Implementation barrier for controlled environment during sample processing and sample detection

Abstract

Various methods for processing and / or detecting a sample are provided herein. One method can include providing a barrier between a first region and a second region, wherein the first region contains a sample, the barrier maintains the first region at a first atmosphere different from a second atmosphere of the second region, and a portion of the barrier contains a fluid in coherent motion, and detecting one or more signals from the sample using a detector at least partially contained in the first region while the first region is maintained at the first atmosphere different from the second atmosphere of the second region. The portion of the barrier containing the fluid can have a lower pressure than the first atmosphere, the second atmosphere, or both.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Patent Application No. 16 / 665,559 (filed October 28, 2019), U.S. Patent Application No. 16 / 665,540 (filed October 28, 2019), U.S. Patent Application No. 16 / 440,026 (filed June 13, 2019), and U.S. Provisional Patent Application No. 62 / 776,866 (filed December 7, 2018), each of which is incorporated by reference herein in its entirety. [Background technology]

[0002] Biological sample processing has various applications in the fields of molecular biology and medicine (e.g., diagnosis).For example, nucleic acid sequencing may provide information that may be used to diagnose certain conditions in subjects and possibly adjust treatment plans.Sequencing is widely used for various molecular biology applications, including vector design, gene therapy, vaccine design, industrial strain design and validation.Biological sample processing may involve fluidics systems and / or detection systems. Summary of the Invention [Problem to be solved by the invention]

[0003] Various samples, including biological and non-biological samples, may be processed in a controlled environment, e.g., in a controlled environment having a controlled temperature, pressure, and / or humidity. Analysis of such samples may involve detecting the sample in the controlled environment. Detection may involve continuous detection (e.g., continuous scanning) in which continuous relative motion exists between a detector (e.g., an optical head) and the sample. Detection may require proximity between an objective lens and a sample, e.g., to achieve direct or indirect contact between the objective lens and the sample. However, detection activities, such as the act of continuously scanning the sample, may disturb the controlled environment. In some cases, efforts to maintain the controlled environment may disturb the continuous motion of one or more detectors. In some cases, moving a detector in the controlled environment while maintaining the controlled environment may not be possible, e.g., because the presence or movement of the detector may make it difficult or impossible to contain or maintain the controlled environment, or because the presence or movement of the detector may affect the sample and thus the detection results. In some cases, the implementation of mechanical seals, such as bellows or sliding gaskets, to maintain a controlled environment from a normal environment (e.g., room environment) may introduce undesirable forces into the detection or may impede or disrupt the relative motion between the detector and the sample. Such problems may result in inaccurate and unclear detection results. It is therefore recognized herein that there is a need for systems, devices and methods that address at least the above-mentioned problems. [Means for solving the problem]

[0004] Provided herein is a barrier that can be implemented between a controlled sample environment and an external environment. Such a barrier may allow low-friction or no-friction relative motion between the detector and the sample while maintaining a controlled sample environment. The barrier may allow the objective lens to contact the sample directly or indirectly (e.g., via immersion in a fluid) during detection and movement. The barrier may allow continuous scanning with relative motion in non-linear directions (e.g., non-linear directions in an R, θ coordinate system) and / or linear directions (e.g., linear directions in an X, Y and / or Z coordinate system). Advantageously, such a barrier may allow continuous scanning in a 100% or substantially 100% relative humidity environment. The barrier may prevent humidity from escaping from the sample environment, which may condense when leaked and affect (e.g., cause corrosion, contamination, etc.) sensitive instruments, such as optical and electronic instruments. Additionally, the barrier may prevent contaminants from the external environment from entering the sample environment, which may contaminate the sample and / or affect fluidics and / or detection (e.g., imaging).

[0005] The barrier may comprise a transition region between the sample environment and the external environment. The barrier may comprise a fluid barrier. The barrier may comprise fluid from the sample environment, the external environment, or both. The barrier may be a low pressure region. The low pressure region may have a lower pressure than the sample environment, the external environment, or both. The barrier may comprise a partial vacuum. The barrier may further comprise a physical barrier. In one aspect, a method for processing a biological analyte is provided, comprising: (a) providing a barrier between a first region and a second region, wherein the first region includes a substrate having a biological analyte immobilized in its vicinity, and wherein the barrier maintains the first region at a first atmosphere that is different from a second atmosphere of the second region; and (b) detecting, using a detector at least partially contained in the first region, one or more signals from the biological analyte, or changes therein, while (i) the detector is undergoing motion relative to the substrate and the substrate and the detector are not in direct mechanical contact, and (ii) the first region is maintained at a first atmosphere that is different from the second atmosphere of the second region.

[0006] In some embodiments, the portion of the barrier comprises a fluid in bulk motion. In some embodiments, the fluid comprises air. In some embodiments, the portion of the barrier comprises a partial vacuum. In some embodiments, the portion of the barrier comprises a fluid from the first region, the second region, or both.

[0007] In some embodiments, the first atmosphere is maintained at a first humidity or a first humidity range that is different from a second humidity or a second humidity range of the second atmosphere, hi some embodiments, the first atmosphere has a relative humidity of greater than 90%.

[0008] In some embodiments, the first atmosphere is maintained at a first temperature or a first temperature range that is different from a second temperature or a second temperature range of the second atmosphere.

[0009] In some embodiments, the first region includes a first portion and a second portion, the first portion being maintained at a first local atmosphere and the second portion being maintained at a second local atmosphere that is different from the first local atmosphere. In some embodiments, the first local atmosphere is maintained at a first local temperature or a first local temperature range that is different from a second local temperature or a second local temperature range of the second local atmosphere. In some embodiments, the first local atmosphere is maintained at a first local humidity or a first local humidity range that is different from a second local humidity or a second local humidity range of the second local atmosphere.

[0010] In some embodiments, the detector is an optical detector and the one or more signals are one or more optical signals or changes in an optical signal.

[0011] In some embodiments, the barrier comprises a first solid component and a second solid component, the first solid component and the second solid component are not in direct mechanical contact, and the first solid component is movable relative to the second solid component. In some embodiments, a portion of the barrier comprises a fluid in bulk motion, the portion being disposed between the first solid component and the second solid component.

[0012] In some embodiments, the detector is fixed relative to a first solid component and the substrate is translationally fixed relative to a second solid component.

[0013] In some embodiments, the substrate is rotatable relative to the second solid component.

[0014] In some embodiments, a first portion of the first solid component is provided between the first region and the second region, and a second portion of the first solid component is provided between the second region and a third region to form a portion of another barrier configured to maintain the third region at a third atmosphere independent of the first and second atmospheres, the portion of the another barrier including a fluid in bulk motion, and the third region is movable relative to the first solid component independent of the first region.

[0015] In some embodiments, the second atmosphere is a room atmosphere or an ambient atmosphere.

[0016] In some embodiments, a first portion of the detector is in a first region and a second portion of the detector is in a second region, In some embodiments, the first portion of the detector includes an optical imaging objective lens that is at least partially immersed in an immersion fluid that is in contact with the substrate in the first region.

