WO2024163782A1

WO2024163782A1 - Robust flow cell design for spatial transcriptomics and in situ hybridization that operates under high pressure with increased flow rates and improved flow dynamics

Info

Publication number: WO2024163782A1
Application number: PCT/US2024/014059
Authority: WO
Inventors: Joseph R. Johnson; Dontario Le'Marcus BEVERLY; Ang Li; Yun-Ching Chang
Original assignee: Applied Materials, Inc.
Priority date: 2023-02-01
Filing date: 2024-02-01
Publication date: 2024-08-08

Abstract

This disclosure provides a technology for the manufacture and use of an improved flow cell for reactive analysis of cell or tissue samples. The flow cell comprises a surface upon which the sample may be mounted or positioned, a reaction chamber surrounding the surface that is designed for exchange of liquid reagents, and an optical viewing window for periodically observing or imaging the sample. The viewing window is considerably thicker than prior art flow cells, enabling the user to operate the flow cell at high pressures and high flow rates to accelerate sample analysis. Optics of the viewing or imaging system are adapted to accommodate the thicker viewing window. Flow cells according to this disclosure may have other features to improve handling characteristics or control flow patterns of aqueous reagents around and about the sample being analyzed.

Description

Robust flow cell design for spatial transcriptomics and in situ hybridization that operates under high pressure with increased flow rates and improved flow dynamics

REFERENCE TO PREVIOUS APPLICATION

[0001] This patent disclosure claims the priority benefit of U.S. provisional patent application 63/442,507 (pending). The priority application is hereby incorporated herein in its entirety for all purposes.

FIELD OF THE INVENTION

[0002] The technology disclosed and claimed below relates generally to the fields of nucleic acid biology and the analysis and characterization of tissue sections in a computer controlled fluid flow apparatus. More specifically, it provides an improved flow cell designed for higher throughput and ease of use.

BACKGROUND

[0003] The spatial distribution of gene transcript in a tissue provides a research window for following interactive cell biology: specifically, what each specialized cell is doing in a heterogeneous population, and how the interaction of individual cells affects tissue biology, tissue homeostasis, and the emerging pathology of adverse conditions.

[0004] Spatial transcriptomics (the subcellular location of newly transcribed mRNA) can be measured and characterized using in situ hybridization and gene synthesis techniques. Single-cell transcriptome imaging allows quantitative measurements of both the gene expression profiles of individual, spatially localized cells and the intracellular distributions of transcription. At the tissue level, cell-specific gene expression defines cell types and cell states, the spatial organization of which is tightly coupled to both the development and function of normal tissues and to the pathogenesis and prognosis of tissue pathology from patients.

[0005] Multiplexed fluorescence in situ hybridization (mFISH) is a powerful imaging technique for subcellular localization of mRNA. A sample is exposed to multiple oligonucleotide probes that target particular RNA species of interest to the user. The probes have different labeling schemes that allow the user to distinguish different RNA species when the complementary, labeled probes are introduced to the sample. Sequential rounds of fluorescence images are acquired with exposure to excitation light of different wavelengths. For each given pixel, fluorescence intensities from the different images for the different wavelengths of excitation light form a signal sequence.

[0006] The detected sequence is then compared to a library of reference codes that associates each code with a gene. The best matching reference code is used to identify an associated gene that is expressed at that pixel in the image. Spatial transcriptomics permits visualization of expression of genes of interest spatially within cells and tissues. Depending on the technology used, mFISH is capable of profiling hundreds to thousands of RNA molecules in single cells. Spatially resolved RNA profiling of individual cells can be done for a range of gene transcripts with high accuracy and high detection efficiency.

[0007] The value of the analysis depends on the fidelity of the reactions that identify expression products, sophistication of data analysis, and ability of automated equipment to process biological samples in a high throughput manner.

SUMMARY

[0008] This disclosure provides a flow cell for analyzing a solid biological sample in an assay, wherein a series of reagents is flowed across the sample, in preparation for imaging or analysis. The flow cell comprises a surface upon which the sample may be mounted or positioned, a reaction chamber surrounding the surface that is designed for exchange of liquid reagents, and an optical viewing window for periodically observing or imaging the sample. The viewing window is considerably thicker than prior art flow cells, enabling the user to operate the flow cell at high pressures and high flow rates to accelerate sample analysis. Optics of the viewing or imaging system are adapted to accommodate the thicker viewing window. Flow cells according to this disclosure may have other features to improve handling characteristics or promote laminar flow of aqueous reagents around and about the sample being analyzed.

