Assembly for forming a sealed chamber

Abstract

Various embodiments of the present disclosure disclose a system for forming a sealed chamber for preparing a sample therein. Specifically, various embodiments propose an assembly for forming a sealed chamber that includes a sample device and a lid that can be used, inter alia, for culturing a sample therein. When the cover is engaged with the sample device, the plurality of snap joint elements of the cover are engaged with a corresponding plurality of snap joint elements to form a seal between the cover and the sample device.

Description

Technical Field

[0001] The present disclosure relates to an assembly for forming a sealed chamber for preparing a sample therein. In particular, the present disclosure describes an assembly of a sample device and a cover for forming a sealed chamber, which can be used, inter alia, for culturing a sample therein. Background Art

[0002] Existing solutions for sealing holes to form sealed chambers utilize sealing tape or other coverings, and are placed on the opening of the hole. In most cases, the tape or covering is flush with the opening of the hole. This is to reduce the space between the sample and the tape so that heat can be effectively transferred to the sample when transferred via the tape. For example, when heat is transferred to the sample via a thermal cycler (thermal cycler) cover of heating that is in contact with or flush with the tape (for example, in the presence of fluid), when the space between the sample and the tape is low, heat can be more effectively transferred to the sample. However, this may be undesirable, because interference with the sample or hole can result in forming a fluid capillary bridge (capillarybridge) between the top surface and the tape at the bottom of the hole. For example, the fluid in the hole may be wicked (wick up) and form a bridge, producing undesirable consequences such as but not limited to the dehumidification, leakage of the sample / hole, etc. Therefore, a system for forming a sealed chamber is needed to at least solve the defect. Summary of the invention

[0003] Various embodiments of the present disclosure disclose a system for forming a sealed chamber for preparing a sample therein. For example, the sample can be an assembly including a sample device and a cover for covering the sample device. In various embodiments, the sample device includes a bottom portion and a top portion. In some cases, the bottom portion is releasably coupled to the top portion. In addition, when the bottom portion is coupled to the top portion, a gap is formed between the bottom portion and the top portion, wherein the gap is configured to receive a sample substrate. In addition, the top portion includes a hole and a plurality of first snap joint elements, wherein at least one pair of snap joint elements in the plurality of first snap joint elements are substantially arranged on the top portion opposite to each other. The cover includes a cover covering and a skirt. In some cases, the covering includes a flat outer surface; and an inner surface, the inner surface being separated from the flat outer surface by the thickness of the covering. In some cases, the skirt extends around the periphery of the inner surface. In addition, the skirt and the inner surface define a recessed portion. In addition, the skirt includes a plurality of second snap joint elements extending therefrom, wherein at least one pair of snap joint elements in the plurality of second snap joint elements are substantially arranged on the skirt opposite to each other. In some cases, when the cover is engaged with the sample device, the plurality of second snap-fit ​​elements respectively engage with the plurality of second snap-fit ​​elements to form a seal between the cover and a perimeter of the aperture of the top portion. BRIEF DESCRIPTION OF THE DRAWINGS

[0004] For a more complete understanding of the principles and advantages disclosed herein, reference is now made to the following description taken in conjunction with the accompanying drawings, in which:

[0005] Figure 1 is an exemplary workflow for analyzing a biological sample (eg, a cell or tissue sample) using an optofluidic instrument according to various embodiments.

[0006] Figure 2 A sample device including a sample well defined by a substrate and a gasket is shown according to various embodiments.

[0007] FIG. 3A to FIG. 3C Shown is a bridge in a sample device sealed with tape according to various embodiments.

[0008] Figure 4A and Figure 4B Shown are top and bottom views of a cover for covering a sample device according to various embodiments.

[0009] Figure 4C and Figure 4D Cross-sectional views and partial views of a cover for covering a sample device according to various embodiments are shown.

[0010] FIG. 5A to FIG. 5D The engagement of the cover with the sample device according to various embodiments is shown ( FIG. 5A to FIG. 5C ), including in the presence of a thermal cycling cover ( Figure 5D ).

[0011] Figure 6 A cross-sectional view of a cover according to various embodiments is shown.

[0012] 7A to 7H A gasket for a sample device according to various embodiments is shown.

[0013] It should be understood that the drawings are not necessarily drawn to scale, nor are the objects in the drawings necessarily drawn to scale relative to each other. The drawings are intended to provide clarity and understanding of the various embodiments of the devices, systems, and methods disclosed herein. Whenever possible, the same reference numerals will be used throughout the drawings to refer to the same or similar components. In addition, it should be understood that the drawings are not intended to limit the scope of the present teachings in any way. DETAILED DESCRIPTION

[0014] I. Overview

[0015] Target molecules (e.g., nucleic acids, proteins, antibodies, etc.) can be detected in biological samples (e.g., one or more cells or tissue samples) using an instrument with integrated optics and a fluidics module ("optical fluidic instrument" or "optical fluidic system"). In the optical fluidic instrument, the fluidics module is configured to deliver one or more reagents (e.g., fluorescent probes) to the biological sample and / or remove the used reagents from the biological sample. In addition, the optical module is configured to irradiate the biological sample with light having one or more spectral emission curves (within a wavelength range), and then capture one or more images of the emitted light signal from the biological sample during one or more detection cycles. In various embodiments, the captured images can be processed in real time and / or later to determine the presence of one or more target molecules in the biological sample, as well as the three-dimensional position information associated with each detected target molecule. In addition, the optical fluidic instrument includes a sample module configured to receive (and optionally fix) one or more biological samples. In some cases, the sample module includes an XY stage configured to move the biological sample along an XY plane (e.g., perpendicular to the objective lens of the optical module).

[0016] In various embodiments, the optofluidic instrument is configured to analyze one or more target molecules at the location where the one or more target molecules naturally occur (i.e., in situ) within a biological sample. For example, the optofluidic instrument can be an in situ analysis system for analyzing a biological sample and detecting target molecules, including but not limited to DNA, RNA, proteins, antibodies, and / or the like.

[0017] Samples disclosed herein can be or are derived from any biological sample. Biological samples can be obtained from any suitable source using any of a variety of techniques, including but not limited to biopsy, surgery and laser capture microscopy (LCM), and generally include cells, tissues and / or other biological materials from a subject. Biological samples can be obtained from prokaryotes (such as bacteria, archaea, viruses or viroids). Biological samples can also be obtained from non-mammalian organisms (e.g., plants, insects, arthropods, nematodes, fungi or amphibians). Biological samples can also be obtained from eukaryotic organisms, such as tissue samples from mammals. Biological samples from organisms can include one or more other organisms or components thereof. For example, in addition to mammalian cells and non-cellular tissue components, mammalian tissue sections can include prions, viroids, viruses, bacteria, fungi or components from other organisms. The subject from which a biological sample can be obtained can be a healthy or asymptomatic subject, a subject suffering from or suspected of having a disease (e.g., an individual suffering from a disease such as cancer) or a subject susceptible to a disease, and / or a subject requiring therapy or suspecting the need for therapy.

[0018] Biological samples can include any number of macromolecules, for example, cellular macromolecules and organelles (e.g., mitochondria and nuclei). Biological samples can be obtained as tissue samples (such as tissue sections, biopsy samples, core needle biopsy samples, needle aspirates, or fine needle aspirates). The sample can be a fluid sample, such as a blood sample, a urine sample, or a saliva sample. The sample can be a skin sample, a colon sample, a cheek swab, a histological sample, a histopathological sample, a plasma or serum sample, a tumor sample, a living cell, a cultured cell, a clinical sample (e.g., whole blood or blood-derived products, blood cells, or cultured tissues or cells, including cell suspensions).

[0019] In some embodiments, the biological sample may include cells or tissue samples deposited on a substrate. As described herein, the substrate may be any support that is insoluble in aqueous liquids, and allows the positioning of biological samples, analytes, features, and / or reagents on the support. In some embodiments, the biological sample is attached to the substrate. In some embodiments, the substrate is optically transparent to facilitate analysis on the optofluidic instrument disclosed herein. For example, in some cases, the substrate is a glass substrate (e.g., a microscope slide, a cover glass, or other glass substrates). The attachment of the biological sample may be irreversible or reversible, depending on the nature of the sample and the subsequent steps in the analytical method. In certain embodiments, by applying a suitable polymer coating to the substrate and contacting the sample with the polymer coating, the sample may be reversibly attached to the substrate. Then, for example, using an organic solvent that at least partially dissolves the polymer coating, the sample may be separated from the substrate. Hydrogels are examples of polymers suitable for this purpose. In some embodiments, the substrate may be coated or functionalized with one or more substances to facilitate sample attachment to the substrate. Suitable substances that may be used to coat or functionalize the substrate include, but are not limited to, lectins, polylysine, antibodies, and polysaccharides.

[0020] Various steps can be performed to prepare or process biological samples for and / or during an assay using the optofluidic instrument disclosed herein. Unless otherwise indicated, the preparation or processing steps described below can generally be combined in any manner and in any order to appropriately prepare or process a particular sample for and / or analysis.