[0017] In some embodiments, the biological analyte is a nucleic acid molecule, further comprising identifying the sequence of the nucleic acid molecule or derivatives thereof based at least in part on one or more signals or changes therein.

[0018] In some embodiments, the motion comprises one or more selected from the group consisting of: (i) substantially linear motion relative to the substrate, and (ii) substantially non-linear motion relative to the substrate.

[0019] In some embodiments, the detector is subjected to a rotational motion relative to the substrate.

[0020] In some embodiments, the detector undergoes translational motion relative to the substrate.

[0021] In some embodiments, the detector undergoes translational and rotational motion relative to the substrate.

[0022] In some embodiments, in (b), the detector scans the substrate along a substantially linear scan path.

[0023] In some embodiments, in (b), the detector scans the substrate along a substantially non-linear scan path. In some embodiments, in (b), the detector scans the substrate along one or more scan paths selected from the group consisting of a loop, a spiral, and an arc.

[0024] In another aspect, a method for processing a biological analyte is provided, the method comprising: (a) providing a barrier between a first region and a second region, the first region containing the biological analyte, the barrier maintaining the first region at a first atmosphere different from a second atmosphere of the second region, and a portion of the barrier containing a fluid in bulk movement; and (b) detecting one or more signals or changes therein from the biological analyte using a detector at least partially contained in the first region while the first region is maintained at the first atmosphere different from the second atmosphere of the second region.

[0025] In some embodiments, a portion of the barrier comprises fluid from the first region, the second region, or both.

[0026] In some embodiments, the first atmosphere is maintained at a first humidity or a first humidity range that is different from a second humidity or a second humidity range of the second atmosphere, hi some embodiments, the first atmosphere has a relative humidity of greater than 90%.

[0027] In some embodiments, the first atmosphere is maintained at a first temperature or a first temperature range that is different from a second temperature or a second temperature range of the second atmosphere.

[0028] In some embodiments, the first region includes a first portion and a second portion, the first portion being maintained at a first local atmosphere and the second portion being maintained at a second local atmosphere that is different from the first local atmosphere. In some embodiments, the first local atmosphere is maintained at a first local temperature or a first local temperature range that is different from a second local temperature or a second local temperature range of the second local atmosphere. In some embodiments, the first local atmosphere is maintained at a first local humidity or a first local humidity range that is different from a second local humidity or a second local humidity range of the second local atmosphere.

[0029] In some embodiments, (b) includes moving the detector relative to the biological analyte while the detection is occurring.

[0030] In some embodiments, the detector is an optical detector and the one or more signals or changes therein are one or more optical signals or changes therein.

[0031] In some embodiments, the barrier comprises a first solid component and a second solid component, the first solid component and the second solid component are not in mechanical contact, and the first solid component is movable relative to the second solid component, hi some embodiments, a portion of the barrier that includes the fluid is disposed between the first solid component and the second solid component.

[0032] In some embodiments, the detector is immobilized relative to a first solid component and the biological analyte is translationally immobilized relative to a second solid component.

[0033] In some embodiments, a first portion of the first solid component is provided between the first region and the second region, and a second portion of the first solid component is provided between the second region and a third region to form a portion of another barrier configured to maintain the third region at a third atmosphere independent of the first and second atmospheres, the portion of the another barrier comprising a fluid, and the third region is movable relative to the first solid component independent of the first region.

[0034] In some embodiments, the second atmosphere is a room atmosphere or an ambient atmosphere.

[0035] In some embodiments, a first portion of the detector is in the first region and a second portion of the detector is in the second region, In some embodiments, the first portion of the detector includes an optical imaging objective lens that is at least partially immersed in an immersion fluid that is in contact with the biological analyte in the first region.

[0036] In some embodiments, the biological analyte is a nucleic acid molecule, further comprising identifying the sequence of the nucleic acid molecule or a derivative thereof based at least in part on one or more signals or signal changes.

[0037] In some embodiments, the fluid comprises air.

[0038] In another aspect, a system for processing an analyte is provided, comprising: (i) a first region configured to contain a substrate having an analyte immobilized in its vicinity; and (ii) at least a portion of a detector; and a barrier disposed between the first region and a second region, the barrier configured to maintain the first region at a first atmosphere different from a second atmosphere of the second region while the detector and substrate undergo relative motion with respect to one another to detect one or more signals or changes therein from the analyte.

[0039] In some embodiments, a portion of the barrier is configured to contain fluid in bulk motion. In some embodiments, a portion of the barrier is configured to be under vacuum. In some embodiments, a portion of the barrier is configured to contain fluid from the first region, the second region, or both the first region and the second region.

[0040] In some embodiments, a portion of the barrier is configured to contain air.

[0041] In some embodiments, the barrier is configured to maintain the first region at a first humidity or first humidity range, the first humidity or first humidity range being different from a second humidity or second humidity range of the second region, hi some embodiments, the first atmosphere has a relative humidity of greater than 90%.

[0042] In some embodiments, the barrier is configured to maintain the first region at a first temperature or first temperature range, which is different from a second temperature or second temperature range of the second region.

[0043] In some embodiments, the first region includes a first portion and a second portion, and the barrier is configured to maintain the first portion at a first local atmosphere and to maintain the second portion at a second local atmosphere different from the first local atmosphere. In some embodiments, the barrier is configured to maintain the first local atmosphere at a first local temperature or a first local temperature range different from a second local temperature or a second local temperature range of the second local atmosphere. In some embodiments, the barrier is configured to maintain the first local atmosphere at a first local humidity or a first local humidity range different from a second local humidity or a second local humidity range of the second local atmosphere.

[0044] In some embodiments, the detector is at least partially contained in the first region. In some embodiments, the detector is an optical detector and the one or more signals are one or more optical signals or optical signal changes. In some embodiments, a first portion of the detector is in the first region and a second portion of the detector is in the second region. In some embodiments, the first portion of the detector comprises an optical imaging objective configured to be at least partially immersed in an immersion fluid in contact with the substrate when the substrate is in the first region. In some embodiments, the detector is configured to undergo motion while the substrate is stationary. In some embodiments, the substrate is configured to undergo motion while the detector is stationary.

[0045] In some embodiments, the barrier comprises a first solid component and a second solid component, the first solid component and the second solid component are not in direct mechanical contact with each other, and the first solid component and the second solid component are movable relative to each other. In some embodiments, a portion of the barrier is configured to contain a fluid in bulk motion, the portion being disposed between the first solid component and the second solid component.

[0046] In some embodiments, the detector is configured to be fixed relative to a first solid component, and the substrate is configured to be fixed relative to a second solid component.

[0047] In some embodiments, the detector is configured to be fixed relative to a first solid-state component, and the substrate is configured to be rotatable relative to a second solid-state component.

[0048] In some embodiments, a first portion of the first solid component is provided between the first region and the second region, and a second portion of the first solid component is provided between the second region and a third region to form part of another barrier configured to maintain the third region at a third atmosphere independent of the first and second atmospheres, the part of the another barrier containing a fluid in bulk movement, and the third region is movable relative to the first solid component independent of the first region.