DRAWINGS

[0009] FIG. l is a component overview of a spatial genomics tool that illustrates the use of the this technology. The flow cell is designed to withstand high pressures, making it suitable for higher flow rates than previous flow cells. The flow cell is shown in the middle, secured on a stage of an imaging apparatus. Aqueous reaction solvents for a series of timed reactions are drawn through a manifold of valve with valve positioners through the flow cell by a down-stream peristaltic pump, introducing fluorescent labels into the sample. Emission from the labels, along with other aspects of the tissue (H&E and/or DAPI staining) are imaged for analysis.

[0010] FIG. 2 is a flowchart showing a typical reaction cycle. The sample is contacted with buffer, a first probe set, a wash solution, an imaging buffer containing fluorescent labels, and a final wash solution. A typical run has at least 4 probe sets to be delivered and imaged, for a processing time of about 5 min.

[0011] FIG. 3 shows an end-to-end cross-section of a flow cell. The sample surface is the bottom surface of a standard glass microscope slide. In this example, the slide itself is the viewing window through which the sample may be monitored, imaged, or otherwise analyzed. It is clamped on the left and right side to a lower surface or backing plate. The flow cell comprises an inlet port and an outlet port, with a reaction chamber in between.

[0012] FIG. 4 shows commercially available lenses that are suitable for obtaining images of processed samples though a thick cover plate.

[0013] FIG. 5A shows an example of a first type of clamping mechanism that can be used to hold the top plate with the viewing window with the backing plate. FIGS. 5B to 5D show an example of a second type of clamping mechanism that can be used to hold the top plate with the viewing window with the backing plate.

[0014] FIG. 6A shows the reflectance that was experimentally observed for black anodized aluminum. FIG. 6B shows a flow cell constructed to minimize reflection, while withstanding high pressure fluid flow.

[0015] FIG. 7A depicts a flow device in which inlet and outlet ports can be placed anywhere on the surface. In FIG. 7B, a machined groove at the top can be included in the design to create horizontal wavefront and/or to trap bubbles. FIG. 7C shows a gasket shape design, which can be used to guide flow patterns. FIG. 7D shows possible score patterns. FIG 7E is a side view showing how to mill the bottom plate to obtain a suitable scoring pattern.

[0016] FIGS. 8 A and 8B show the adaptation of a commercially available flow cell to accord with the technology put forth here. As currently distributed, the flow cell comprises a 170 pm coverslip, which means that pumping pressure must remain low. This is adapted by using a 1 mm thick surface for the lower plate.

[0017] FIG. 9 shows the adaptation of a second technology that may be adapted in accordance with the technology described here by using a 1 mm thick surface for the upper plate.

[0018] FIG. 10 shows that with minimal surface area required for clamping, the flow cell can be tightly packed to increase tool throughput.

[0019] FIG. 11 depicts a work flow for LISH Lock ’n ’Roll (LnR), an in situ hybridization technique that hybridizes split probes to target nucleic acids in a sample that are of interest to the user.

[0020] FIG. 12A shows the results of flow cells that were tested at high pressures. The flow cells showed no cracks or breaks at 500, 750, or even 1,000 mbar. FIG. 12B is a graph showing the flow profile for 19 mm wide channel. The height of the channel (the distance between the viewing window and the backing plate) was modeled at 200 or 300 pm. Even at 10 mm/s, the Reynold number (Re) was well below 2100. In this implementation of the technology, the flow remains laminar.

DETAILED DESCRIPTION

[0021] Current methods for analyzing a cell or tissue sample for spatial transcriptomics are typically complex, using a series of sophisticated biotechnology reagents with highly advanced automated devices. This disclosure provides a technology to improve handling characteristics, processing speed, and image quality.

Advantages of this technology

[0022] As illustrated below, implementing the flow cell technology of this disclosure into an in situ hybridization method provides the user with important advantages:

• Reagent exchange can be done at pressures of 1 bar or more;

• Flow rates for a standard 1 >< 3 inch flow cell can be 2 mL/min or more, speeding up the pace of sample analysis;

• The flow cell is more robust and resistant to damage during handling, sample preparation, and use;

• The reaction surface can be open during sample preparation, and simply clamped together with a gasket or other sealing surface and a backing plate to complete the flow cell;

• Reduced surface area is required for clamping the cell together, increasing the imageable area;

• With minimal surface area required for clamping, flow cells can be tightly packed to increase tool throughput;

• The thickness of the viewing window reduces warping of the surface during fluid flow;

• Various backing materials and surfaces can be used to control the pattern of flow of liquid through the cell (for example, laminar or turbulent flow);

• The pattern of fluid flow through the cell can be chosen to promote uniform reactivity across the surface of the cell or tissue sample and/or to promote mixing of reagents, thereby improving quality of the analysis;

