the respective well includes a number of fixed cells and / or nuclei predicted to cover a target percentage of total area of the respective sample region with a monolayer of the fixed cells and / or nuclei, wherein the suspension comprises the target concentration.

37. The method of claim 36, wherein the target percentage is between approximately 50% and 100%.Attorney Docket #: 243734.000229SJ-25 -0018-0238. The method of claim 37, wherein the target percentage is between 70% and 100%.

39. The method of any one of claims 36-38, wherein the target cell concentration is between approximately 1.000 cells per microliter and approximately 3,000 cells per microliter.

40. The method of claim 39, wherein the target cell concentration is approximately 2,500 cells per microliter.

41. The method of any one of claims 36-40. wherein the one or more openings of the compartmentalizing apparatus comprises a first opening comprising a first area and a second opening comprising a second area different than the first area such that applying the compartmentalizing apparatus to the surface of the substrate forms a first well and a second well and such that the area of the respective sample region of the first well is different than the area of the respective sample region of the second well and the volume of the first well is different than the volume of the second well, and wherein calculating the target concentration comprises calculating a first target concentration based at least in part on the volume of the first well and the area of the respective sample region of the first well and calculating a second target concentration based at least in part on the volume of the second well and the area of the respective sample region of the second well.

42. The method of any one of claims 36-41, wherein preparing the suspension comprises preparing a first suspension of fixed cells, nuclei, or combination thereof comprising a first average size and preparing a second suspension of fixed cells, nuclei, or combination thereof of a second average size different from the first average size, wherein calculating the target concentration comprises calculating a first target concentration based at least in part on the first average size and calculating a second target concentration based at least in part on the second average size.

43. The method of any one of claims 3-5 and 10-42, wherein the volume of the respective well is between approximately 5 microliters and approximately 500 microliters.

44. The method of claim 43, wherein the volume of the respective well is approximately 10 microliters.

45. The method of any one of claims 3-5 and 10-44, wherein the quantity of suspension loaded into each well of the one or more wells includes a respective number of the fixed cells and / or nucleiAttorney Docket #: 243734.000229SJ-25 -0018-02 determined to cover a target percentage of the respective sample region of the bottom of each well of the one or more wells.

46. The method of any one of claims 3-5 and 10-44, wherein the quantity of suspension loaded into each well of the one or more wells is approximately equal to the volume of the respective well.

47. The method of any one of claims 3-5 and 10-46, wherein loading the quantity of the suspension into each well of the one or more wells comprises entirely filling the volume of each of the respective wells.

48. The method of claim 47, wherein loading the quantity of the suspension into each well of the one or more wells comprises forming a surface tension dome of the suspension of individual fixed cells such that the surface tension dome extends above the sidewall.

49. The method of any one of claims 3-5 and 6-48, loading a quantity of the suspension of individual fixed cells into each well of the one or more wells comprises loading at least one well of the one or more wells with a known control sample.

50. The method of any one of claims 1-49, wherein evaporating water from the one or more wells comprises: placing a loaded substrate into a thermocycler and cooling in an initial ambient temperature of between about 4 °C and about 15 °C.

51. The method of claim 50, wherein the initial ambient temperature is about 4 °C.

52. The method of claim 50 or 51, wherein evaporating water from the one or more wells comprises: placing a loaded substrate into a thermocycler and initially cooling in the initial ambient temperature for about 30 minutes.

53. The method of any one of claims 50-52, wherein evaporating water from the one or more wells comprising: increasing temperature of the thermocycler from the initial ambient temperature to an intermediate ambient temperature between about 20 °C to about 30 °C for between about 2 minutes and about 10 minutes; and increasing temperature of the thermocycler from the intermediate ambient temperature to a drying temperature of between about 35 °C and about 50 °C and holding at least until water of the suspension is essentially entirely evaporated.Attorney Docket #: 243734.000229SJ-25 -0018-0254. The method of claim 53, wherein evaporating water from the one or more wells comprises: placing a loaded substrate into a thermocycler and initially cooling in the initial ambient temperature for about 30 minutes, wherein the initial ambient temperature is about 4 °C, and wherein the loaded substrate comprises the substrate, gasket, and suspension in each well of the one or more wells; increasing temperature of the thermocycler from the initial ambient temperature to the intermediate ambient temperature for about 5 minutes, wherein the intermediate ambient temperature is about 25 °C; and increasing temperature of the thermocycler from the intermediate ambient temperature to the drying temperature and holding for approximately 2 hours after water of the suspension is essentially entirely evaporated, wherein the drying temperature is about 42 °C.