[0021] For example, a biological sample can be obtained from a subject (e.g., via surgical biopsy, whole subject sectioning) or grown in vitro as a cell population on a growth matrix or culture dish, and prepared for analysis as a tissue slice or tissue section (e.g., fresh frozen, fixed frozen, or formalin fixed paraffin embedded (FFPE) tissue section). The thickness of the tissue section generally depends on the method used to prepare the section and the physical properties of the tissue, so sections with various thicknesses can be prepared and used.

[0022] In some cases, biological samples are fixed in any of a variety of suitable fixatives to preserve the biological structure of the sample prior to analysis. Exemplary fixatives include formalin, formaldehyde, ethanol, methanol, acetone, paraformaldehyde (PFA)-Triton, and combinations thereof.

[0023] In some embodiments, a biological sample can be permeabilized to facilitate transfer of an analyte from the sample and / or to facilitate transfer of species (such as a probe or probe set) into the sample. Typically, a biological sample can be permeabilized by exposing the sample to one or more permeabilizing agents. Suitable reagents for this purpose include, but are not limited to, organic solvents (e.g., acetone, ethanol, and methanol), cross-linking agents (e.g., paraformaldehyde), detergents (e.g., saponin, Triton X-100, TM or Tween-20 TM ) and enzymes (e.g., trypsin, proteases).

[0024] In some embodiments, biological samples are embedded in polymers and / or cross-linked matrices (e.g., hydrogel matrices). Embedding samples in this way generally involves contacting biological samples with hydrogels so that the biological samples become surrounded by hydrogels. For example, samples can be embedded (e.g., tissue sections on substrates such as glass substrates) by contacting the sample with a suitable polymer material and activating the polymer material to form a hydrogel. In some embodiments, hydrogels are formed so that the hydrogels are internalized in biological samples. In some embodiments, biological samples (including biological analytes) are fixed in hydrogels by crosslinking of the polymer material forming the hydrogel. Crosslinking can be performed chemically and / or photochemically, or alternatively by any other suitable hydrogel formation method. In some cases, biomolecules (or their derivatives) are crosslinked or otherwise covalently attached to the hydrogel. For example, in some embodiments, nucleic acid molecules (or their derivatives, such as amplification products or probes attached to cell nucleic acid molecules) in tissue samples are crosslinked or otherwise covalently attached to the hydrogel.

[0025] Any suitable method can be used to make the hydrogel embedded in the biological sample transparent.For example, electrophoretic tissue clearing method or surfactant-based (e.g., sodium dodecyl sulfate (SDS)) clearing method can be used to remove biomacromolecules from the hydrogel embedded sample.

[0026] Tissue clearing is a process of optically analyzing samples or complex biological materials (such as complete organs, large tissues, and cell models) with minimal changes to morphology and without compromising the ability of immunolabeling or fluorescence imaging detection. In various embodiments, refractive index matching is used to obtain fluorescence images. Mismatches between media can lead to loss of imaging resolution because light may need to pass through the sample itself, mounting media, glass coverslips, oils, and / or microscope objectives. In various embodiments, various methods described herein are used to reduce (e.g., minimize) the amount of variable scattering of light from the cell membrane, lipids, and / or molecules of the sample. The inhomogeneity of scattering between cell components may lead to an increase in image opacity. In various embodiments, denser compositions of lipids, transport organelles, and other subcellular molecules may increase lateral or non-forward light scattering. In various embodiments, non-forward light scattering in situ may not pass through the sample because the continuous, pinball-like interaction of scattered light with neighboring molecules exacerbates this situation. In various embodiments, the energy of light may be reduced or ultimately lost through multiple scattering, refraction, and absorption, resulting in a distorted white non-translucent image. In various embodiments, the clearing agent and mounting medium optically clear the sample by matching refractive indices to minimize light scattering through the sample and to the microscope objective.

[0027] In various embodiments, optical clearing can be performed by a variety of different methods, mainly divided into chemical methods and matrix-based methods. In various embodiments, chemical methods include water-based or solvent-based methods to obtain high-resolution 3D images for immunolabeling, immunocytochemistry, immunohistochemistry and / or immunofluorescence. In various embodiments, water-based clearing methods are generally used to avoid dehydration and toxicity, which can destroy the integrity of the sample.

[0028] In various embodiments, the passive clearing technique (PACT) is a passive tissue clearing and immunolabeling protocol. In various embodiments, PACT is used for intact thick organs. In various embodiments, RIMS includes a protocol for passive tissue clearing and immunostaining of intact organs that is suitable for long-term storage and has an imaging medium that preserves fluorescent labels for months.

[0029] In various embodiments, a refractive index matching solution (RIMS) can be produced with sugar or glycerol for simple passive infiltration. This may be preferred for thinner or smaller samples because they are easier to transparentize and can maintain fluorescent protein emission. In various embodiments, this infiltration technique can achieve a refractive index of less than 1.5, and it may take several days to achieve transparency, resulting in reduced image quality compared to solvent methods due to the refractive index mismatch between the transparent sample, glass coverslip, and immersion oil (glass and oil have an RI of 1.51). Since sugar or glycerol solutions may take a long time to transparentize, the sample may experience considerable shrinkage while losing lipid content. In various embodiments, commercially available solutions control morphological changes and loss of lipid content while achieving a higher refractive index of 1.52. In various embodiments, considerations for transparentization include sample type and thickness, so that shrinkage of the sample is minimized and lipid content and fluorescence are retained.

[0030] In various embodiments, perfusion assist agent in situ release (PARS) includes whole body tissue clearing and phenotyping methods compatible with endogenous fluorescence. In various embodiments, all steps for PARS including retention, clearing and labeling are performed in situ before tissue extraction. In various embodiments, PARS, together with RIMS, converts opaque, intact, whole organisms into optically transparent, fluorescently labeled samples for visualization with conventional confocal microscopy and phenotyping at the cellular, subcellular and / or single molecule transcript levels, as described by Yang et al. in "Single-Cell Phhenotyping within Transparent Intact Tissue through Whole-Body Clearing", Cell. Vol. 158, No. 4, pp. 945 to 958, August 14, 2014 (available online at https: / / doi.org / 10.1016 / j.cell.2014.07.017).

[0031] The biological sample may contain one or more analytes of interest. The optofluidic instrument disclosed herein can be used to detect and analyze a variety of different analytes. In some aspects, the analyte may include any biological substance, structure, part or component to be analyzed. For example, the analyte may include any biomolecule or chemical compound, including macromolecules (such as proteins or peptides, lipids or nucleic acid molecules) or small molecules (including organic or inorganic molecules). The analyte may be a cell or microorganism, including a virus or a fragment or product thereof. The analyte may be any substance or entity for which a specific binding partner (e.g., affinity binding partner) may be developed and detected (e.g., using the optofluidic instrument disclosed herein).

[0032] Analytes of particular interest may include nucleic acid molecules such as DNA (e.g., genomic DNA, mitochondrial DNA, plastid DNA, viral DNA, etc.) and RNA (e.g., mRNA, microRNA, rRNA, snRNA, viral RNA, etc.); and synthetic and / or modified nucleic acid molecules (e.g., including nucleic acid domains comprising synthetic or modified nucleotides such as LNA, PNA, morpholino, etc. or consisting of these substances); protein molecules such as peptides, polypeptides, proteins or prions, or any molecule comprising a protein or polypeptide component, etc., or a fragment thereof. The analyte may be a single molecule or a complex comprising two or more molecular subunits, such as, but not limited to, a complex between a protein or peptide and a nucleic acid molecule (such as DNA or RNA), such as an interactor between a protein and a nucleic acid (e.g., a regulatory factor (such as a transcription factor) and a DNA or RNA).

[0033] In some embodiments, the optofluidic instrument described herein can be used for in situ detection and analysis of cell analytes (such as nucleic acid sequences), such as methods based on fluorescence in situ hybridization (FISH), in situ transcriptome analysis, or in situ sequencing, the cell analytes are, for example, from intact tissues or samples in which spatial information has been preserved. In some aspects, the embodiments can be applied to imaging or detection methods for multiple nucleic acid analysis. In some aspects, the provided optofluidic instrument can be used to detect signals associated with detectable labels of nucleic acid probes that hybridize to target sequences of target nucleic acids in biological samples.

[0034] In some aspects, disclosed herein is a labeling agent (e.g., nucleic acid probe and / or probe group), which is introduced into a cell or used to detect an analyte in a biological sample such as a tissue sample in another way. Labeling agents include probes based on nucleic acids (e.g., primary probes disclosed herein and / or any detectable probes disclosed herein), and may include any of a variety of entities that can be hybridized with nucleic acids by Watson-Crick base pairing, such as DNA, RNA, LNA, PNA, etc. Nucleic acid probes may include a hybridization region that can directly or indirectly bind to at least a portion of a target sequence in a target nucleic acid. Nucleic acid probes may be able to bind to specific target nucleic acids (e.g., mRNA or other nucleic acids disclosed herein).