• The flow cell can be manufactured with the same as a standard microscope slide (75 mm x 25 mm), RMS standard (ISO 8037-1 : 1986) This is compatible with equipment for processing ordinary microscope slides. The sample can be mounted directly on the slide using standard pathological tissue sectioning and fixation procedures. Features of the technology

[0023] FIG. l is a component overview of a spatial genomics tool that illustrates the use of the technology put forward in this disclosure. The flow cell is shown in the middle, secured on a stage of an imaging apparatus. Aqueous reaction solvents for a series of timed reactions are drawn through a manifold of valve with valve positioners through the flow cell by a down-stream peristaltic pump, and into a waste container. Periodically or after all reactions are complete, the sample is imaged. Laser light at a plurality of wavelengths corresponding to the activation energy of labels in the sample is impinged upon the sample. Emission from the labels, along with other aspects of the tissue (such as H&E and/or DAPI staining) is collected for analysis.

[0024] Referring to the flow chart shown in FIG. 2, a typical reaction cycle includes contacting the sample with buffer (10 mL, 500 pL/min), a first probe set (2.5 mL, 300 pL/min), a wash to remove unbound probes (4 mL, 600 pL/min), an imaging buffer containing fluorescent labels (3 mL, 600 pL/min), and final wash and preparation for imaging (6 mL, 600 pL/min).

Using a standard flow cell, these five steps typically take 20, 8, 7, 5, and 10 min respectively. The total pump time is thus ~1 hour per probe set. A typical run has at least 4 probe sets to be delivered and imaged, for a processing time of at least about 5 min. A higher plex (# of RNAs identified) will have more probe sets to image, requiring a commensurate increase in processing time.

[0025] In the current state of art, sample processing speed and throughput are limited by the 170 pm coverslip that overlays the sample. This limits the analysis in several ways. First, a low pumping pressure is required to prevent breaking the coverslip. A 170 pm glass coverslip will crack at > 350 mbar. A large surface area is needed to clamp coverslip, which limits the imageable area of the sample. Furthermore, the 170 pm coverslip can easily break during handling.

[0026] However, in the current state of art, 170 pm cover slips are always used, because the optics of objective lenses require that the sample being visualized be within no more than 230 pm of the lens (excluding the 170 pm cover slip thickness).

[0027] FIG. 3 shows an end-to-end cross-section of a flow cell in accordance with this disclosure. The sample surface is the bottom surface of a standard glass microscope slide. In this example, the slide itself is the viewing window through which the sample may be monitored, imaged, or otherwise analyzed. It is clamped on the left and right side to a lower surface or backing plate. The flow cell comprises an inlet port and an outlet port, with a reaction chamber in between where reagents react with the sample. The clamping mechanism includes a spacer that maintains a specified distance between the lower surface of the slide and the backing plate, typically selected to optimize flow characteristics. The flow cell optionally includes a sealing surface to prevent leakage of fluid outside the cell. In this configuration, the microscope slide serves as a viewing window, whereby the sample may be monitored, measured, and/or imaged. [0028] In this example, higher pressures can be used to increase flow rate. Only one piece of glass needs to be prepared, and there is little to no warping during high pressure flow. High force clamping can be used to secure slide as there is little risk of shattering glass. The clamping surface area can be minimized. By using a metal plate underneath, custom glass is not needed.

[0029] More generally, the technology of this invention provides a flow cell for analyzing a cell or tissue sample, comprising a sample surface, a reaction chamber around the surface configured for aqueous reagent exchange, and an optical viewing window for observing a sample on the sample surface. The viewing window is made of any material that is optically transparent at the wavelengths used for the imaging or analysis. Suitable materials for many applications are glass, such as silicate or borosilicate glass, or specialty plastics such as NUNC™ Brand Thernanox®. Fused quartz slides or plates may also be used where ultraviolet transparency is desired, for example, for fluorescence microscopy.

[0030] The viewing window is typically at least 0.5, 0.8, 1.0, or 1.2 mm thick, or between 0.4 and 2 mm, or between 0.6 and 1.5 mm, or between 0.8 and 1.2 mm. Where depicted in the drawings, the viewing window is 1.0 mm thick. The flow cell configured to operate at a fluid pressure of at least 500, 750, 1000, or 1500 mbar without cracking or separating. Possible flow rates are at least 1, 2, 3, 5, or 8 mL/min, or between 0.5 and 5 or between 0.8 and 3 mL/min, depending on such features as the vertical span of the reaction chamber. The sample surface may be on the opposite side of the viewing window, or on a coplanar surface underneath the viewing window. The sample surface is clamped to a sealing surface above a backing plate, thereby forming the reaction chamber. The sealing surface could be a gasket, an O-ring, a rubberized surface on an adjacent component, or other means of reliably preventing leakage or intercompartmental fluid flow.