55. The method of any one of claims 11-54, wherein removing the compartmentalizing apparatus from the substrate comprises peeling the compartmentalizing apparatus away from the substrate.

56. The method of any one of claims 11-49, wherein removing the compartmentalizing apparatus from the substrate results in the sample comprising the substrate and one or more sample regions each comprising a respective monolayer of the fixed cells, nuclei, or combination thereof.

57. The method of claim 56, further comprising: hydrating the sample in a flow cell assembly such that fluid flows over each of the one or more sample regions.

58. A kit comprising : a substrate; fixative and permeabilization buffer;RNAse inhibitor; resuspension reagent; a coating configured to provide an adhesive coating to the substrate; and a compartmentalizing apparatus comprising a plurality of openings and shaped to cover a predetermined area of a surface of the substrate.

59. The kit of claim 58, wherein the coating comprises poly-D-lysine.

60. The kit of claim 58 or 59, wherein the compartmentalizing apparatus comprises silicone, plastic, metal, glass, or combination thereof.Attorney Docket #: 243734.000229SJ-25 -0018-0261. The kit of any one of claims 58-60, wherein the compartmentalizing apparatus comprises one or more openings, and wherein the compartmentalizing apparatus is configured to be applied to a surface of a substrate to form one or more wells each comprising a bottom and a sidewall such that the bottom comprises a respective sample region on the surface of the substrate and the sidewall comprises a surface of the gasket within a respective opening of the one or more openings.

62. The kit of claim 61 , wherein each of the openings comprises an area greater than 700 square millimeters.

63. The kit of claim 61, wherein each of the openings comprises an area between approximately 3 square millimeters and approximately 700 square millimeters.

64. The kit of claim 63, wherein at least one of the openings comprises an area of between approximately 3 square millimeters and approximately 15 square millimeters.

65. The kit of claim 61, wherein at least one of the openings comprises an area between approximately 0.2 square millimeters and about 3 square millimeters.

66. The kit of any one of claims 61-65, wherein at least one of the openings has a circular shape.

67. The kit of any one of claims 61-66, wherein at least one of the openings has a rounded square shape.

68. The kit of any one of claims 58-67, wherein the compartmentalizing apparatus comprises a height of approximately 1 millimeters to approximately 10 millimeters.

69. The kit of claim 68, wherein the compartmentalizing apparatus comprises a height of approximately 1 millimeters to approximately 3 millimeters.

70. The kit of any one of claims 58-67, wherein the compartmentalizing apparatus comprises a height of approximately 0.5 millimeters to approximately 1 millimeters.

71. The kit of any one of claims 58-70, wherein the compartmentalizing apparatus is sized according to a viewing area of a spatial molecular imager and a predetermined arrangement of sample regions within the viewing area.

72. The kit of any one of claims 58-71, wherein the substrate comprises a glass slide or coverslip.

73. A sample preparation comprising: a substrate; andAttorney Docket #: 243734.000229SJ-25 -0018-02 a plurality of sample regions disposed at predetermined locations on the substrate, each sample region of the plurality of sample regions comprising a respective monolayer of fixed cells, nuclei, or combination thereof.

74. The sample preparation of claim 73, wherein the respective monolayer comprises between approximately 1,000 fixed cells and / or nuclei per square millimeter and approximately 20,000 fixed cells and / or nuclei per square millimeter within each sample region of the plurality of sample regions.

75. A sample preparation comprising: a substrate; and at least one sample region on the substrate, each sample region of the at least one sample regions comprising a respective monolayer of fixed cells, nuclei, or combination thereof, wherein the respective monolayer comprises between approximately 1,000 fixed cells and / or nuclei per square millimeter and approximately 20,000 fixed cells and / or nuclei per square millimeter within each sample region.