[0035] Specific probe design can be changed according to application, and any suitable probe or probe group can be utilized, and optical fluid instrument as described herein is used for detection. In some respects, probe as described herein or probe group or intermediate probe (for example, secondary probe and / or higher-level probe) can be selected from the group consisting of circular probe, circularized probe and linear probe. In some embodiments, circular probe is pre-circularized before hybridization with target nucleic acid and / or one or more other probes. In some embodiments, circularized probe is circularized (for example, by connection) when hybridizing with target nucleic acid and / or one or more other probes such as splint. In some embodiments, linear probe can be a probe comprising a target recognition sequence and a sequence not hybridized with target nucleic acid, such as 5' protrusion, 3' protrusion and / or joint or spacer (which can include nucleic acid sequence such as one or more barcode sequences, or non-nucleic acid part). In some embodiments, sequence (for example, 5' protrusion, 3' protrusion and / or joint or spacer) is not hybridized with target nucleic acid, but can hybridize with each other and / or with one or more other probes such as detectably labeled probe hybridization.

[0036] In some embodiments, the primary probes, secondary probes, and / or higher-level probes disclosed herein may include padlock-like probes or probe sets, such as described in one of U.S. Patents 8,551,710, US2020 / 0224244, US2019 / 0055594, US2021 / 0164039, US2016 / 0108458, or US2020 / 0224243, each of which is incorporated herein by reference in its entirety. Any suitable combination of the probe designs described herein may be used.

[0037] In some embodiments, a probe or set of probes described herein (eg, a primary probe or secondary probe, and / or a higher order probe disclosed herein) can comprise two or more moieties. In some cases, the probe can include one or more features and / or be modified based on the following: a split FISH probe or probe set described in WO 2021 / 167526A1 or Goh et al., "Highly specific multiplexed RNA imaging in tissues with split-FISH," Nat Methods 17(7):689-693 (2020), the entire text of which is incorporated herein by reference; a Z-probe or probe set, such as US 7,709,198 B2, US 8,604,182 B2, US 8,951,726 B2, US 8,658,361 B2, or Tripathi et al., "Z Probe, An Efficient Tool for Characterizing Long Non-Coding RNA in FFPE Tissues," Noncoding RNA 4(3):20 (2018), the entire text of which is incorporated herein by reference; an HCR initiator or amplifier, such as US 7,632,641 B2, US2017 / 0009278 A1, US10,450,599 B2, or Choi et al., "Third-generation in situ hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust," Development 145(12): dev165753 (2018), the entire text of which is incorporated herein by reference; PLAYR probes or probe sets, such as US2016 / 0108458 A1 or Frei et al., "Highly multiplexed simultaneous detection of RNAs and proteins in single cells," Nat Methods 13(3): 269-75 (2016), the entire text of which is incorporated herein by reference;PLISH probes or probe sets, such as the PLISH probes or probe sets described in US2020 / 0224243 A1 or Nagendran et al., "Automated cell-type classification in intact tissues by single-cell molecular profiling," eLife 7:e30510 (2018), which are incorporated herein by reference in their entirety; RollFISH probes or probe sets, such as the RollFISH probes or probe sets described in Wu et al., "RollFISH achieves robust quantification of single-molecule RNA biomarkers in paraffin-embedded tumor tissue samples," Commun Biol 1, 209 (2018), which are incorporated herein by reference in their entirety; MERFISH probes or probe sets, such as US2022 / 0064697 A1 or Chen et al., "Spatially resolved, highly multiplexed RNA profiling in single cells,” Science 348(6233): aaa6090 (2015), the entire text of which is incorporated herein by reference; primer exchange reaction (PER) probes or probe sets, such as the PER probes or probe sets described in US2019 / 0106733 A1, the entire text of which is incorporated herein by reference.;

[0038] In some cases, probes and / or probe groups are directly labeled with one or more detectable labels (e.g., optically detectable labels, such as fluorescent moieties), which are detected on the optofluidic instrument disclosed herein. In other cases, probes and / or probe groups comprise a target binding region and one or more nucleic acid barcode sequences that identify an analyte. In these embodiments, the barcode sequence can be detected on the optofluidic instrument disclosed herein to identify the analyte in the sample. In some cases, the probes or probe groups disclosed herein are circularizable probes or probe groups (e.g., padlock probes or padlock-like probes) comprising a barcode region that comprises one or more barcode sequences.

[0039] The probes and / or probe groups described herein may include any suitable number of barcode sequences. In some embodiments, the probe or probe group may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more, 20 or more, 32 or more, 40 or more, or 50 or more barcode sequences. As an illustrative example, a first probe may contain a first target binding sequence, a first barcode sequence, and a second barcode sequence, while a second different probe may contain a second target binding sequence (which is different from the first target binding sequence in the first probe), the same first barcode sequence as in the first probe, but a third barcode sequence instead of a second barcode sequence. Such probes may thus be distinguished by determining various barcode sequence combinations present at a given position in a sample or associated with a given probe.

[0040] In some embodiments, the marker may include an analyte binding portion that interacts with an analyte (e.g., a protein) in a sample (e.g., a cell or tissue sample), and a reporter oligonucleotide comprising one or more barcode sequences associated with the analyte and / or the analyte binding portion. For example, a marker specific for one type of cell feature (e.g., a first protein) may have a first reporter oligonucleotide coupled thereto, while a marker specific for a different cell feature (e.g., a second protein) may have a different reporter oligonucleotide coupled thereto. In some embodiments, the analyte binding portion includes, but is not limited to, proteins, peptides, antibodies (or their epitope binding fragments), lipophilic moieties (such as cholesterol), cell surface receptor binding molecules, receptor ligands, small molecules, bispecific antibodies, bispecific T cell adapters, T cell receptor adapters, B cell receptor adapters, antibody prodrugs, aptamers, monomers, affimers, darpins, and protein scaffolds, or any combination thereof. For a description of exemplary labeling agents, reporter oligonucleotides, and methods of use, see, e.g., U.S. Patent 10,550,429; U.S. Patent Publication 20190177800; and U.S. Patent Publication 20190367969, each of which is incorporated herein by reference in its entirety.

[0041] In some embodiments, nucleic acid probes, probe groups, reporter oligonucleotides, barcode sequences, etc. can be directly detected on the optical fluid instrument disclosed herein (for example, the primary probe includes a detectable label, such as a fluorescent moiety), and / or by using a secondary (or higher order) nucleic acid probe that can bind to the primary probe. In some embodiments, nucleic acid probes (for example, primary probes and / or secondary probes) are compatible with one or more biological and / or chemical reactions. For example, nucleic acid probes disclosed herein can serve as templates or primers for polymerases (for example, circularization probes in rolling circle amplification (RCA) reactions), templates or substrates for ligases, substrates for click chemistry reactions, and / or substrates for nucleases (for example, endonucleases or exonucleases for cutting or digestion). In some cases, a labeling agent (such as a primary probe group) is added to a biological sample (for example, a cell or tissue sample) using an optical fluid instrument, and then detected using an optical fluid instrument (for example, using a detectably labeled primary probe, a detectably labeled oligonucleotide and a sequential hybridization of the primary probe, in situ sequencing (for example, SBS, SBL, SBH), etc.). In some cases, labeling reagents (such as a primary probe set) are added to a biological sample (e.g., a cell or tissue sample) outside of an optofluidic instrument, and the sample is loaded onto an optofluidic instrument disclosed herein for detection (e.g., sequential hybridization using detectably labeled oligonucleotides, in situ sequencing (e.g., SBS, SBL, SBH), etc.).

[0042] In some embodiments, the detection of analytes, probes, probe sets, barcodes, etc. described herein can be performed in situ on an optofluidic instrument disclosed herein. In situ sequencing generally involves incorporating labeled nucleotides (e.g., fluorescently labeled mononucleotides or dinucleotides) or hybridizing labeled primers (e.g., labeled random hexamer) with nucleic acid templates in a sequential, template-dependent manner so that the identity (e.g., nucleotide sequence) of the incorporated nucleotides or labeled primer extension products can be determined, and thus the nucleotide sequence of the corresponding template nucleic acid is determined. Aspects of in situ sequencing methods are described in, for example, Mitra et al., (2003) Anal. Biochem. 320, 55-65 and Lee et al., (2014) Science, 343 (6177), 1360-1363. In addition, examples of methods and systems for performing in situ sequencing are described in US 2016 / 0024555, US 2019 / 0194709, and in US 10,138,509, US 10,494,662, and US 10,179,932.

[0043] In some embodiments, sequencing can be performed by sequencing by synthesis (SBS). In some embodiments, sequencing primers are complementary to sequences at or near a target (e.g., one or more barcodes) to be detected. In such embodiments, sequencing by synthesis can include reverse transcription and / or amplification to generate a template sequence to which a primer sequence can be combined. Exemplary SBS methods include, for example, but not limited to, those described in US2007 / 0166705, US2006 / 0188901, US 7,057,026, US2006 / 0240439, US 2006 / 0281109, US2011 / 005986, US2005 / 0100900, US 9,217,178, US2009 / 0118128, US2012 / 0270305, US2013 / 0260372, and US2013 / 0079232.