[0031] To accommodate the thickness of the viewing window to provide sufficient detail, the optics of the system are changed. This can be done, for example, by interposing corrective lenses between the surface of the viewing window and the objective lens, or by building the correction into the viewing window by variation in surface configuration or density. The user may also obtain special purpose objective lenses. FIG. 4 shows suitable lenses in the Nikon® CFI S Plan Fluor series, available from Nikon Instruments Inc.

[0032] FIG. 5A shows an example of a first type of clamping mechanism that can be used to hold the top plate with the viewing window with the backing plate. A clamping plate is positioned above the viewing window. The area left covered can be used for labeling. The viewing window in this example has a 52 * 24 mm scannable area.

[0033] FIGS. 5B to 5D show an example of a second type of clamping mechanism that can be used to hold the top plate with the viewing window with the backing plate. FIG. 5B is an oblique view; FIG. 5C is a side-to-side cross section; FIG. 5D is a detail showing positioning of a clip. Clips are used on either side that partially but do not entirely follow the perimeter of the glass slide. The glass slide can withstand the concentrated forces applied to these clips to seal the flow cell, so minimal area needs to be taken up by the clips. As shown in FIG. 5C, the very edge of the glass slide (0.5 mm) can be clamped without worry of warping the slide or cracking the glass. The total clamping area is 26 * 63 - 24 * 62 = 7 mm² out of the 132 mm² of the entire slide. The clamping area of the clips is ~10 mm² (FIG. 5C). The objective lens can be moved between the clips to image that area of the slide. If a gasket or O-ring is used, it can be modified (with a large flow region) to allow more scannable area.

Modulating the pattern of fluid flow

[0034] The backing plate of the flow device opposing the viewing window may be, for example, metal, glass, or a polysynthetic. The backing plate may be made with the anti-reflective materials across a wide spectra, such as glass, anodized metal, transparent polysynthetic, nano- structural or AR coated surfaces. Advantages of using a material other than glass are as follows:

1. The user can machine the surface to guide fluid flow (micro grooves, channels, machine lay pattern);

2. The user can also create grooves to trap or guide bubbles away from the center of the flow cell;

3. The user can heat the metal during processing to raise the temperature of the flow cell liquid to reduce incubation times.

[0035] It is beneficial to use anti-reflective materials, because a reflected signal from the backing plate may increase the background, narrowing the working range to characterize signals from a tissue or cell sample in the flow cell. A reflected signal may also reduce the range of selection on imaging optics and autofocus techniques.

[0036] FIG. 6A shows the reflectance experimentally observed for black anodized aluminum, adapted from JL Marshall et al., arXiv: 1407.8265, 2014. The surface was tested across a spectrum of wavelengths, either in raw form (top line), bead blasted (second line), machined (third line), or polished (lowest line). Flow cells having backing plates that are metal or black may generate an undesirable amount of reflected radiation. [0037] FIG. 6B shows a flow cell constructed to minimize reflection, while withstanding high pressure fluid flow. The cell has a partially transparent back plate with a quartz insert. Other types of ceramics may be used as an alternative, as long as they are optically transparent at the desired operating wavelength.

[0038] FIGS. 7A to 7E illustrate patterns that can be etched or otherwise manufactured into the backing plate, whatever material is used. In most applications, the optimal flow pattern is one where probes are evenly distributed across the samples. As shown in FIG. 7A, inlet and outlet ports can be placed anywhere on the surface to promote flow over the sample. As shown in FIG. 7B, a machined groove at the top (near the inlet) can be included in the design to create horizontal wavefront and/or to trap bubbles. FIG. 7C shows a gasket shape design, which can be used to guide flow patterns. FIG. 7D shows possible score patterns. As illustrated in FIG 7E, the user can choose the predominant surface pattern by telling the machine shop which direction to mill the metal plate. This can help give fluids directionality on a micron scale.

Alternative confi urations

[0039] Other configurations of this technology can be used to accommodate or improve devices and methods provided by other manufacturers.

[0040] FIGS. 8 A and 8B show adaptation of a first technology available in the art. As currently distributed, the flow cell comprises a 170 pm coverslip, which means that pumping pressure must remain low. As shown, the top surface of the lower plate constitutes the viewing window, whereas the upper glass aqueduct constitutes the backing plate. This is adapted in accordance with the technology described here by using a 1 mm thick surface for the lower plate. The sample surface is the opposite side of the viewing window, which is positioned above a gasket or sealing surface and a glass aqueduct, thereby forming the reaction chamber.