[0049] In some embodiments, the second atmosphere is a room atmosphere or an ambient atmosphere.

[0050] In another aspect, a system for processing or analyzing an analyte is provided, the system including a chamber including a first region configured to contain (1) a substrate having an analyte immobilized in its vicinity and (2) at least a portion of a detection unit, and a lid configured to be positioned in the vicinity of the chamber, and a fluid flow unit configured to provide a fluid in bulk movement to a location disposed between the chamber and the lid when the lid is positioned in the vicinity of the chamber, such that the first region is maintained at a first atmosphere that is different from a second atmosphere of a second region outside the first region.

[0051] In some embodiments, the bulk moving fluid is configured to create a partial vacuum between the chamber and the lid.

[0052] In some embodiments, the fluid flow unit is configured to use fluid from the first region, the second region, or both, to provide fluid in bulk motion.

[0053] In some embodiments, the fluid comprises air.

[0054] In some embodiments, the fluid flow unit is configured to maintain the first region at a first humidity or first humidity range, where the first humidity or first humidity range is different from the second humidity or second humidity range of the second region, hi some embodiments, the first atmosphere has a relative humidity of greater than 90%.

[0055] In some embodiments, the fluid flow unit is configured to maintain the first region at a first temperature or first temperature range, where the first temperature or first temperature range is different from a second temperature or second temperature range of the second region.

[0056] In some embodiments, the first region includes a first portion and a second portion, and the fluid flow unit is configured to maintain the first portion at a first local atmosphere and to maintain the second portion at a second local atmosphere different from the first local atmosphere. In some embodiments, the fluid flow unit is configured to maintain the first local atmosphere at a first local temperature or a first local temperature range different from a second local temperature or a second local temperature range of the second local atmosphere. In some embodiments, the fluid flow unit is configured to maintain the first local atmosphere at a first local humidity or a first local humidity range different from a second local humidity or a second local humidity range of the second local atmosphere.

[0057] In some embodiments, the detection unit is at least partially contained in the first region. In some embodiments, the detection unit is an optical detection unit. In some embodiments, a first portion of the detection unit is in the first region and a second portion of the detection unit is in the second region. In some embodiments, the first portion of the detection unit comprises an optical imaging objective configured to be at least partially immersed in an immersion fluid in contact with the substrate in the first region. In some embodiments, the detection unit is configured to undergo motion while the substrate is stationary. In some embodiments, the substrate is configured to undergo motion while the detection unit is stationary. In some embodiments, the relative motion comprises one or more selected from the group consisting of: (i) substantially linear motion, and (ii) substantially non-linear motion. In some embodiments, the detection unit is configured to be fixed relative to the lid. In some embodiments, the substrate is configured to be rotatable relative to the chamber.

[0058] In some embodiments, the detection unit includes one or more optical systems.

[0059] In some embodiments, the detection unit includes a sensor configured to capture a signal from the analyte.

[0060] In some embodiments, the chamber is not in mechanical contact with the lid.

[0061] In some embodiments, the lid is configured to move relative to the chamber, or vice versa, the chamber is configured to move relative to the lid.

[0062] In some embodiments, the fluid flow unit is configured to maintain the first region at a first atmosphere while the detection unit and the substrate are undergoing movement relative to one another.

[0063] In some embodiments, the fluid flow unit is configured to create a negative pressure at a location disposed between the chamber and the lid.

[0064] In some embodiments, a first portion of the lid is provided between the first region and the second region, a second portion of the lid is provided between the second region and a third region, and a second fluid flow unit is configured to provide fluid in bulk movement to maintain the third region at a third atmosphere independent of the first atmosphere and the second atmosphere, and the third region is movable relative to the lid independent of the first region.

[0065] In some embodiments, the second atmosphere is a room atmosphere or an ambient atmosphere.

[0066] In some embodiments, the system further includes a controller operably coupled to the fluid flow unit, the controller configured to instruct the fluid flow unit to cause the fluid to undergo bulk movement.

[0067] In another aspect, a system is provided that includes an imaging objective configured to detect a signal or a signal change from an analyte coupled to a substrate; a housing configured to contain a volume of fluid between the imaging objective and the substrate; a fluid source configured to contain an aqueous solution; and a fluid flow unit configured to deliver a volume of fluid from the fluid source to the housing.

[0068] In some embodiments, in this case, the aqueous solution comprises a cleaning solution.

[0069] In some embodiments, the aqueous solution comprises an immersion buffer that includes a salt, a surfactant and a buffering agent.

[0070] In some embodiments, the aqueous solution has a pH between 8.0 and 9.0.

[0071] In some embodiments, the system further comprises a substrate. In some embodiments, the substrate comprises a fluid layer comprising a second aqueous solution. In some embodiments, the aqueous solution and the second aqueous solution comprise different compositions. In some embodiments, the aqueous solution and the second aqueous solution comprise the same composition.

[0072] In another aspect, a method is provided that includes the steps of: (a) bringing an imaging objective into fluid contact with a substrate through a volume of fluid, the fluid comprising a first aqueous solution, the substrate comprising (i) an analyte immobilized near the substrate, and (ii) a layer of fluid near the substrate, the layer of fluid comprising a second aqueous solution; and (b) imaging the analyte with the imaging objective through the volume of fluid.

[0073] In some embodiments, the method further includes moving the imaging objective relative to the substrate while maintaining fluid contact between the imaging objective and the substrate.

[0074] In some embodiments, the method further includes moving the substrate relative to the imaging objective while maintaining fluid contact between the imaging objective and the substrate.

[0075] In some embodiments, the volume of fluid is between about 200 micrometers (μm) and 500 μm thick.

[0076] In some embodiments, the fluid layer is between about 5 μm and 50 μm thick.

[0077] In some embodiments, the method further includes (i) interrupting fluid contact between the imaging objective and the substrate, and (ii) bringing the imaging objective and the substrate into second fluid contact. In some embodiments, after (i), at least a portion of the quantity of fluid remains in fluid contact with the imaging objective. In some embodiments, after (i), at least a portion of the quantity of fluid remains in fluid contact with the substrate.

[0078] In some embodiments, the first aqueous solution comprises a washing solution.

[0079] In some embodiments, the first aqueous solution comprises an soaking buffer comprising a salt, a surfactant and a buffering agent.

[0080] In some embodiments, the first aqueous solution has a pH between 8.0 and 9.0.

[0081] In some embodiments, the first aqueous solution and the second aqueous solution comprise different compositions.

[0082] In some embodiments, the first aqueous solution and the second aqueous solution comprise the same composition.

[0083] In another aspect, a method is provided that includes the steps of: (a) bringing an imaging objective into fluid contact with an analyte immobilized near a substrate, the substrate including a layer of fluid that includes a second aqueous solution, and (b) imaging the analyte with the imaging objective through the volume of fluid.

[0084] In some embodiments, the method further includes moving the imaging objective lens relative to the analyte while maintaining fluid contact between the imaging objective lens and the analyte.