[0044] In some embodiments, sequence analysis of nucleic acids (e.g., nucleic acids comprising barcode sequences, such as RCA products) can be performed by sequential hybridization (e.g., sequencing while hybridizing and / or sequential in situ fluorescent hybridization). Sequential fluorescent hybridization can involve sequential hybridization of detection probes comprising oligonucleotides and detectable labels. In some embodiments, methods disclosed herein include sequential hybridization of detectable probes disclosed herein, including detectably labeled probes (e.g., fluorophore-conjugated oligonucleotides) and / or probes that are not detectably labeled themselves but are capable of binding to detectably labeled probes (e.g., via nucleic acid hybridization) and being detected by detectably labeled probes. Exemplary methods comprising sequential fluorescent hybridization of detectable probes are described in US2019 / 0161796, US2020 / 0224244, US2022 / 0010358, US2021 / 0340618 and WO 2021 / 138676, MERFISH (e.g., described in Moffitt, (2016) Methods in Enzymology, 572, 1-49), and hybridization-based in situ sequencing (HybISS) (e.g., described in Gyllborg et al., Nucleic Acids Res (2020) 48(19):e112), all of which are incorporated herein by reference.

[0045] In some embodiments, sequencing can be performed using sequencing by ligation (SBL). Such technology utilizes DNA ligase to incorporate oligonucleotides and identify the incorporation of such oligonucleotides. Oligonucleotides generally have different tags related to the identity of specific nucleotides in the sequence to which the oligonucleotides hybridize. Aspects and features involved in sequencing by ligation are described in, for example, Shendure et al., Science (2005), 309: 1728-1732, and US 5,599,675; US 5,750,341; US ​​6,969,488; US 6,172,218; and US 6,306,597. Exemplary techniques for in situ SBL include, but are not limited to, STARmap (e.g., described in Wang et al., (2018) Science, 361 (6499) 5691) and US 2021 / 0164039).

[0046] In some embodiments, a probe barcode (e.g., a plurality of probes or probe sets comprising one or more barcode sequences) or its complement or product is targeted by a detectably labeled detection oligonucleotide (e.g., a fluorescently labeled oligonucleotide). In some embodiments, one or more decoding schemes (e.g., sequential rounds of fluorescent probe hybridization) are used on an optofluidic instrument disclosed herein to decode a signal such as fluorescence for sequence identification. In any embodiment herein, any suitable method or technique may be used to analyze (e.g., detect or sequence using an optofluidic instrument disclosed herein) a barcode (e.g., a primary and / or secondary barcode sequence), including those described herein, such as RNA target sequential detection (RNASPOT), sequential fluorescence in situ hybridization (seqFISH), single molecule fluorescence in situ hybridization (smFISH), multiple error robust fluorescence in situ hybridization (MERFISH), hybridization-based in situ sequencing (HybISS), in situ sequencing, targeted in situ sequencing, fluorescence in situ sequencing (FISSEQ), or spatially resolved transcript amplicon readout mapping (STARmap). In some embodiments, the methods provided herein include analyzing barcodes by sequential hybridization and detection with multiple labeled probes (e.g., detection oligonucleotides or detectable probes). Exemplary decoding schemes are described in Eng et al., "Transcriptome-scale Super-Resolved Imaging in Tissues by RNA SeqFISH+," Nature 568(7751):235-239(2019); Chen et al., Science; 348(6233):aaa6090(2015); Gyllborg et al., Nucleic Acids Res (2020) 48(19):e112; US10,457,980B2; US2016 / 0369329 A1; WO 2018 / 026873A1; and US2017 / 0220733 A1, all of which are incorporated by reference in their entirety. In some embodiments, these assays enable simultaneous signal amplification, combined decoding, and error correction schemes.

[0047] It should be understood that although the above discussion relates to optofluidic instruments that can be used for in situ target molecule detection via probe hybridization, the discussion here is equally applicable to any optofluidic instrument that employs any imaging or target molecule detection technology. That is, for example, an optofluidic instrument may include a fluidics module that includes fluids required to establish experimental conditions required to detect target molecules in a sample. In addition, such an optofluidic instrument may also include a sample module configured to receive a sample, and an optical module including an imaging system for irradiating (e.g., exciting one or more fluorescent probes within the sample) and / or imaging light signals received from the detected sample. The optofluidic instrument may also include other auxiliary modules configured to facilitate the operation of the optofluidic instrument, such as but not limited to a cooling system, a motion calibration system, and the like.

[0048] In some embodiments, the aforementioned sample preparation or processing steps may have to be performed in a sealed hole or chamber of the optofluidic instrument. For example, the sample preparation or processing steps may include culturing the sample in a sample device of the optofluidic instrument (e.g., in the presence of a reagent), and the culturing may be performed in a sealed chamber, for example, to avoid evaporation. The heat for culturing may be provided by a heat source that is thermally coupled to the sample device. For example, a sealed sample device that holds the sample may be placed in a thermal cycler, for example, to amplify molecules to be detected in the sample (e.g., DNA, RNA, etc.). In this case, the heated cover of the thermal cycler may be in direct contact with the side of the sample device or otherwise thermally coupled to the side of the sample device, thereby transferring heat to the sample to be cultured. Other heat sources for culturing samples may include, but are not limited to, thermoelectric coolers (TECs). For example, the hot side of the TEC may be in direct contact with the side of the sample device or otherwise thermally coupled to the side of the sample device, to provide heat for sample culture.

[0049] Existing solutions for sealing holes to form sealed chambers utilize sealing tape or other coverings, and are placed on the opening of the hole. In most cases, the tape or covering is flush with the opening of the hole. This is to reduce the space between the sample and the tape so that heat can be effectively transferred to the sample when transferred via the tape. For example, when heat is transferred to the sample via the thermal cycler cover of the heating that is in contact with the tape or flush (for example, in the presence of fluid), when the space between the sample and the tape is low, heat can be more effectively transferred to the sample. However, this may be undesirable because interference with the sample or hole can result in forming a fluid capillary bridge between the top surface and the tape at the bottom of the hole. For example, the fluid in the hole may be wicked (wick up) and form a bridge, producing undesirable consequences such as but not limited to dehumidification, leakage, etc. of the sample / hole. Therefore, a system for forming a sealed chamber is needed to at least solve the defect.

[0050] These and other aspects and implementations are discussed in detail below. The foregoing information and the following detailed description include illustrative examples of various aspects and implementations, and provide an overview or framework for understanding the nature and characteristics of the claimed aspects and implementations. The accompanying drawings provide illustration and further understanding of the various aspects and implementations, and are incorporated into and constitute a part of this specification.

[0051] Example description of terms

[0052] As used in the specification, "a" or "an" may mean one or more. As used in the claims, when used in conjunction with the word "comprising", the word "a" or "an" may mean one or more than one. Some embodiments of the present disclosure may consist of or consist essentially of one or more elements, method steps, and / or methods of the present disclosure. It is contemplated that any method or composition described herein may be implemented with respect to any other method or composition described herein, and different embodiments may be combined.

[0053] As used herein, "substantially" means sufficiently effective for the intended purpose. Thus, the term "substantially" allows for minor, inconsequential variations from an absolute or perfect state, dimension, measurement, result, etc., such as would be expected by one of ordinary skill in the art but which would not significantly affect overall performance. When used with respect to a value or a parameter or characteristic that can be expressed as a value, "substantially" means a deviation within ten percent.

[0054] The use of the term "or" in the claims is intended to mean "and / or" unless it is expressly indicated to refer only to alternatives or the alternatives are mutually exclusive, although the present disclosure supports definitions referring only to alternatives and "and / or". For example, "x, y, and / or z" may refer to "x" alone, "y" alone, "z" alone, "x, y and z", "(x and y) or z", "x or (y and z)", or "x or y or z". It is specifically contemplated that x, y, or z may be specifically excluded from the embodiments. As used herein, "another" may refer to at least a second or more.

[0055] The term "ones" means more than one.

[0056] As used herein, the term "plurality" may be 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.

[0057] As used herein, the term "a set" means one or more. For example, a set of items includes one or more items.

[0058] As used herein, when used with a list of items, the phrase "at least one of" means that different combinations of one or more of the listed items may be used, and only one of the items in the list may be required. The item may be a specific object, thing, step, operation, process, or category. In other words, "at least one of..." means that any combination of the items in the list or any number of the items may be used, but not all of the items in the list are required. For example, but not limited to, "at least one of item A, item B, or item C" means item A; item A and item B; item B; item A, item B, and item C; item B and item C; in some cases, "at least one of item A, item B, or item C" means, but is not limited to, two of item A, one of item B, and ten of item C; four of item B, and seven of item C; or some other suitable combination

[0059] As used herein, the term "approximately" refers to the usual error range of the corresponding value that is easily known. References to "about" values ​​or parameters herein include (and describe) embodiments involving the value or parameter itself. For example, a description involving "about X" includes a description of "X". In some embodiments, as understood by those skilled in the art, "approximately" can refer to ± 15%, ± 10%, ± 5% or ± 1%.

[0060] As used herein, in some cases, the term "thermally coupled" or variations thereof refers to a configuration of two or more components that allow them to exchange heat directly (e.g., direct contact) or indirectly with each other, such that the temperature of one or both of them is increased or decreased.

[0061] Although the present teachings are described in conjunction with various embodiments, the present teachings are not intended to be limited to such various embodiments. On the contrary, as will be appreciated by those skilled in the art, the present teachings encompass various alternatives, modifications and equivalents.

[0062] When describing various embodiments, this specification may present a method and / or process as a specific sequence of steps. However, to the extent that the method or process does not rely on the specific order of the steps set forth herein, the method or process should not be limited to the specific sequence of steps described, and those skilled in the art can readily appreciate that the sequence can be varied and still remain within the spirit and scope of the various embodiments.