[0041] FIG. 9 shows the adaptation of a second technology that is commercially available to incorporate features of the technology described here. The flow cell comprises an upper plate which constitutes the viewing window, and a lower plate (a microscope slide) that constitutes the backing plate. The sample surface is coplanar with and faces the viewing window, wherein the viewing window and the sample surface are separated by a gasket or sealing surface, thereby forming said reaction chamber. This second technology uses custom (fluidic holes drilled) coverslip of standard 170 pm thickness over a standard 1 x 3” glass slide, which is susceptible to cracking under rapid or high pressure fluid flow. The clamping along all edges of coverslip limits the imageable area. This flow cell may be adapted in accordance with the technology described here by using a 1 mm thick surface for the upper plate. [0042] FIG. 10 shows that with minimal surface area required for clamping, the flow cell can be tightly packed to increase tool throughput. Any of the flow cells illustrated in this disclosure or otherwise falling within the bounds of this technology and other adaptations to technologies available in the art can be manufactured or distributed as a cassette configured to be exchangeably secured to and operated by a fluid flow apparatus configured for the type of in situ analysis to be conducted by the user.

Types of in situ hybridization or in situ sequencing that are compatible with the technology in this disclosure

[0043] Although various aspects of the technology of this disclosure are exemplified in the context of LISH Lock’n’Roll in situ hybridization, they may be implemented or adapted into other forms of in situ hybridization analysis. Other ways of doing in situ hybridization include the following:

[0044] Fluorescence in situ hybridization (TISH) is a molecular cytogenetic technique in which fluorescent oligonucleotide probes hybridize to nucleic acid sequences in a tissue sample to detect and localize specific RNA targets (mRNA, IncRNA, miRNA) in tissue samples and cells. Multiplexed fluorescence in situ hybridization (mFISH) is a technique that uses a range of probes that each bind specifically to different nucleic acid targets having different sequences, with a sequential or simultaneous labeling strategy that separately identifies mRNA species having different sequences.

[0045] Single-molecule fluorescent in situ hybridization (smFISH) implements short (50 bp) oligonucleotide probes conjugated with five fluorophores to obtain quantitative information about expression of certain genes in the cell. RNAscope employs probes of the specific Z-shaped design to simultaneously amplify hybridization signals and suppress background noise.

[0046] Single-molecule RNA detection at depth by hybridization chain reaction (smHCR) is an advanced seqFISH technique in which a set of short DNA probes attach to a defined subsequence of the target, followed by fluorophore-labeled DNA HCR hairpins that penetrate the sample and assemble into fluorescent amplification polymers attached to the initiating probes. Cyclic-ouroboros smFISH (osmFISH) visualizes transcripts in the manner of smFISH, and an image is acquired before the probe is stripped and the sample is reprobed. Multiplexed error- robust fluorescence in situ hybridization (MERFISH) is a single-cell transcriptome imaging method that encodes RNA target molecules with error-robust binary barcodes. The readout sequences are detected using fluorophore-labelled secondary probe, and the fluorescence signal is extinguished via photobleaching before subsequent rounds of imaging. DNA microscopy generates cDNA in fixed tissue, following which randomized nucleotides are used to tag and amplify target cDNAs in situ, thereby generating unique labels for each molecule. SeqFISH Plus resolves optical issues related to spatial crowding using a primary probe anneals to targeted mRNA, followed by readout probes that bind to flanking regions in a way that can be captured as an image and collapsed into a super-resolved image.

[0047] LISH Lock’n ’Roll (LnR) is an in situ hybridization technique that hybridizes split probes to target nucleic acids in a sample that are of interest to the user. The split probes are then circularized, amplified, and used to create image spots using labeled detection reagents.

[0048] FIG. 11 depicts an LnR workflow. In Step 1 and 2, the LnR acceptor and donor probes anneal to the target RNA sequence, followed by ligation with T4 RNA Ligase 2 (Rnl2). Excess probes are then washed away. After adjacent donor and acceptor probe have become ligated, will the two probe halves present the complete 34-nucleotide bridge sequence (17 nucleotides from each probe), which is subsequently hybridized by the bridge primer (Step 3) and ligated by T4 DNA ligase (Step 4).

[0049] At this stage, the probe sets have been ligated at both ends, forming a closed-circular DNA molecule. Due to the twist of the double helix, locks it into place around the target mRNA. A suitable DNA polymerase is then added to the tissue (shown as a grey oval). This initiates rolling circle amplification (RCA) to take place in situ, as it is primed by the annealed bridge primer that was used for circularization (Step 5). Preferred DNA polymerase for rolling circle amplification is an enzyme capable of multiple displacement amplification of DNA: such as 029 (Phi 29) DNA Polymerase or its equivalent (available, for example, from NxGen or ThermoFisher). Desirable features are high processivity and strand displacement activity, capable of synthesizing DNA up to 70 kb long, highly accurate DNA synthesis, high yields of amplified DNA even from minute amounts of template, amplification products suitable for hybridizing detection probes.