[0085] In some embodiments, the method further includes moving the analyte relative to the imaging objective while maintaining fluid contact between the imaging objective and the analyte.

[0086] In some embodiments, the volume of fluid is between about 200 μm and 500 μm thick.

[0087] In some embodiments, the fluid layer is between about 5 μm and 50 μm thick.

[0088] In some embodiments, the method further includes (i) disrupting fluid contact between the imaging objective and the analyte, and (ii) bringing the imaging objective and the analyte into second fluid contact. In some embodiments, after (i), at least a portion of the volume of fluid remains in fluid contact with the imaging objective. In some embodiments, after (i), at least a portion of the volume of fluid remains in fluid contact with the analyte.

[0089] In some embodiments, the first aqueous solution comprises a washing solution.

[0090] In some embodiments, the first aqueous solution comprises an soaking buffer comprising a salt, a surfactant and a buffering agent.

[0091] In some embodiments, the first aqueous solution has a pH between 8.0 and 9.0.

[0092] In some embodiments, the first aqueous solution and the second aqueous solution comprise different compositions.

[0093] In some embodiments, the first aqueous solution and the second aqueous solution comprise the same composition.

[0094] In another aspect, a system for processing or analyzing an analyte includes a chamber configured to include an interior region and a substrate configured to immobilize an analyte in its vicinity, and a lid configured to be disposed in the vicinity of the chamber, and an environmental unit configured to maintain a first local environment, a second local environment, and a third local environment within the interior region, (i) to maintain the first local environment at a first temperature or temperature range, (ii) to maintain the second local environment at a second temperature or temperature range, and (iii) to maintain the third local environment. A system is provided that includes an environmental unit configured to maintain a local environment at a third temperature or temperature range, wherein the first local environment is disposed above the second local environment and the third local environment, and the first local environment is located at or in close proximity to the lid, the second local environment is disposed at or near the surface of the substrate, and the third local environment is disposed below the first local environment and the second local environment, and the first temperature or temperature range is higher than the second temperature or temperature range and the third temperature or temperature range, and the second temperature or temperature range is lower than the third temperature or temperature range.

[0095] Further aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description, in which only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modification in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

[0096] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained herein, it is intended that the specification supersede and / or take precedence over any such conflicting material.

[0097] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also referred to herein as "Figures"), in which: [Brief description of the drawings]

[0098] [Figure 1A] FIG. 1 illustrates a cross-sectional side view of an example barrier system. [Figure 1B] FIG. 1B illustrates a perspective view of FIG. 1A. [Figure 1C] FIG. 1 illustrates a cross-sectional view of an example immersion optical system. [Figure 2A] FIG. 1 illustrates a partial cross-sectional view of a barrier system that maintains a fluid barrier. [Figure 2B] FIG. 2B illustrates a close-up view of the barrier system of FIG. 2A. [Figure 2C] FIG. 2B illustrates a perspective view of a chamber of the barrier system of FIG. 2A. [Diagram 3] FIG. 1 illustrates a barrier system having multiple sample environments. [Figure 4] FIG. 1 illustrates an example barrier system including different local environments. [Diagram 5] FIG. 1 illustrates a processing system including an example barrier system. [Figure 6] FIG. 1 illustrates various examples of arrays on a substrate. [Figure 7] FIG. 1 illustrates a computer system that is programmed or otherwise configured to perform the methods provided herein. [Figure 8] FIG. 1 shows an example of an image produced by imaging a substrate on which a biological analyte is immobilized in the sample environment of a barrier system of the present disclosure. [Figure 9] FIG. 1 shows signal data processed by imaging a substrate on which a biological analyte is immobilized in the sample environment of a barrier system of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0099] While various embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous changes, modifications and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be used.

[0100] Provided herein is a barrier that may be implemented between a controlled sample environment and an external environment. The barrier may comprise a transition region between the sample environment and the external environment. The barrier may comprise a fluid barrier. The barrier may comprise fluid from the sample environment, the external environment, or both. The barrier may be a low pressure region. The low pressure region may have a lower pressure than the sample environment, the external environment, or both. The barrier may comprise a partial vacuum. The barrier may further comprise a physical barrier. Beneficially, such a barrier may allow frictionless or low-friction relative motion between the detector and the sample while maintaining a controlled sample environment. The barrier may allow continuous scanning with relative motion in non-linear directions (e.g., non-linear directions in an R, θ coordinate system) and / or linear directions (e.g., linear directions in an X, Y and / or Z coordinate system). The barrier may allow continuous scanning in a 100% or substantially 100% relative humidity environment. The barrier may prevent humidity from escaping the sample environment, which may condense and affect (e.g., cause corrosion, contamination, etc.) sensitive instruments, such as optical instruments. Additionally, the barrier may prevent contaminants from the external environment from entering the sample environment, which may affect fluidics and / or detection (e.g., imaging).

[0101] The term "fluid" as used herein generally refers to a gas or liquid or a mixture thereof. A fluid may include solid particles, liquid particles (e.g., water droplets), gas particles (e.g., inert gas atoms or non-inert gas molecules), or a mixture thereof. A fluid may include steam. A fluid may include water content. A fluid may include air, such as ambient air, room air, atmospheric air, and / or pressurized air. The fluid may include concentrated elements or compounds in an isolated state or in a mixture, for example, in a concentration of at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% in a mixture. Alternatively, or in addition, the fluid may contain a concentrated element or compound in a mixture at a concentration of at most about 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less. The fluid may contain any suspension or mixture of particles in a gaseous medium. A fluid may include any suspension or mixture of particles in a liquid medium. A fluid may include mist, fog, water vapor, or aerosols. In some cases, a fluid may include plasma. A volume of fluid may be capable of flowing, for example, with random motion, coherent motion, and / or bulk motion. A volume of fluid may have a net mean motion that is oriented along one or more directions or toward a reference destination.In some cases, a volume of fluid undergoing coherent or bulk motion may have streamlines oriented along the same general direction. A volume of fluid undergoing coherent or bulk motion may be distinguished from a fluid undergoing random motion (e.g., a fluid that is not in coherent motion, is not in bulk motion, or has no net average motion). A volume of fluid may have turbulent and / or laminar flow.

[0102] The term "sample" as used herein generally refers to a biological sample. The systems, devices and methods provided herein may be particularly useful for analyzing biological samples that may be highly sensitive to the environment, such as the temperature, pressure and / or humidity of the environment. The biological sample may be derived from any subject or living organism. For example, the subject may be an animal, a mammal, a bird, a vertebrate, a rodent (e.g., mouse), a primate, an ape, a human, or other organism, such as a plant. The animal may include, but is not limited to, livestock, sport animals and pets. The subject may be a healthy or asymptomatic individual, an individual having or suspected of having a disease (e.g., cancer) or a predisposition to the disease, and / or an individual in need of treatment or suspected of needing treatment. The subject may be a patient. The subject may be a microorganism or a microorganism (e.g., bacteria, fungi, archaea, viruses).