[0063] Optofluidic Instrumentation for Analysis of Biological Samples

[0064] Figure 1An exemplary workflow for analyzing a biological sample 110 (e.g., a cell or tissue sample) using an optofluidic instrument 120 according to various embodiments is shown. In various embodiments, the sample 110 can be a biological sample (e.g., a tissue) containing molecules such as DNA, RNA, proteins, antibodies, etc. For example, the sample 110 can be a sliced ​​tissue that is processed to obtain its RNA for labeling with a circularizable DNA probe. The ligation of the probe can produce a circular DNA probe that can be enzymatically amplified and combined with a fluorescent oligonucleotide, which can produce a bright signal that is convenient for imaging and has a high signal-to-noise ratio.

[0065] In various embodiments, the sample 110 can be placed in an optofluidic instrument 120 for analyzing and detecting molecules in the sample 110. In various embodiments, the optofluidic instrument 120 can be a system configured to promote experimental conditions that are conducive to detecting target molecules. For example, the optofluidic instrument 120 can include a fluidics module 140, an optical module 150, a sample module 160, and an auxiliary module 170, and these modules can be operated by a system controller 130 to generate experimental conditions for detecting molecules in the sample 110 by a selected probe (e.g., a circularizable DNA probe), and to facilitate imaging of the detected sample (e.g., by an imaging system of the optical module 150). In various embodiments, the various modules of the optofluidic instrument 120 can be independent components that communicate with each other, or at least some of them can be integrated together.

[0066] In various embodiments, the sample module 160 can be configured to receive the sample 110 into the optofluidic instrument 120. For example, the sample module 160 can include a sample interface module (SIM) configured to receive a sample device (e.g., a cartridge) on which the sample 110 can be deposited. That is, the sample 110 can be placed in the optofluidic instrument 120 by depositing the sample 110 (e.g., a sliced ​​tissue) on the sample device and then inserting the sample device into the SIM of the sample module 160. In some cases, the sample module 160 can also include an XY stage on which the SIM is mounted. The XY stage can be configured to move the SIM mounted thereon (e.g., and thus move the sample device containing the sample 110 inserted therein) in a vertical direction along the two-dimensional (2D) plane of the optofluidic instrument 120. Additional discussion related to SIM can be found in U.S. Provisional Application No. 63 / 348,879, filed on June 3, 2022, entitled “Methods, Systems, and Devices for Sample Interface,” the entire contents of which are incorporated herein by reference.

[0067] Experimental conditions that are beneficial to detecting molecules in sample 110 can depend on the target molecule detection technology adopted by optofluidic instrument 120. For example, in various embodiments, optofluidic instrument 120 can be a system configured to detect molecules in sample 110 via hybridization of probes. In this case, the experimental conditions can include molecular hybridization conditions, which cause the hybridization intensity of target molecule (e.g., nucleic acid) to probe (e.g., oligonucleotide) to be significantly higher than the hybridization intensity when there is a single base mismatch when the probe sequence is complementary to the target molecule. Hybridization conditions include preparing sample 110 using reagents such as washing / stripping reagents, probe reagents, etc., and such reagents can be provided by fluidics module 140. Examples of washing buffers include, but are not limited to, deionized water, phosphate buffered saline (PBS), PBS containing dimethyl sulfoxide (DMSO), etc. Stripping buffer can be, but are not limited to, DMSO, surfactants, and / or the like. In some cases, the surfactant can be or include polysorbate 20. In some cases, the stripping buffer can include a surfactant in a weight ratio of about 0.1%. The probe reagent can be a fluorescent probe, such as, but not limited to, an oligonucleotide probe.

[0068] In various embodiments, the fluidics module 140 may include one or more components that can be used to store reagents, and to transport the reagents to and from a sample device containing a sample 110. For example, the fluidics module 140 may include a reservoir configured to store reagents, and a waste container configured to collect reagents (e.g., and other wastes) after use of the optofluidic instrument 120 to analyze and detect molecules of the sample 110. In addition, the fluidics module 140 may also include pumps, tubes, pipettes, etc. configured to facilitate the transport of reagents to the sample device (e.g., and thus to the sample 110). For example, the fluidics module 140 may include a pump ("reagent pump") configured to pump a cleaning / stripping reagent to the sample device for cleaning / stripping the sample 110 (e.g., and other cleaning functions, such as cleaning the objective lens of the imaging system of the optical module 150). In some cases, a stage (e.g., a YZ stage) can be configured to move a pipette in one or more directions toward and away from a sample device containing sample 110 so that various reagents can be dispensed in the sample device and spent reagents can be extracted from the sample device.

[0069] In various embodiments, the auxiliary module 170 may be a cooling system of the optofluidic instrument 120, and the cooling system may include a network of coolant delivery pipes configured to deliver coolant to various modules of the optofluidic instrument 120 to regulate their temperatures. In this case, the fluidics module 140 may include a coolant reservoir for storing coolant and a pump (e.g., a "coolant pump") for generating a pressure difference, thereby forcing the coolant to flow from the reservoir to the various modules of the optofluidic instrument 120 via the coolant delivery pipe. In some cases, the fluidics module 140 may include a return coolant reservoir, which may be configured to receive and store return coolant, i.e., heated coolant that flows back into the return coolant reservoir after absorbing heat discharged by the various modules of the optofluidic instrument 120. In this case, the fluidics module 140 may also include a cooling fan configured to force air (e.g., cold air and / or ambient air) into the return coolant reservoir to cool the heated coolant stored therein. In some cases, the fluidics module 140 may also include a cooling fan configured to force air directly into components of the optofluidic instrument 120 to cool the components. For example, the fluidics module 140 may include a cooling fan configured to direct cool or ambient air into the system controller 130 to cool the system controller.

[0070] As described above, the optofluidic instrument 120 may include an optical module 150 that includes various optical components of the optofluidic instrument 120, such as, but not limited to, a camera, an illumination module (e.g., an LED), an objective lens, and / or the like. The optical module 150 may include a fluorescence imaging system configured to image fluorescence emitted by a probe (e.g., an oligonucleotide) in the sample 110 after the probe is excited by light from an illumination module of the optical module 150.

[0071] In various embodiments, the system controller 130 can be configured to control the operation of the optofluidic instrument 120 (e.g., and the operation of one or more modules thereof). In some cases, the system controller 130 can take various forms, including a processor, a single computer (or computer system), or multiple computers in communication with each other. In various embodiments, the system controller 130 can be communicatively coupled to a data storage device, a set of input devices, a display system, or a combination thereof. In some cases, some or all of these components can be considered part of the system controller 130 or otherwise integrated with the system controller, can be independent components that communicate with each other, or can be integrated together. In other examples, the system controller 130 can be a cloud computing platform, or can communicate with a cloud computing platform.

[0072] In various embodiments, the optofluidic instrument 120 can analyze the sample 110 and can generate an output 190 that includes an indication of the presence of target molecules in the sample 110. For example, with respect to the exemplary embodiment discussed above where the optofluidic instrument 120 employs hybridization techniques to detect molecules, the optofluidic instrument 120 can subject the sample 110 to successive rounds of fluorescent probe hybridization (using two or more sets of fluorescent probes, where each set of fluorescent probes is excited by a different color channel) and imaged to detect target molecules in the probed sample 110. In this case, the output 190 can include an optical signature (e.g., a codeword) specific to each gene, which allows the target molecules to be identified.

[0073] Sample Holder and Cover Assembly

[0074] Figure 2 A sample device 200 is shown for receiving a sample to be probed to detect molecules therein. The sample device 200 is configured to be inserted into or otherwise placed in a sample module (e.g., such as Figure 1 The sample device 200 may be a sample module 160 of the optofluidic instrument 120. In some cases, the sample device 200 may be a cartridge. In various embodiments, the sample device 200 may include a top portion 210 and a bottom portion 220 configured to releasably engage or couple to each other with the substrate 230 located therebetween such that the hole 240 is formed. That is, when the top portion 210 and the bottom portion 220 are releasably coupled to each other, a gap configured to receive the substrate 230 is formed therebetween.

[0075] In some cases, the top portion 210 can have one or more snap joints 270 (e.g., cantilevered snap joints), and the bottom portion 220 can have one or more lugs 260 (e.g., cantilevered lugs). In yet other cases, the top portion 210 can have one or more lugs, and the bottom portion 220 can have one or more snap joints. In various embodiments, the one or more snap joints 270 and the one or more lugs 260 can be engaged or coupled to each other, with a substrate sandwiched therebetween to lock the top portion 210 and the bottom portion 220 together, thereby forming a hole 240 having a substrate 230 (e.g., particularly an exposed area of ​​the substrate 230) as a base. That is, the hole 240 is formed in the top portion 210 of the sample device 200 and is defined by the substrate 230 (e.g., a base forming the hole 240) and the gasket 250 (e.g., a wall forming the hole 240). In some cases, the substrate 230 can be a glass slide.