[0050] The RCA product formed from the circular DNA is a nanoball of single-stranded DNA containing multiple copies of the detector sequences. Due to the extensive crosslinking of the surrounding tissue, the nanoball remains trapped in a position that approximates the position of the templating RNA molecule. Following completion of RCA, fluorescently labeled oligonucleotides (detector probes) are annealed to the complementary detector sequences, of which there are now many spatially localized copies (Step 6). The tissue is now ready to be processed for imaging. [0051] More generally, the technology of this invention includes a method of analyzing a cell or tissue sample, typically comprising the following steps: (a) placing a cell or tissue sample on the sample surface of a flow cell according to this disclosure; (b) assembling the flow cell such that the reaction chamber is formed around the sample; (c) flowing one or more reagent solutions through the flow cell such that reagents in the solutions contact the cell or tissue sample on the sample surface, thereby forming an optically observable reaction product; and (d) observing said reaction product. The assembling in step (b) may include clamping the sample surface, a gasket or other sealing surface, and a fluid flow manifold together to form said reaction chamber. The chemical analysis may include DNA analysis or amplification, RNA analysis or amplification, nucleic acid sequencing, protein analysis, antigen retrieval, hematoxylin and eosin (H&E) staining, immunofluorescence (IF) staining, immunohistochemical (IHC) staining, or a combination thereof. There may be multiple cycles of contacting the cell or tissue sample with reagents in a reagent solution, observing a reaction product, and stripping reaction product from the sample to prepare for the next cycle of reagent solutions.

Devices for carrying out mFISH analysis of tissue samples

[0052] The flow cells of this disclosure can be used with any handling apparatus for any purpose. FIG. 1 illustrates general principles of a device configured for spatial transcriptomics or in situ hybridization.

[0053] The Veranome VSA-1 system is an apparatus and computerized control system for conducting mFISH. Incorporated herein by reference in its entirety as part of this disclosure is US 2021/0181111 Al. The system comprises the following components:

• a flow cell to contain a sample to be exposed to fluorescent probes in a reagent;

• a valve to control flow from one of a plurality of reagent sources the flow cell;

• a pump to cause fluid flow through the flow cell;

• a fluorescence microscope including a variable frequency excitation light source and a camera positioned to receive fluorescently emitted light from the sample;

• an actuator to cause relative vertical motion between the flow cell and the fluorescence microscope;

• a motor to cause to cause relative lateral motion between the flow cell and the fluorescence microscope; and

• a control system.

[0054] The control system operates the components of the apparatus by performing the following actions as nested loops:

• cause the valve to sequentially couple the flow cell to a plurality of different reagent sources to expose the sample to a plurality of different reagents,

• for each reagent of the plurality of different reagents, cause the motor to sequentially position the fluorescence microscope relative to sample at a plurality of different fields of view, • for each field of view of the plurality of different fields of view, cause the variable frequency excitation light source to sequentially emit a plurality of different wavelengths,

• for each wavelength of the plurality of different wavelengths, cause the actuator to sequentially position the fluorescence microscope relative to sample at a plurality of different vertical heights, and

• for each vertical height of the plurality of different vertical heights, obtain an image at the respective vertical height covering the respective field of view of the sample having respective fluorescent probes of the respective regent as excited by the respective wavelength.

[0055] The flow cell technology of this disclosure can be integrated with a flow cell handling device as follows. In general terms, the apparatus or device for performing analysis of a tissue or cell sample in a flow cell comprises a platform adapted to receive, secure, and operate said flow cell; an assembly of reservoirs, fluid conduits and pumps arranged and connected to contact a cell or tissue sample in a flow cell on the platform with said reagents, thereby generating said reaction products; a microprocessor connected and programmed to control the components and conduct an analysis of a cell or tissue sample. The apparatus comprises an optical system positioned to focus on and observe reaction products associated with the sample through the optical viewing window, optically adapted to accommodate the thickness of the optical window. The optical system may be adapted to detect and localize fluorescence signals in the sample after in situ hybridization. The system may further comprise a viewing and recordation system adapted to detect and locate optical signals in the sample through the thickness of the viewing window.