76. The sample preparation of any one of claims 73-75, wherein at sample region of the at least one sample region comprises a known control sample.

77. The sample preparation of claim 76, further comprising a test sample mounting region configured to receive a test sample for comparison against the known control sample.

78. A sample preparation comprising: a substrate; and a first sample region comprising a known control sample comprising a respective monolayer of fixed cells, nuclei, or combination thereof on the substrate; and a test sample mounting region configured to receive a test sample on the substrate for comparison against the known control sample.

79. The sample preparation of any one of claims 75-78, comprising: a tissue sample affixed to the substrate on the test sample mounting region.

80. The sample preparation of any one of claims 73-79, wherein the substrate comprises a coated surface comprising a poly-D-lysine coating.

81. The sample preparation of any one of claims 73-80, wherein a majority of the fixed cells and / or nuclei in each sample region are accessible for hybridization with a probe configured to bind cellular mRNA while remaining fixed to the substrate.Attorney Docket #: 243734.000229SJ-25 -0018-0282. The sample preparation of any one of claims 73-81 , wherein a respective monolayer of at least one sample region comprises between approximately 10,000 and approximately 20,000 fixed cells and / or nuclei per square millimeter.

83. The sample preparation of claim 82, wherein the respective monolayer comprises between approximately 18,000 and approximately 20,000 fixed cells and / or nuclei per square millimeter.

84. The sample preparation of any one of claims 73-83. wherein the sample region(s) comprise a total sum of between approximately 1,000,000 and approximately 4,000,000 total fixed cells and / or nuclei.

85. The sample preparation of any one of claims 73-84, wherein at least one sample region comprises an area greater than 700 square millimeters.

86. The sample preparation of any one of claims 73-84, wherein each sample region comprises an area of between approximately 3 square millimeters and approximately 700 square millimeters.

87. The sample preparation of claim 86, wherein at least one sample region comprises an area of between approximately 3 square millimeters and approximately 15 square millimeters.

88. The sample preparation of any one of claims 73-87, wherein the sample regions are arranged in a grid.

89. The sample preparation of any one of claims 73-88, wherein at least one sample has a circular shape.

90. The sample preparation of any one of claims 73-89, wherein at least one sample regions has a rounded square shape.

91. The sample preparation of any one of claims 73-90, wherein the fixed cells and / or nuclei within each sample region are uniformly distributed over a majority of a respective area of each sample region.

92. The sample preparation of any one of claims 73-91, wherein the majority of the fixed cells and / or nuclei in each sample region are configured to remain affixed to the substrate during transcriptomics and / or proteomics imaging.

93. The sample preparation of any one of claims 73-92, wherein the respective monolayer of at least one sample region comprises peripheral blood mononuclear cells, dissociated cancer cells, dissociated cells from tissues, differentiated embryonic stem cell cultures, cells from a perturbation assay or any combination thereof.Attorney Docket #: 243734.000229SJ-25 -0018-0294. Non-transitory computer readable medium with instructions thereon that, when executed by one or more processors cause the one or more processors to: receive an identification of a multi-sample substrate preparation; partition a viewing area into a plurality of predetermined sample regions based at least in part on the identification; and image at least a portion of the viewing area inside the plurality of predetermined sample regions while excluding at least a portion of the viewing area outside of the predetermined sample regions.

95. The non-transitory computer readable medium of claim 94, wherein the instructions, when executed by the one or more processors cause the one or more processors to: distinguish data from each of the plurality of predetermined sample regions as individual, separable data sets.

96. The non-transitory computer readable medium of claim 95, wherein the instructions, when executed by the one or more processors cause the one or more processors to: save data from each of the plurality of predetermined sample regions as separate data files.

97. The non-transitory computer readable medium of claim 95 or 96, wherein the separable data sets comprise a plurality of gene counts per cell / nuclei datasets each associated with a respective predetermined sample region of the plurality of predetermined sample regions.