[0076] In various embodiments, the sample device 200 can also include a gasket 250 that can serve as a wall for the hole 240. In some cases, the gasket 250 is configured to form a seal between the base 230 and the top portion 210. In some cases, the gasket 250 can provide a "stadium seat" wall for the hole 240. That is, the gasket 250 can have an angled inner surface between the first opening and the second opening, wherein the cross-sectional area of ​​the first opening is smaller than the cross-sectional area of ​​the second opening. When positioned in the sample device 200, the first opening of the gasket 250 contacts the base 230 to form a seal that is a boundary of the exposed area of ​​the base 230. In addition, the angled inner surface of the gasket 250 forms a receding wall (e.g., corresponding to the "stadium seat" wall of the hole 240) that terminates at the larger second opening of the gasket.

[0077] In various embodiments, substrate 230 may include a fiducial or marking configured to guide placement of a sample on the substrate. In some cases, substrate 230 may include a sample slice boundary 290 that indicates an area of ​​the substrate where the sample may be positioned. Sample slice boundary 290 may be positioned within an imageable slice boundary 295 that surrounds an imaging system (e.g., such as Figure 1 The imaging system of the optical module 150 of the optofluidic instrument 120 can access and image the area of ​​the substrate 230 when the sample device is inserted into the sample module of the optofluidic instrument. The substrate 230 can have a linear dimension (e.g., length, width) of about 1 cm to about 3 cm, about 1.5 cm to about 2.5 cm, including values ​​and subranges therebetween. For example, the sample slice boundary 280 can have a linear dimension of approximately 1 cm.

[0078] In various embodiments, the top portion 210 can have a plurality of snap-fit ​​joint elements 280a to 280c (alternatively referred to as 280). The snap-fit ​​joint element 280 of the top portion 210 can be any suitable feature configured to engage with another snap-fit ​​joint element (e.g., the snap-fit ​​joint element of the cover of the sample device 200) to form a snap-fit ​​joint. For example, the snap-fit ​​joint element 280 of the top portion can be an orifice, a recessed portion, etc., and can be configured to form a snap-fit ​​joint when being coupled to another snap-fit ​​joint such as but not limited to (e.g., the sample device cover) clip, hook, etc. A plurality of snap-fit ​​joint elements 280 can be arranged on the top portion 210 so that at least one pair of multiple snap-fit ​​joint elements 280 are positioned substantially relative to each other. In some cases, the term "substantially relative" used herein can refer to at least one pair of multiple snap-fit ​​joint elements 280 positioned relative to each other across the hole 240.

[0079] For example, Figure 2As shown, the pair of snap-fit ​​joint elements 280a and 280c (or 280b and 280c) are positioned across the hole 240 from each other and can be described as being positioned "substantially opposite" to each other. In other cases, the term "substantially opposite" can refer to at least one pair of multiple snap-fit ​​joint elements 280 being spaced apart from each other by at least half of the length of the hole 240. That is, for example, if one snap-fit ​​joint element (e.g., 280a or 280b) is located on one side of the hole 240, then another snap-fit ​​joint element located on the top portion 210 facing away from the other snap-fit ​​joint element 280a or 280b by at least half of the length of the hole 240 can be described as being positioned substantially opposite to the other snap-fit ​​joint element 280a or 280b. The number of multiple snap-fit ​​joint elements 280 can be 2, 3, 4, 5, etc.

[0080] FIG. 3A to FIG. 3C Figure 1 shows a bridge in a sample device sealed with tape according to various embodiments. Figure 2 The sample device 310 that is the same as or substantially similar to the sample device 200 may include a substrate 350 that may be Figure 2 The sample device 310 may be the same or substantially similar to the substrate 230 of the sample device 310. In various embodiments, the sample device 310 may be used to prepare or process a sample to detect molecules therein, and part of the sample preparation or processing may be culturing the sample in the sample device 310 when the sample device 310 is sealed with the cover. As described above, existing solutions for sealing a well containing a sample for culturing purposes include using a flat polymer tape 320 (e.g., a polyethylene tape, a polypropylene tape, a polyester tape, a polyolefin tape, etc.) to reduce the space between the sample therein and the tape 320 (e.g., so that heat provided by a heat source above the tape 320 is effectively transferred to the sample).

[0081] However, when the sample is in the fluid or reagent 330, such a solution may have undesirable results, such as forming bridges 360 (e.g., capillary bridges) between the strip 310 and the substrate 350, as shown in FIG. Figure 3B This bridging problem may occur when the sample device 310 is disturbed, for example, due to vibrations from a vibration source, which is located inside the optofluidic instrument (e.g., a pump, a cooling fan, etc.) or outside the optofluidic instrument (e.g., the background environment, pedestrians moving near the optofluidic instrument, etc.). In this case, the reagent 330 in the sample device 310 can be wicked and contact the inner surface of the belt 320, thereby forming a bridge 360. Figure 3CAn exemplary illustration of a bridge 370 formed in a sample device 380 when the sample device 380 sealed with a polymer band 390 is temporarily tilted is shown. Bridges 360, 370 may be a source of leakage or may cause dewetting of the sample in the sample device 310, 380 and are therefore undesirable. Compared to existing solutions that reduce the space between the sample in the well and the cover or band sealing the well, various embodiments of the present disclosure disclose a sample device cover with a raised cover that can reduce or eliminate the formation of bridges covered by the cover in the sample device.

[0082] FIG. 4A to FIG. 4D A method for covering a sample device such as Figure 2 View of an exemplary cover 400 of a sample device 200 ). Figure 4A and Figure 4B An isometric view and a bottom view of the cover 400 in various embodiments are shown. In addition, in various embodiments, Figure 4C A cross-sectional view of the cover 400 is shown, and Figure 4D A close-up view of the snap-fit ​​element 410 of the cover 400 is shown. In some embodiments, the cover 400 includes a cover 402 having an outer surface 405 and an inner surface 425 separated by a thickness 435 of the cover 402. The outer surface 405 can be shaped to fully engage with the thermal cycler cover. For example, the outer surface 405 can be a flat outer surface, such as FIG. 4A to FIG. 4DAs shown. The cover 400 also includes a skirt 415 extending around the periphery 430 of the cover 402. That is, the skirt 415 can extend around the periphery 430 of the inner surface 425 of the cover 400. In some embodiments, the skirt 415 and the inner surface 425 define a recessed portion 427, which has a height 440 between the edge or periphery of the inner surface 425 and the skirt 415. In some embodiments, the cover 400 is made of a polymer. In various embodiments, the cover is made of at least one of the following materials: polyethylene (PE), polyethylene terephthalate (PET), high-density PET, polyurethane (PU), polystyrene (PS), polypropylene (PP), polycarbonate (PC), acrylonitrile butadiene styrene (ABS), acetal, acrylic, PEEK, polyvinyl chloride (PVC), polyamide, polyimide, polyamideimide, fluorinated ethylene propylene (FEP), ethylene tetrafluoroethylene (ETFE), polyphenylene oxide and / or polyphenylene sulfone (PPSU). In some embodiments, the cover 400 is manufactured using injection molding. In some embodiments, the cover 400 is manufactured using 3D printing technology. In various embodiments, the cover 400 is manufactured using vacuum thermoforming. In some embodiments, the skirt 415 can be angled relative to the inner surface 425, that is, the skirt 415 can extend around the periphery 430 of the inner surface 425 at an angle 445. In some embodiments, the angle 445 is less than 90 degrees. In some embodiments, the angle 445 is greater than 90 degrees. For example, the angle 445 can be in the range of from about 75° to about 120°, from about 90° to about 120°, from about 100° to about 120°, from about 110° to about 120°, including values ​​and sub-ranges therebetween. In some embodiments, an angle 445 greater than 90° is used for design for manufacturability purposes (e.g., for injection molding).

[0083] In various embodiments, the skirt 415 includes a plurality of snap-fit ​​elements 410a, 410b, 410c (collectively 410) that are arranged substantially opposite to each other on the skirt 415. In some cases, the term "substantially opposite" as used herein may refer to at least one pair of the plurality of snap-fit ​​elements 410 being positioned opposite to each other across the outer surface 405 of the cover 402 or cap 400. For example, Figure 4AAs shown, the pair of snap-fit ​​joint elements 410a and 410c (or 410b and 410c) are positioned across the cover 402 from each other and can be described as being positioned substantially opposite to each other. In other cases, the term "substantially opposite" can refer to at least one pair of multiple snap-fit ​​joint elements 410 being spaced apart from each other by at least half of the length of the cover 402. That is, for example, if one snap-fit ​​joint element (e.g., 410a or 410b) is located on one side of the cover 402, then another snap-fit ​​joint element located on the skirt 415 facing away from the other snap-fit ​​joint element 410a or 410b by at least half of the length of the cover 402 can be described as being positioned substantially opposite to the other snap-fit ​​joint element 410a or 410b. The number of the multiple snap-fit ​​joint elements 410 can be 2, 3, 4, 5, etc. In some cases, only one snap joint element 410c of the plurality of snap joint elements 410 may be on one side of the cover 402, while the remainder (e.g., two snap joint elements 410a, 410b, or three snap joint elements of the plurality of snap joint elements 410) may be positioned substantially opposite to one snap joint element 410c of the plurality of snap joint elements 410.