[0056] In general terms, this disclosure provides an improved apparatus for analyzing Spatial transcriptomics in a cell or tissue sample. The apparatus includes a flow cell that comprises a sample surface, a reaction chamber around the surface configured for aqueous reagent exchange, and an optical viewing window for observing a sample on the sample surface. The apparatus further includes a viewing and recordation system configured for detecting and locating optical signals in a sample located on the sample surface. The apparatus is improved by adapting the flow cell so that any optically transparent surfaces are at least 500, 750, or 100 pm thick, and adapting the viewing and recordation system to detect and locating optical signals in the sample through the thickness of the viewing window. The reaction chamber around the sample is configured to operate at a flow rates and pressures as hereintofore described. Effectiveness of the technology

[0057] FIG. 12A shows the results of flow cells in accordance with this disclosure that were tested at high pressures. Typical pumping pressures or prior art technologies are between 100 and 300 mbar. In this example, the flow cells showed no cracks or breaks at 500, 750, or even 1 ,000 mbar.

[0058] Laminar flow helps preserve tissue integrity and allows for even distribution of probes. The extent of laminar flow can be estimated by determining the Reynold’s number. This helps predict flow patterns in different fluid flow situations by measuring the ratio between inertial and viscous forces. At low Reynolds numbers, flows tend to be dominated by laminar (sheet-like) flow, while at high Reynolds numbers flows tend to be turbulent. This creates unfavorable differences in the fluid's speed and direction, which may sometimes intersect or even move counter to the overall direction of the flow (eddy currents). The Reynold’s number can be calculated using the following equation.

[0059] In the example shown in FIG. 3, the viewing window (the upper plate) and the backing plate are parallel. Typical distance between the two plates is 200 or 300 pm. This is substantially larger than the width and length of the flow cell (19 mm and 47 mm, respectively). Assuming that there parallel plate flows because the relative height of the flow chamber, Cl = %, C2 = 1, n = 1, and K = p (dynamic viscosity), the equation simplifies to:

PUDH

Re =

1.5p where p = density of the liquid, U = velocity of the liquid, DH = hydraulic diameter of the flow cross section, and p = dynamic viscosity of the liquid. For water, p = 1,000 kg/m³ and p = 8.9 * 1 O'⁴ N/m² For rectangular flow channels, DH = 2 a b / (a + b), where b = the height of the flow channel (200 pm) and a = width of the flow channel (19 mm). Then DH = 0.362 mm. The remaining variable U is flow velocity.

[0060] FIG. 12B is a graph showing the flow profile for 19 mm wide channel. The height of the channel (the distance between the viewing window and the backing plate) was modeled at 200 or 300 pm. Even at 10 mm/s, the Reynold number (Re) was well below 2100. Flow transitions from laminar to turbulent between Re = 2100 and Re = 4000. In this implementation of the technology, the flow remains laminar, which is good for filling the flow cell uniformly with liquids, probes, and reagents. It helps ensure uniform exposure across the width and breadth of the cell or tissue sample to each of the reagents in the reaction cycle.

[0061] Alternatively, the flow cell can be adapted to have flow patterns that are not entirely laminar. For example, turbulence can be induced in fluid passing through the flow cell by roughening or etching the surface of the back plate. This would promote mixing, for example, where two liquids are being flowed through the cell concurrently. As illustrated in FIG. 7B, grooves and troughs can be included in the backing plate to promote flow from an inlet port across the entire width of the flow cell, and then collected together for common egress through an outlet port.

* * * * *

Practice of the invention

[0062] The technology provided in this disclosure and its use are described within a hypothetical understanding of general principles of fluid flow dynamics, nucleic acid chemistry, tissue analysis, and device electronics. Particular discussions and examples are provided for the edification and entertainment of the reader, and are not intended to limit the practice of the claimed invention. All of the products and methods claimed in this application may be used for any suitable purpose without restriction, unless otherwise indicated or required.

[0063] While the invention has been described with reference to the specific examples and illustrations, changes can be made and equivalents can be substituted to adapt the technology to a particular context or intended use as a matter of routine development and optimization and within the purview of one of ordinary skill in the art, thereby achieving benefits of the invention without departing from the scope of what is claimed and their equivalents.

Claims

CLAIMS The invention claimed is:

1. A flow cell for analyzing a cell or tissue sample, comprising a sample surface, a reaction chamber around the surface configured for aqueous reagent exchange, and an optical viewing window for observing a sample on the sample surface that is at least 500 pm thick.

2. The flow cell of the preceding claim, configured to operate at a fluid pressure of at least 750 mbar without cracking or separating.

3. The flow cell of either claim 1 or 2, wherein the sample surface is the opposite side of the viewing window, which is clamped to a sealing surface above a backing plate, thereby forming said reaction chamber.