[0103] The biological sample may include many macromolecules, for example many cellular macromolecules. The biological sample may be a cell sample. The biological sample may be a cell line or cell culture sample. The biological sample may include one or more cells. The biological sample may include one or more microorganisms. The biological sample may be a nucleic acid sample or a protein sample. The biological sample may also be a carbohydrate sample or a lipid sample. The biological sample may be derived from another sample. The sample may be a tissue sample, such as a biopsy, a core biopsy, a needle aspirate or a fine needle aspirate. The sample may be a fluid sample, such as a blood sample, a urine sample or a saliva sample. The sample may be a skin sample. The sample may be an oral mucosal swab. The sample may be a plasma sample or a serum sample. The sample may be a cell-free sample or a cell-free sample. The cell-free sample may include extracellular polynucleotides. The extracellular polynucleotides may be isolated from a body sample, which may be selected from the group consisting of blood, plasma, serum, urine, saliva, mucosal discharge, sputum, stool and tears.

[0104] The biological sample may include one or more biological particles. The biological particle may be a macromolecule. The biological particle may be a small molecule. The biological particle may be a virus. The biological particle may be a cell or a derivative of a cell. The biological particle may be an organelle. The biological particle may be a rare cell from a population of cells. The biological particle may be any type of cell, including but not limited to, a prokaryotic cell, a eukaryotic cell, a bacterial cell type, a fungal cell type, a plant cell type, a mammalian cell type or other animal cell type, mycoplasma, a normal tissue cell, a tumor cell, or any other cell type, whether derived from a single-celled or multicellular organism. The biological particle may be a component (e.g., a macromolecular component) of a cell, such as deoxyribonucleic acid (DNA), ribonucleic acid (RNA), a nucleus, an organelle, a protein, a peptide, a polypeptide, or any combination thereof. The RNA may be coding or non-coding. The RNA may be, for example, messenger RNA (mRNA), ribosomal RNA (rRNA) or transfer RNA (tRNA). The RNA may be a transcript. The RNA may be a small RNA less than 200 nucleobases in length or a large RNA more than 200 nucleobases in length. Small RNA may include 5.8S ribosomal RNA (rRNA), 5S rRNA, transfer RNA (tRNA), microRNA (miRNA), small interfering RNA (siRNA), small nucleolar RNA (snoRNA), Piwi-interacting RNA (piRNA), small tRNA-derived RNA (tsRNA) and small rRNA-derived RNA (srRNA). The RNA may be double-stranded or single-stranded RNA. The RNA may be circular RNA. The biological particle may be a hardened cell. Such hardened cells may include a cell wall or cell membrane or may not include a cell wall or cell membrane. Alternatively or in addition, the sample of the present disclosure may include a non-biological sample.

[0105] The term "analyte" as used herein generally refers to an object that is analyzed, i.e., one or more properties are measured, determined, or otherwise assayed. An analyte may be, for example, a biological analyte that is a biological sample, or a biological analyte derived from a biological sample. An analyte may be, for example, a non-biological analyte that is a non-biological sample, or a non-biological analyte derived from a non-biological sample.

[0106] The term "motion relative to" or similar variations ("movable relative to," "moves relative to"), when used herein in reference to a relationship between a first object and a second object (e.g., motion of the first object relative to the second object), generally refers to motion by the first object relative to the other, motion by the second object relative to the other, or both.

[0107] The term "detector," as used herein, may refer to any device or collection of devices configured to detect a signal. The detector may include an objective lens. The detector may include multiple objective lenses. The detector may include an imaging system.

[0108] Whenever the term "at least," "more than," or "greater than or equal to" precedes the first number in a series of two or more numbers, the term "at least," "more than," or "greater than or equal to" applies to each and every number in the series. For example, 1, 2, or 3 or more is equivalent to 1 or more, 2 or more, or 3 or more.

[0109] Whenever the term "at most," "less than," or "equal to or less than" precedes the first number in a series of two or more numbers, the term "at most," "less than," or "equal to or less than" applies to each and every number in the series. For example, "equal to or less than 3, 2, or 1" corresponds to 3 or less, 2 or less, and 1 or less.

[0110] Fluid Barrier Various methods for processing and / or detecting a sample are provided herein. In some cases, the method can include providing a barrier between a first region (e.g., a sample-containing region) and a second region (e.g., an external region). The barrier can maintain the first region at a first atmosphere different from the second atmosphere of the second region. A portion of the barrier can include a fluid in coherent or bulk motion. The first region can include a sample. A detector at least partially contained in the first region can then detect one or more signals from the sample while the first region is maintained at a first atmosphere different from the second atmosphere of the second region. The detector can be in direct mechanical contact with a substrate contained in the first region. The substrate can include a sample thereon. The detector can be in fluidic contact (or in fluidic contact) with the substrate (e.g., the detector can be in direct mechanical contact with the substrate, but can be coupled to the substrate via a fluid). The detector can be in liquid contact with the substrate. The detector may be in gas contact with the substrate.

[0111] In some cases, the method can include providing a barrier between a first region (e.g., a sample-containing region) and a second region (e.g., an external region) such that the barrier maintains the first region in a first atmosphere different from the second atmosphere of the second region. The first region can contain a sample. A detector at least partially contained in the first region can then detect one or more signals from the sample (i) while the detector undergoes a detection event, such as when (1) the detector undergoes continuous low-friction or frictionless motion relative to the first region, (2) the detector undergoes discrete motion relative to the first region at different time intervals (e.g., in a discontinuous manner), and (ii) while the first region is maintained in a first atmosphere different from the second atmosphere of the second region. The detection event can include imaging or scanning during a period of relative motion between the detector and the sample. The detection event can include imaging or scanning while the detector and the sample are stationary relative to each other. The detector may not be in direct mechanical contact with the substrate containing the sample thereon and contained in the first region. The detector may be in fluid contact with the substrate. The detector may be in liquid contact with the substrate. The detector may be in gas contact with the substrate.

[0112] Various systems for processing and / or detecting a sample are provided herein. In some cases, the system can include a barrier disposed between a first region (e.g., a sample-containing region) and a second region (e.g., an external region). The first region can be configured to contain a sample. The barrier can be configured to maintain the first region at a first atmosphere different from a second atmosphere of the second region. A portion of the barrier can include a fluid in coherent or bulk motion. The system can include a detector at least partially contained in the first region. The detector can be configured to detect one or more signals from the sample while the first region is maintained at a first atmosphere different from the second atmosphere of the second region. In some cases, the detector can be configured to detect one or more signals from the sample while the detector undergoes a detection event. For example, the detection event can include a continuous low-friction or frictionless motion of the detector relative to the first region. For example, the detection event can include discrete motions of the detector relative to the first region at different time intervals (e.g., in a discontinuous manner). The detection event may include imaging or scanning during a period of relative motion between the detector and the sample. The detection event may include imaging or scanning while the detector and the sample are stationary relative to one another. In some cases, the first region may include a substrate that includes the sample on its surface. For example, the sample may be immobilized near the substrate. In some cases, the detector may not be in direct mechanical contact with the substrate. In some cases, the detector may be in fluid contact with the substrate. The detector may be in liquid contact with the substrate. The detector may be in gas contact with the substrate.