[0084] In various embodiments, the skirt 415 can include a flange 420. In some cases, the flange 420 can extend around the edge or perimeter of the skirt 415 and can also extend outwardly parallel to the outer surface 405 of the cover 402. In some cases, the skirt 415 can have the shape of a closure strip, wherein one edge of the closure strip corresponds to an edge that intersects the inner surface 425 of the cover 400, and the other edge includes a flange 420 that extends around the cover 402 and extends outwardly parallel to the outer surface 405 of the cover 402 or cover 400. In various embodiments, the aforementioned plurality of snap-fit ​​elements 410 of the skirt 415 can be located on the flange 420 of the skirt 415.

[0085] In various embodiments, the plurality of snap-fit ​​elements 410 of the cover 400 can be configured to engage or couple to a corresponding plurality of snap-fit ​​elements of a sample device, such as a Figure 2 The plurality of snap-fit ​​elements 280 of the cover 400 can be provided. For example, the snap-fit ​​element 410 of the cover 400 can be a clip. For example, the snap-fit ​​element 410 can be a cantilevered clip configured to be clamped on an aperture or recess on the top portion 210 of the sample device 200. Figure 4DA close-up view of a snap-fit ​​element 410 in various embodiments is shown. The snap-fit ​​element 410 includes a tab segment 450 and a support segment 475. In some cases, the support segment 475 can extend from the skirt 415. In some cases, the support segment 475 can extend from the flange 420 and in some cases can have the same thickness as the flange 420. In addition, the support segment 475 can support the tab segment 450.

[0086] In various embodiments, the tab segment 450 of the snap connector element 410 may include a first segment 460 and a second segment 455, each of which is angled relative to the support tab 475. For example, the first segment 460 may be at a first angle 485 relative to the support tab 475, and the second segment 455 may be at a second angle 480 relative to the support tab 475. In some cases, the first angle 485 and the second angle 480 are equal to each other. For example, each of the first angle 485 and the second angle 480 is equal to about 90°. In other cases, the first angle 485 and the second angle 480 are different from each other. In some cases, the first angle 485 is greater than about 90°, and the second angle 480 is less than about 90°. In some cases, the second angle 480 is greater than about 90°, and the first angle 485 is less than about 90°. In some cases, the first angle 485 and / or the second angle 480 can be in a range from about 70° to about 110°, from about 70° to about 90°, from about 75° to about 90°, from about 90° to about 110°, from about 95° to about 110°, from about 95° to about 115°, including values ​​and sub-ranges therebetween.

[0087] In some cases, the first portion 460 and / or the second portion 455 of the tab segment 450 include a locking lip 470 and / or a nob 465 at their respective ends located distally of the support segment 475. That is, as described above, the support segment 475 supports the tab segment 450, that is, the support segment 475 supports the first portion 460 and the second portion 455. In this case, the first portion 460 includes a locking lip 470 at one of its ends located distally of the position where the support segment 475 supports the first portion 460. Similarly, the second portion 455 includes a nob 465 at one of its ends located distally of the position where the support segment 475 supports the second portion 455. In some cases, the locking lip 470 may extend from the first portion 460 toward the cover 402. For example, the locking lip 470 may extend from the first portion 460 at an angle equal to or greater than about 90° but less than about 105°. In some cases, the contact 465 may extend from the second portion 455 away from the cover 402 (eg, at an angle of approximately 90° relative to the second portion 455 ).

[0088] FIG. 5A to FIG. 5D The engagement of the cover with the sample device according to various embodiments is shown ( FIG. 5A to FIG. 5C ), including in the presence of a thermal cycling cover ( Figure 5D ). In various embodiments, as described above, the sealed chamber of the optofluidic instrument can be used to prepare or process a sample to detect molecules in the sample using a probe. For example, the sample can be cultured in the presence of a reagent in the sealed chamber. In various embodiments, a sealed chamber suitable for this purpose can be formed by assembling a sample device 530 and a sample device cover 510. In some cases, the sample device 530 can be the same or substantially similar to the sample device 200, and the cover 510 can be the same or substantially similar to the cover 400.

[0089] In various embodiments, the snap-fit ​​elements of the cover 510 and the snap-fit ​​elements of the sample device 530 can be configured such that when the cover 510 is engaged or in contact with the sample device 530, the corresponding snap-fit ​​elements of the cover 510 and the sample device 530 align and couple with each other to lock the cover 510 in place on the sample device 530. That is, for example, the number of snap-fit ​​elements 520a, 520b, 520c (collectively 520) of the cover 510 can be the same as the number of snap-fit ​​elements 560a, 560b, 560c (collectively 560) of the sample device 530. In some cases, there may be two snap-fit ​​elements on one side (snap-fit ​​elements 520a, 520b for cover 510 and snap-fit ​​elements 560a, 560b for sample device 530) and one snap-fit ​​element on the opposite side (snap-fit ​​element 520c for cover 510 and snap-fit ​​element 560c for sample device 530) for both cover 510 and sample device 530. This configuration is particularly desirable because it allows a person to attach and detach cover 510 from sample device 530 using only one hand. For example, a person may place their index and middle fingers on snap-fit ​​elements 520a, 520b of cover 510 and their thumb on snap-fit ​​element 520c of cover 510 to place cover 510 on sample device 530 and remove the cover therefrom.

[0090] The placement of the cover 510 on the sample device 530 may be described below with reference to the specific configuration and Figure 4D 4. However, it should be understood that similar discussions will apply for any other configurations of snap-fit ​​elements of the cover 510 and sample device 530 (e.g., different numbers, positions, etc.). In some cases, a person can pull the corresponding contacts 465 of the two snap-fit ​​elements 520a, 520b with their index and middle fingers, while pushing the contact 465 of the snap-fit ​​element 520c toward the other snap-fit ​​element with their thumb. This can pull the tab section 450 of the snap-fit ​​element 520 toward the support section 475, thereby causing the second angle 480 to decrease and the first angle 485 to increase, further causing the first portion 460 to pull away from the support section 475. When the cover approaches the sample device with the snap-fit ​​elements aligned, the locking lip 470 is pulled away from the support section 475 far enough to enter the corresponding aperture or recess of the sample device and lock in the aperture or recess when the contact is released.

[0091] In some cases, the cover 402, the snap connector element 410, or the tab segment 450 may be made of a material that is flexible enough to allow the tab segment 450 to be pulled or pushed without breaking. For example, the cover 402, the snap connector element 410, or the tab segment 450 may be made of a polymer material, such as, but not limited to, a polyphenylsulfone material, a polyethylene material, a polyurethane material, a polyethylene terephthalate material, a polystyrene material, a polycarbonate material, a polypropylene material, or a combination thereof. The cover 402, the snap connector element 410, or the tab segment 450 may be made of any one or more of these materials using any suitable manufacturing technology including, but not limited to, 3D printing, injection molding, rapid casting, and the like.

[0092] In various embodiments, when the cover 510 is placed on the sample device 530, the flange of the cover 510 contacts the perimeter of the hole 550 of the sample device 530. For example, when the cover 510 contacts the sample device 530, the flange of the cover 510 contacts and remains flush with the outer perimeter of the gasket 540. In addition, as described above, the corresponding snap-fit ​​elements 520 and 560 of the covers 510 and 530 lock with each other, thereby forming a seal between the outer perimeter of the cover 510 and the gasket 540 (e.g., the outer perimeter of the hole 550). For example, the snap-fit ​​element 520 of the cover 510 can be a cantilevered clip, and the snap-fit ​​element 560 of the sample device 530 can be an aperture or a recess. In this case, when the cover 510 is placed on the sample device 530 (e.g., the flange of the cover 510 contacts the outer periphery of the gasket 540), the cantilevered clamp can be clamped into the corresponding hole or recess of the sample device 530 to lock the cover 510 to the sample device 530. In this case, a sealed chamber is formed in the hole 550, which is defined by the inner surface of the cover of the cover 510 as a top plate, the base 555 as a bottom plate, and the inner surface of the gasket 540 and the inner surface of the skirt of the cover 510 sealed together as a wall. Due to the recessed cover of the cover 510 (e.g., the recessed Figure 4C The top plate of the sealed chamber is higher than the hole 550 if a sealed sample device is used. Figure 5B (Isometric view), Figure 5C (side view) and Figure 5D The sealed chamber of the assembly of sample device 530 and cover 510 shown in (side view of thermal cycler cover 570 pressing on the assembly) will reduce or eliminate any bridging problems that may be caused by disturbances (e.g., vibrations) to the assembly when fluids (e.g., reagents) are present in the assembly.

[0093] Figure 6 Illustrative examples of cross-sectional views of covers including snap-fit ​​connector elements according to various embodiments are provided. Figure 4DAs described, the snap-fit ​​joint element includes a tab segment 450 connected to a support segment 475. In various embodiments, the tab segment 450 of the snap-fit ​​joint element 410 includes a first portion 460 and a second portion 455 connected to the support segment 475. In some cases, the first portion 460 and / or the second portion 455 extend substantially perpendicular to the support segment 475. In some cases, each of the first portion 460 and the second portion 455 has a proximal end connected to the support segment 475 and extends away from the support segment 475 to the distal end. In some cases, the first portion 460 includes a contact 465 located at the distal end. In some cases, the second portion 455 includes a locking lip 470 located at the distal end. That is, as described above, the support segment 475 supports the tab segment 450, that is, the support segment 475 supports the first portion 460 and the second portion 455. In some cases, the tab segment 450 is configured to actuate (e.g., bend, pivot, hingedly rotate, etc.) at the location where the first portion 460 and the second portion 455 are connected to the support segment 475. For example, a user can apply a force to the contact 465 to rotate the tab segment 450 about the connection point with the support segment 475.