4. The flow cell of either claim 1 or 2, wherein the sample surface is the opposite side of the viewing window, which is positioned above a sealing surface and a glass aqueduct, thereby forming said reaction chamber.

5. The flow cell of either claim 1 or 2, wherein the sample surface is coplanar with and faces the viewing window, wherein the viewing window and the sample surface are separated by a sealing surface, thereby forming said reaction chamber.

6. The flow cell of any preceding claim, wherein the viewing window is glass that is 1 ± 0.2 mm thick.

7. The flow cell of any preceding claim, wherein the flow cell has been adapted so that liquid flows from an inlet port to an outlet port in the reaction chamber in a laminar or other controlled flow pattern.

8. A flow cell for analyzing a cell or tissue sample, comprising a sample surface and a reaction chamber around the surface configured for aqueous reagent exchange, wherein the reaction chamber is bounded by coplanar plates, the first plate comprising an optical window configured for observing a sample on the sample surface, the second plate having a surface that is etched or otherwise configured to control the flow pattern of liquid flowing from an inlet port to an outlet port in the reaction chamber.

9. The flow cell of any preceding claim, comprising a backing plate that is optically transparent or partly transparent, thereby decreasing reflection of incident light at wavelengths over 750 nm..

10. The flow cell of claim 9, wherein the backing plate consists essentially of quartz or another transparent ceramic.

11. The flow cell of any preceding claim, wherein the reaction chamber around the sample is configured to operate at a flow rate of up to at least 2 mL per min.

12. The flow cell of any preceding claim, configured for spacial transcriptomics of a sample on the sample surface by in situ hybridization or in situ sequencing of mRNA transcripts in the sample.

13. A method of analyzing a cell or tissue sample, comprising:

(a) placing a cell or tissue sample on the sample surface of a flow cell according to any preceding claim;

(b) assembling the flow cell such that the reaction chamber is formed around the sample;

(c) flowing one or more reagent solutions through the flow cell such that reagents in the solutions contact the cell or tissue sample on the sample surface, thereby forming an optically observable reaction product; and

(d) observing said reaction product.

14. The method of claim 13, wherein the assembling in step (b) comprises clamping the sample surface, a sealing surface, and a fluid flow manifold together to form said reaction chamber.

15. The method of claim 13 or 14, wherein the analysis comprises DNA analysis or amplification, RNA analysis or amplification, nucleic acid sequencing, protein analysis, antigen retrieval, hematoxylin and eosin (H&E) staining, immunofluorescence (IF) staining, immunohistochemical (IHC) staining, or a combination thereof.

16. The method of any of claims 11 to 15, wherein the analysis comprises spatial transcriptomics by in situ hybridization or in situ sequencing of individual mRNA transcripts.

17. The method of any of claims 11 to 16, comprising multiple cycles of contacting the cell or tissue sample with reagents in a reagent solution, observing a reaction product, and stripping reaction product from the sample to prepare for the next cycle of reagent solutions.

18.. An apparatus for performing analysis of a tissue or cell sample in a flow cell according to any of claims 1 to 12, comprising: a platform adapted to receive, secure, and operate said flow cell; an assembly of reservoirs, fluid conduits and pumps arranged and connected to contact a cell or tissue sample in a flow cell on the platform with said reagents, thereby generating said reaction products; a microprocessor connected and programmed to cause the apparatus to carry out the aforesaid steps of the analysis method; and an optical system positioned to focus on and observe reaction products associated with the sample through the optical viewing window, optically adapted to accommodate the thickness of the optical window.

19 The apparatus of claim 18, further comprising a viewing and recordation system adapted to detect and locate optical signals in the sample through the thickness of the viewing window.

20. A cassette configured to be exchangeably secured to and operated by an apparatus according to claim 18 or 19, the cassette comprising at least four of said flow cells.

21. An improved apparatus for analyzing spatial transcriptomics in a cell or tissue sample, wherein the apparatus includes a flow cell that comprises a sample surface for mounting or securing a cell or tissue sample, a reaction chamber around the surface configured for aqueous reagent exchange, and an optical viewing window for observing a sample on the sample surface; wherein the apparatus further includes a viewing and recordation system configured for detecting and locating optical signals in a sample located on the sample surface; wherein the apparatus is improved by adapting the flow cell so that the viewing window and any other optically transparent surfaces have a thickness of at least 500 pm, and adapting optics of the viewing and recordation system to detect and locating optical signals in the sample through the thickness of the viewing window.

22. The improved apparatus of claim 21, wherein the reaction chamber around the sample is configured to operate at a flow rate of up to at least 2 mL per min and/or at a pressure of at least 750 mbar.