[0113] 1A and 1B illustrate an example barrier system 100, showing a cross-sectional side view and a perspective view, respectively. A fluid barrier 113 may be implemented between a sample environment 105 (e.g., a first region) and an external environment 107 (e.g., a second region). The sample environment 105 may be a controlled environment that contains one or more samples therein. The external environment 107 may be a closed or open environment. In some cases, the external environment 107 may be a room environment or an ambient environment. In some cases, the external environment 107 may also be a controlled environment.

[0114] The region of the sample environment 105 may be defined by the chamber 115, the plate 103 and the fluid barrier 113. The fluid barrier 113 may be maintained between a physical gap between the chamber 115 and the plate 103. The term "plate" as used herein may be interchangeably referred to herein as a lid. In some cases, the physical gap may be large enough to allow fluid communication between the sample environment 105 and the external environment 107 when the fluid barrier 113 is not otherwise in place. The chamber 115 and the plate 103 may be independent such that the chamber 115 and the region of the sample environment 105 defined thereby are movable relative to the plate 103. For example, the region of the sample environment 105 may be defined by different portions of the plate 103 with different locations of the chamber 115 relative to the plate 103. The relative motion between the chamber 115 and the plate 103 can be in any direction, such as non-linear (e.g., non-linear in an R, θ coordinate system) and / or linear (e.g., linear in an X, Y and / or Z coordinate system). For example, the relative motion can be rotational about a central axis or linear along any linear axis. In some cases, the relative motion between the chamber 115 and the plate 103 can be constrained by actuator units (e.g., linear stages, motors, etc.) and / or structural units (e.g., beams, supports, tracks, etc.).

[0115] The plate 103 and the chamber 115 may not be in direct mechanical contact such that a minimum distance exists between the plate and the chamber. The minimum distance between the plate 103 and the chamber 115 may be at least about 100 micrometers (μm), 150 μm, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 550 μm, 600 μm, 650 μm, 700 μm, 750 μm, 800 μm, 850 μm, 900 μm, 950 μm, 1 millimeter (mm), 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 1 centimeter (cm) or more. In the alternative, or in addition, the minimum distance may be at most about 1 cm, 9 mm, 8 mm, 7 mm, 6 mm, 5 mm, 4 mm, 3 mm, 2 mm, 1 mm, 950 μm, 900 μm, 850 μm, 800 μm, 750 μm, 700 μm, 650 μm, 600 μm, 550 μm, 500 μm, 450 μm, 400 μm, 350 μm, 300 μm, 250 μm, 200 μm, 150 μm, 100 μm or less. In the alternative, or in addition, the minimum distance may be within a range defined by any two of the foregoing values.

[0116] The fluid barrier 113 may act as a transition region between the sample environment 105 and the external environment 107. The fluid barrier 113 may contain fluid (e.g., air) from the sample environment, the external environment, or both. The fluid barrier 113 may be a low pressure region. The fluid barrier 113 may have a lower pressure than the sample environment, the external environment, or both. The barrier may include a partial vacuum. The barrier may contain a fluid volume(s) that is subjected to a negative pressure. In some cases, the fluid barrier 113 may be a high pressure region. For example, the fluid barrier may have a higher pressure than the sample environment, the external environment, or both. The fluid barrier 113 may be in coherent motion, such as with a coherent flow direction. The fluid barrier 113 may be in bulk motion. The fluid barrier may contain a volume of fluid that has a net average motion that is oriented along one or more directions or toward a reference destination. In some cases, a volume of fluid in coherent or bulk motion may have streamlines that are oriented along the same general direction. Fluids with coherent or bulk motion may be distinguished from fluids with random motion that are not part of a fluid barrier (e.g., fluids that are not in coherent motion, fluids that are not in bulk motion, fluids that do not have a net average motion). Fluids in a fluid barrier may have turbulent and / or laminar flow.

[0117] The sample environment 105 may include a substrate. One or more samples may be immobilized on or near the substrate. Alternatively, or in addition, one or more samples may be otherwise disposed on the substrate. In some cases, at least a portion of the chamber 115 may be or may include the substrate. In other examples, the chamber 115 may be coupled to the substrate. In some cases, the substrate may be fixed relative to the chamber 115. In the alternative, the substrate may be movable relative to the chamber 115, for example, in a linear and / or non-linear direction (e.g., rotational). For example, the substrate may be fixed in XY coordinates (and / or Z coordinates) to the chamber 115, but rotatable relative to the chamber 115. If the chamber 115 is movable relative to the plate 103 as well as the substrate is movable relative to the chamber 115, these two relative movements may or may not be actuated by the same actuator unit.

[0118] The detector 101 may protrude from the external environment 107 into the sample environment 105 through the plate 103, such as through an opening in the plate 103. The fit between the detector 101 and the opening may be fluid-tight such that there is no fluid communication through the opening when the detector 101 fits through it. In the alternative, or in addition, the opening may be sealed. In the alternative, the plate 103 may be integral with the detector 101. In the alternative, the detector 101 may be fully contained in the sample environment 105, for example by attaching the end not facing the sample to the plate 103.

[0119] At least a portion of the detector 101 may be fixed relative to the plate 103. In some cases, the detector 101 may be capable of translating independently of the plate 103 along an axis substantially perpendicular to the plane of the plate 103 (e.g., through an aperture). In some cases, at least a portion of the detector 101 (e.g., the portion of the detector that is within the sample environment region) may be capable of moving (e.g., linearly or non-linearly, e.g., rotating, etc.) independently of the plate 103.

[0120] Within the sample environment 105, the detector 101 may be configured to detect one or more samples disposed on a substrate using an immersion optical system. A portion of the detector (e.g., an optical imaging objective, etc.) within the sample environment 105 may be in optical communication with the substrate via a liquid fluid 131 medium. In some cases, the liquid fluid medium may be disposed in a localized region of the substrate. In other examples, the liquid fluid medium may be disposed across a given surface of the substrate (e.g., across a given base of the chamber 115). In the alternative, the detector may be in optical communication with the substrate without a liquid fluid medium.

[0121] FIG. 1C illustrates a cross-sectional view of an example immersion optical system 1100. The system 1100 may be used to optically image a substrate as described herein. The system 1100 may be integrated with any barrier system described elsewhere herein. The system may include an optical imaging objective 1110 (e.g., detector 101). For example, the objective may protrude into the sample environment (e.g., through plate 103) or may be contained within the sample environment (e.g., attached to the surface of plate 103). The optical imaging objective may be an immersion optical imaging objective. The optical imaging objective may be configured to be in optical communication with the substrate 1160. The optical imaging objective may be partially or completely surrounded by a housing 1120. The housing may partially or completely surround the sample-side end of the optical imaging objective. The housing may be fixed to the optical imaging objective and / or the plate. The housing may have a generally cup-like shape or form. The housing may be any container. The housing may be configured to contain a fluid 1140 (e.g., water or an aqueous solution or an oil or an organic solution, etc.) in which the optical imaging objective is to be immersed. The fluid may be in contact with the substrate 1160. Thus, the objective and the substrate may be in fluid contact, e.g., in liquid contact.