[0094] FIG. 5A to FIG. 5D FIG. 5 shows the engagement of the cover 510 with the sample device according to various embodiments ( FIG. 5A to FIG. 5C ), including in the presence of a thermal cycling cover ( Figure 5D ). In various embodiments, as described above, the sealed chamber of the optofluidic instrument can be used to prepare or process a sample to detect molecules in the sample using a probe. For example, the sample can be cultured in the presence of a reagent in the sealed chamber. In various embodiments, a sealed chamber suitable for this purpose can be formed by assembling a sample device 530 and a sample device cover 510. In some cases, the sample device 530 can be the same or substantially similar to the sample device 200, and the cover 510 can be the same or substantially similar to the cover 400.

[0095] In addition, Figure 6, the angle "θ1" formed by the second section 455 and the support section 475 is equal to or substantially equal to the angle "θ2" formed by the intersection of the first section 460 and the support section 475. In various embodiments, θ1 and θ2 are equal, or approximately equal to 90°. In addition, as shown, the width w1 (or thickness) of the first portion 460 at the root 790 can be reduced to a second width w2 (or thickness) at the base 491 (that is, the first portion 460 can taper from a larger width to a smaller width at the base 491). In various embodiments, the width w1 is linearly reduced to the width w2. In various embodiments, the width w2 can be half of the width w1. In various embodiments, the width w1 can be linearly reduced to the width w2, and the width w2 is half of the width w1. In various embodiments, the first portion 460 can have a length l1, and the locking lip 470 can have a length l2 less than l1. In various embodiments, l2 is approximately one-sixth of the length l1.

[0096] 7A to 7H A gasket for a sample device according to an embodiment of the present disclosure is shown.

[0097] FIG. 7A to FIG. 7G A gasket 700 is shown for use with the sample device 200. FIG. 7A to FIG. 7G As shown, the gasket 700 includes a substantially flat surface defined between a first perimeter 701a and a second perimeter 701b. In various embodiments, the gasket 700 includes a tapered portion 702 between the second perimeter 701b and the third perimeter 701c. In various embodiments, the tapered portion 702 has a constant taper. In various embodiments, the tapered portion 702 has a variable taper (e.g., a curved taper). In various embodiments, the first perimeter 701a has a first width, the second perimeter 701b has a second width that is less than the first width, and the third perimeter 701c has a third width that is less than the second width. Figure 7G A cross section of a gasket 700 is shown, wherein the gasket 700 includes a substantially flat upper portion with a height h1 (e.g., thickness). In various embodiments, the upper portion includes a gap 703. In various embodiments, the gasket 700 includes one or more vertical ribs 704 disposed within the gap 703. In various embodiments, the tapered portion 702 tapers over a height h2 and has an angle θ relative to a horizontal axis. In various embodiments, the angle θ corresponds to (e.g., is equal to) an angle outside the objective lens to optimize the travel distance of the objective lens and thereby maximize the possible imaging area within the box.

[0098] Figure 7HA cross section of an exemplary gasket device 710 for sample device 200 according to various embodiments is shown. Gasket 710 may include a substantially flat surface defined between first perimeter 711a and second perimeter 711b. In various embodiments, gasket 710 includes a tapered portion 712 between second perimeter 711b and third perimeter 711c. In various embodiments, tapered portion 712 has a constant taper. In various embodiments, tapered portion 712 has a variable taper (e.g., a curved taper). In various embodiments, first perimeter 711a has a first width, second perimeter 711b has a second width less than the first width, and third perimeter 711c has a third width less than the second width. First perimeter 711a, second perimeter 711b, tapered portion 712, and third perimeter 711c together form an upper portion 713 that merges with lower portion 715 around base portion 717.

[0099] As shown in the figure, for example, Figure 7H , the upper portion 713 and the lower portion 715 together form a gap 719 . Figure 7H It is also shown that the lower portion 715 can include a fourth perimeter 721a having a first width, a fifth perimeter 721b having a second width, and a tapered portion 722. The tapered portion 722 can have a constant taper. In various embodiments, the tapered portion 722 can have a variable taper (e.g., a curved taper). In various embodiments, the slope (or angle θ relative to the horizontal axis) of the tapered portion 722 is the same or substantially similar to the tapered portion 712. Similarly, in various embodiments, the lower portion 715 can be coplanar with the upper portion 713.

[0100] Figure 7H It is further shown that the upper portion 713 can further include an O-ring 723. The O-ring 723 can be configured to extend from an upper surface 725 of the upper portion 713. The O-ring can be disposed along any portion of the surface 725. For example, FIG. 4H shows an O-ring 723 positioned closer to the first perimeter 711a than the second perimeter 711b. The O-ring 723 can be configured to extend from the upper surface 725 along the entire or substantially the entire perimeter of the gasket 700.

[0101] Although the present teachings are described in conjunction with various embodiments, the present teachings are not intended to be limited to such various embodiments. On the contrary, as will be appreciated by those skilled in the art, the present teachings encompass various alternatives, modifications and equivalents.

[0102] When describing various embodiments, this specification may present a method and / or process as a specific sequence of steps. However, to the extent that the method or process does not rely on the specific order of the steps set forth herein, the method or process should not be limited to the specific sequence of steps described, and those skilled in the art can readily appreciate that the sequence can be varied and still remain within the spirit and scope of the various embodiments.

Claims

1. A component comprising: A sample device, the sample device comprising a bottom portion and a top portion, wherein: The bottom portion is releasably coupled to the top portion; When the bottom portion is coupled to the top portion, a gap is formed between the bottom portion and the top portion, the gap being configured to receive a sample substrate; and the top portion comprising a hole and a plurality of first snap-fit ​​elements, at least one pair of snap-fit ​​elements of the plurality of first snap-fit ​​elements being arranged substantially opposite each other on the top portion; and A cover comprising a cover and a skirt, wherein: The covering comprises: A flat outer surface; and an inner surface separated from the planar outer surface by a thickness of the covering; and The skirt extends around the periphery of the inner surface, wherein: The skirt and the inner surface define a recessed portion; The skirt includes a plurality of second snap-fit ​​elements extending therefrom, at least one pair of the plurality of second snap-fit ​​elements being arranged substantially opposite each other on the skirt, wherein: When the cover is engaged with the sample device, the plurality of first snap-fit ​​elements respectively engage with the plurality of second snap-fit ​​elements to form a seal between the cover and a perimeter of the aperture of the top portion.

2. The assembly of claim 1 further comprising a gasket disposed about the periphery of the aperture, wherein when the cover is engaged with the sample device, the gasket couples with the periphery of the skirt to form the seal.

3. The assembly of claim 2, wherein the hole is defined by the sample base and the gasket.

4. An assembly according to claim 2 or 3, wherein the well is a sealed chamber defined by the sample substrate, the gasket and the cover.

5. An assembly according to any one of claims 2 to 4, wherein the gasket comprises an angled inner surface.

6. A component according to any of the preceding claims, wherein one of the plurality of second snap-fit ​​joint elements comprises a cantilevered clip, and one of the plurality of first snap-fit ​​joint elements comprises an orifice or a recess configured to receive the cantilevered clip when the cover is engaged with the sample device.

7. An assembly according to any one of the preceding claims, wherein the skirt extends around the periphery of the inner surface at an angle greater than 90°.

8. An assembly according to any one of the preceding claims, wherein the skirt has a flange parallel to the planar outer surface. 9 . The assembly of claim 8 , wherein one of the plurality of second snap-fit ​​fitting elements has a support section and a tab portion, the support section extending from the flange and having the same thickness as the flange.

10. The assembly of claim 9, wherein the tab portion includes a first portion and a second portion, the first portion and the second portion being angled at less than 90° and greater than 90°, respectively, relative to the flange.

11. The assembly of claim 10, wherein the first portion is angled relative to the flange at an angle greater than about 75°.

12. An assembly according to any one of claims 9 to 11, wherein the first portion of the tab portion comprises a locking lip at an end of the first portion distal to the support section, the locking lip extending towards the cover.

13. An assembly according to any one of claims 9 to 12, wherein the second portion of the tab portion comprises a contact at an end of the second portion distal to the support section, the contact extending away from the cover.

14. An assembly according to any of the preceding claims, wherein the plurality of second snap-fit ​​joint elements include three cantilevered clips, two of the three cantilevered clips are located on one side of the skirt, and another of the three cantilevered clips is located on an opposite side of the skirt.

15. An assembly according to any preceding claim, wherein the cover is formed using injection moulding.

16. An assembly according to any preceding claim, wherein the cover is made of a polyphenylsulfone material.

17. The assembly of any one of the preceding claims, wherein the cover is made of a polyethylene material, a polyurethane material, a polyethylene terephthalate material, a polystyrene material, a polycarbonate material, a polypropylene material, or a combination thereof.

18. An assembly according to any one of the preceding claims, wherein the sample substrate is a glass slide.

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