98. The non-transitory computer readable medium of any one of claims 94-97, wherein the instructions, when executed by the one or more processors cause the one or more processors to: identify a control sample region among the plurality of predetermined sample regions.

99. The non-transitory computer readable medium of claim 98, wherein the instructions, when executed by the one or more processors cause the one or more processors to: calibrate, based at least in part on a dataset associated with the control sample region, respective dataset(s) associated with a portion of the plurality of predetermined sample regions excluding the control sample region.

100. The non-transitory computer readable medium of any one of claims 94-99, wherein the instructions, when executed by the one or more processors cause the one or more processors to: provide, to a user via a user interface, the identification as one of a plurality of predetermined sample identifications; andAttorney Docket #: 243734.000229SJ-25 -0018-02 receive, from the user interface, the identification as a selection of one of the plurality of predetermined sample identifications.

101. The non-transitory computer readable medium of any one of claims 94-100, wherein the instructions, when executed by the one or more processors cause the one or more processors to: image the entirety of each of the predetermined sample regions.

102. The non-transitory computer readable medium of claim 94-101, wherein the instructions, when executed by the one or more processors cause the one or more processors to: image at least a majority of the viewing area inside the predetermined sample regions while excluding at least a majority of the viewing area outside of the predetermined sample regions.

103. A method of preparing a sample, the method comprising: growing or culturing at least one of cells or nuclei on a surface of a substrate; fixing the at least one of cells or nuclei on the substrate to form a monolayer of fixed cells or nuclei within one or more sample regions; and analyzing the fixed cells or nuclei using one of (i) imaging-based transcriptomics or (ii) proteomics or sequencing technologies.

104. The method of claim 103 further comprising applying a compartmentalizing apparatus comprising a plurality of openings to a surface of the substrate to form a plurality of wells each comprising a bottom and a sidewall such that the bottom comprises a respective sample region of the one or more sample regions on the surface of the substrate, and the sidewall comprising a surface of the gasket within a respective opening of the plurality of openings.

105. A method of analyzing samples comprising: depositing a plurality of distinct samples onto a single slide comprising a plurality of spatial regions, a first spatial region comprising a first sample, and a second spatial region comprising a second sample comprising a different type of cell or nuclei than the first sample; applying a multiplex panel of target-binding reagents to the first sample and the second sample; and performing multi-cycle imaging on the single slide.

106. The method of claim 105 further comprising associating each of the first sample and the second sample with a unique identifier comprising at least one of a spatial region map or a nucleic-acid sample barcode readable during cyclic imaging.Attorney Docket #: 243734.000229SJ-25 -0018-02107. The method of claim 106 further comprising demultiplexing the per-cell feature measurements to assign each cell identified to its originating first sample or second sample using the unique identifier.

108. The method of any of claims 105 to 107, wherein the type of cell or nuclei of the first sample and the second sample comprises at least one of peripheral blood mononuclear cells, dissociated cancer cells, dissociated cells from tissues, differentiated embryonic stem cell cultures, cells from a perturbation assay or any combination or nuclei thereof.

109. The method of any of claims 105 to 108, wherein performing multi-cycle imaging generates per-cell feature measurements.

110. The method of any of claims 105 to 109, wherein depositing the plurality of distinct samples comprises depositing the first sample and the second sample into a compartmentalizing apparatus comprising a plurality of wells, each well comprising a bottom and a sidewall such that the bottom comprises a respective spatial region of the plurality of spatial regions, and the sidewall comprising a surface of a gasket.

111. The method of claim 110 wherein depositing the plurality of distinct samples comprises: preparing a suspension of fixed cells, nuclei, or combination thereof; depositing the suspension within the first spatial region or the second spatial region; and evaporating water from the suspension leaving a monolayer of the first sample or the second sample.

112. The method any of claims 105 to 111, wherein performing multi-cycle imaging comprises using at least two different imaging platforms.

113. The method of claim 112, wherein the at least two different imaging platforms are configured to support one of RNA profiling or protein profiling.

114. The method of claim 112, wherein the at least two different imaging platforms are configured to support RNA profiling and protein profiling.

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