[0122] In some cases, the objective lens 1110 and the substrate 1160 undergo relative motion (e.g., linear motion, non-linear motion, rotational motion, etc.) substantially in the XY plane such that the objective lens and the substrate remain in fluid contact (e.g., liquid contact) via the fluid 1140.

[0123] In some cases, at least a portion of the amount of fluid 1140 from a previous fluidic contact between the objective lens and the substrate may remain in contact with the objective lens as the objective lens 1110 and the substrate 1160 undergo relative motion along the Z axis or another axis (having a Z component), such as when the objective lens is removed from fluidic (e.g., liquid) contact with the substrate, for example, between different scans. In some cases, at least a portion of the amount of fluid 1140 from a previous fluidic contact between the objective lens and the substrate may remain on the substrate. In some cases, such fluid may become part of the aqueous interface or aqueous environment of one or more layers of an aqueous solution or mixture disposed near the substrate. At the next fluidic contact between the objective lens and the substrate, a new amount of fluid may be brought into the housing 1120, such as with the aid of a fluid flow tube 1130 described elsewhere herein.

[0124] The housing 1120 may be configured to maintain a minimum distance 1150 between the substrate and housing to avoid contact between the housing and the substrate 1160 during periods when the substrate is moving relative to the plate. The minimum distance may be at least about 100 nanometers (nm), 200 nm, 300 nm, 400 nm, 500 nm, 1 micrometer (μm), 2 μm, 3 μm, 4 μm, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 1 millimeter (mm) or more. Alternatively or in addition, the minimum distance may be at most about 1 mm, 500 μm, 400 μm, 300 μm, 200 μm, 100 μm, 50 μm, 40 μm, 30 μm, 20 μm, 10 μm, 5 μm, 4 μm, 3 μm, 2 μm, 1 μm, 500 nm, 400 nm, 300 nm, 200 nm, 100 nm or less. Alternatively or in addition, the minimum distance may be within a range defined by any two of the aforementioned values. Even with a minimum distance, the enclosure may contain fluid due to surface tension effects. The system may include a fluid flow tube 1130 configured to deliver the fluid 1140 to the inside of the enclosure. A certain amount of fluid 1140 in the enclosure may be continuously or intermittently replenished and / or washed during the period of the detection event via the fluid flow tube. Beneficially, any contaminants in the fluid volume may be washed away during such refilling and / or cleaning. For example, in some cases, the volume of fluid may be continuously refilled. During refilling, a new volume of fluid may be directed into the housing, and at least a portion of the existing volume of fluid in the housing may wet the surface of the substrate, and in some cases may break off from the edge of the surface of the substrate. The existing volume of fluid may become part of the aqueous interface or fluid layer of the substrate. The fluid flow tube may be connected to the housing via an adapter 1135. The adapter may include a threaded adapter, a compression adapter, or any other adapter.

[0125] An electric field application unit (not shown) can be configured to adjust the hydrophobicity of one or more surfaces of the container, for example by applying an electric field, to retain at least a portion of the fluid in contact with the immersion objective lens and the open substrate.

[0126] The fluid 1140 may include water or an aqueous solution. The fluid may include an oil or an organic solution. The fluid may include a mixture of aqueous and non-aqueous solutions. When the fluid includes water or an aqueous solution(s), such fluid may be advantageously particularly compatible with maintaining the continuity of an aqueous interface or environment near the substrate 1160 and facilitating interaction between the objective lens and the substrate. For example, the substrate 1160 may include one or more layers of an aqueous solution or mixture near the substrate, such as for chemical processing operations and / or maintenance of an analyte or sample disposed on the substrate. The volume(s) of fluid 1140 contacted with and / or moved relative to one or more layers of an aqueous solution or mixture near the substrate may not disturb such one or more layers of an aqueous solution or mixture near the substrate or may minimize or reduce disturbance to such one or more layers of an aqueous solution or mixture near the substrate. For example, if the volume of fluid 1140 is immiscible with the composition of one or more layers near the substrate, the optical path from the objective lens to the sample on the substrate surface may be disrupted, the relative motion between the objective lens and the substrate may be disrupted, the aqueous interface or environment near the substrate may be disrupted, unwanted residue may be generated or left behind in one or more layers near the substrate or fluid volume, or both, and / or any combination of the above may occur. In some cases, the fluid and one or more layers of the aqueous solution or mixture may comprise the same aqueous solution or mixture. In some cases, the fluid and one or more layers of the aqueous solution or mixture may comprise different aqueous solutions or mixtures.

[0127] In some examples, the fluid 1140 includes a soaking buffer. The soaking buffer may have the same composition as the cleaning fluid used during the chemical operation. The soaking buffer and / or cleaning fluid may include a composition of buffers, salts and surfactants. The buffer may have a pH between about 8.0-9.0. In some examples, the fluid may have a pH of at least about 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5 or more. In the alternative, or in addition, the fluid may have a pH of at most about 9.5, 9.4, 9.3, 9.2, 9.1, 9.0, 8.9, 8.8, 8.7, 8.6, 8.5, 8.4, 8.3, 8.2, 8.1, 8.0, 7.9, 7.8, 7.7, 7.6, 7.5, 7.4, 7.3, 7.2, 7.1, 7.0, 6.9, 6.8, 6.7, 6.6, 6.5 or less. In some examples, the soaking buffer comprises 20 millimolar (mM) tris(hydroxymethyl)aminomethane, 110 mM NaCl, and 0.1% Triton-X100.

[0128] In some cases, the optical imaging objective 1110 and the housing 1120 may provide a physical barrier between a first location on the substrate where a chemical processing operation is performed and a second location on the substrate where a detection operation is performed. In this way, the chemical processing operation and the detection operation may be performed using independent operating conditions and contamination of the detector may be avoided. The first and second locations may have different humidity, temperature, pressure or atmospheric admixtures.

[0129] One method of detecting one or more signals or changes therein from an analyte may include using an immersion optical system. The method may include bringing an optical imaging objective lens 1110 into fluidic contact with a substrate 1160 having an analyte disposed thereon by providing a fluid 1140 in a housing 1120 between the objective lens and the substrate. The fluid may be continuously or intermittently replenished or washed using a fluid flow tube 1130. The method may further include washing a surface of the substrate before bringing the objective lens into fluidic contact with the substrate. The method may further include contacting a reaction mixture with a surface of the substrate to perform one or more chemical processing operations before washing the surface of the substrate. The washing operation may prevent contamination (e.g., contamination from a reaction mixture) between one or more chemical processing operations and a detection operation. For example, such a washing operation may prevent nucleotides or other reagents from a chemical processing operation from being carried over to an imaging or scanning operation.

[0130] The method may further include releasing fluid contact between the objective lens and the substrate, such as by, for example, lifting the objective lens relative to the substrate and / or pressing the substrate down relative to the objective lens.

[0131] The method further includes repeating the detection operation (e.g., bringing the optical imaging objective into fluid contact with the substrate and removing the fluid contact) multiple times on the same substrate. For example, the detection operation may be repeated at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more times on the same substrate.

[0132] Fluid 1140 may include water or an aqueous solution or aqueous mixture. Fluid may include oil, a non-aqueous solution, and / or an organic solution or mixture. Substrate may include one or more layers of an aqueous solution or aqueous mixture in its vicinity. Substrate may include one or more layers of an oil, a non-aqueous solution or mixture, and / or an organic solution or mixture in its vicinity. Fluid 1140 and one or more layers of a solution or mixture in its vicinity may each include a miscible composition.

[0133] Various systems and methods for detection may include an immersion objective lens system as described herein.

[0134] A system may include an imaging objective configured to detect a signal or signal change from an analyte coupled to a substrate, a housing configured to contain a volume of fluid between the imaging objective and the substrate, a fluid source including an aqueous solution, and a fluid flow unit configured to deliver a volume of fluid from the fluid source to the housing. The housing may be a physical housing. The aqueous solution may include an immersion buffer. The substrate may include one or more fluid layers. The one or more fluid layers may include a second aqueous solution. The second aqueous solution and the aqueous solution may include different compositions (e.g., different salts or concentrations thereof, different surfactants or concentrations thereof, different buffers or concentrations thereof, different compounds or mixtures or concentrations thereof). The second aqueous solution and the aqueous solution may include the same solution. The analyte and the objective may be in fluid contact through the aqueous solution and the second aqueous solution. The system may further include a substrate. The substrate may be any substrate as described elsewhere herein.

[0135] A method is provided that includes bringing an imaging objective into fluid contact with a substrate through a volume of fluid. The volume of fluid may include a first aqueous solution. The substrate may include (i) an analyte immobilized in its vicinity, and (ii) a layer of fluid in its vicinity. The layer of fluid may include a second aqueous solution. The method may include imaging the analyte through the volume of fluid with the imaging objective. The method may further include moving the imaging objective relative to the substrate, or moving the substrate relative to the imaging objective, or both, while maintaining fluid contact between the imaging objective and the substrate. The method may further include (i) disrupting fluid contact between the imaging objective and the substrate, and (ii) bringing the imaging objective and the substrate into a second fluid contact. After disrupting fluid contact, at least a portion of the volume of fluid may remain in fluid contact with the imaging objective and / or the substrate.

[0136] A method is provided that includes bringing an imaging objective into fluid contact with an analyte immobilized near a substrate through a volume of fluid that includes a first aqueous solution. The substrate may include a fluid layer that includes a second volume of fluid. The method may include imaging the analyte through the volume of fluid by the imaging objective. The method may further include moving the imaging objective relative to the substrate, or moving the substrate relative to the imaging objective, or both, while maintaining fluid contact between the imaging objective and the substrate. The method may further include (i) disrupting fluid contact between the imaging objective and the analyte, and (ii) bringing the imaging objective and the analyte into a second fluid contact. After disrupting fluid contact, at least a portion of the volume of fluid can remain in fluid contact with the imaging objective and / or the analyte.

[0137] The volume of fluid may have a thickness (e.g., the minimum distance between the objective lens and the substrate and / or analyte) of approximately 10 μm, 100 μm, 1000 μm (i.e., 1 millimeter (mm)), 10 mm, 100 mm, or more. In some cases, the thickness of the fluid volume is at least about 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm, 200 μm, 210 μm, 220 μm, 230 μm, 240 μm, 250 μm, 260 μm, 270 μm, 280 μm, 290 μm, 300 μm, 310 μm, 320 μm, 330 μm, 340 μm, 350 μm, 360 μm, 370 μm, 380 μm, 390 μm, 400 μm, 410 μm, 420 μm, 430 μm, 440 μm, 450 μm, 460 μm, 470 μm, 480 μm, 490 μm, 500 μm, 510 μm, 520 μm, 530 μm, 540 μm, 550 μm, 560 μm, 570 μm, 580 μm, 590 μm, 600 μm, 610 μm, 620 μm, 630 μm, 640 μm, 0 μm, 330 μm, 340 μm, 350 μm, 360 μm, 370 μm, 380 μm, 390 μm, 400 μm, 410 μm, 420 μm, 430 μm, 440 μm, 450 μm, 460 μm, 470 μm, 480 μm, 490 μm, 500 μm, 550 μm, 600 μm, 650 μm, 700 μm, 750 μm, 800 μm, 850 μm, 900 μm, 950 μm, 1000 μm or more. Alternatively, or in addition, the thickness of the volume of fluid is at most about 1000 μm, 950 μm, 900 μm, 850 μm, 800 μm, 750 μm, 700 μm, 650 μm, 600 μm, 550 μm, 500 μm, 490 μm, 480 μm, 470 μm, 460 μm, 450 μm, 440 μm, 430 μm, 420 μm, 410 μm, 400 μm, 390 μm, 380 μm, 370 μm, 360 μm, 350 μm, 340 μm, 360 μm, 380 μm, 390 μm, 380 μm, 370 μm, 360 μm, 350 μm, 360 μm, 37 ... 0 μm, 330 μm, 320 μm, 310 μm, 300 μm, 290 μm, 280 μm, 270 μm, 260 μm, 250 μm, 240 μm, 230 μm, 220 μm, 210 μm, 200 μm, 190 μm, 180 μm, 170 μm, 160 μm, 150 μm, 140 μm, 130 μm, 120 μm, 110 μm, 100 μm, 90 μm, 80 μm, 70 μm, 60 μm, 50 μm or less. Alternatively, or in addition, the thickness of the amount of fluid may range between any two of the aforementioned values. The substrate may include one or more fluid layers, where each layer has the same or different fluid composition. The fluid layer may include an aqueous solution. The fluid layer may include a non-aqueous solution.The fluid layer may be a thin film. In some cases, the thickness of the fluid layer is at least about 10 nanometers (nm), 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 16 μm, 17 μm, 18 μm, 19 μm, 20 μm, 21 μm, 22 μm, 23 μm, 24 μm, 25 μm, 26 μm, 27 μm, 28 μm, 29 μm, 30 μm, 31 μm, 32 μm, 33 μm, 34 μm, 35 μm, 36 μm, 37 μm, 38 μm, 39 μm, 40 μm, 41 μm, 42 μm, 43 μm, 44 μm, 45 μm, 46 μm, 47 μm, 48 μm, 49 μm, 50 μm, 51 μm, 52 μm, 53 μm, 54 μm, 55 μm, 56 μm, 57 μm, 58 μm, 59 μm, 60 μm, 61 μm, 62 μ m, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 16 μm, 17 μm, 18 μm, 19 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1000 μm or more. Alternatively, or in addition, the thickness of the fluid layer is at most about 1 mm, 900 μm, 800 μm, 700 μm, 600 μm, 500 μm, 400 μm, 300 μm, 200 μm, 100 μm, 90 μm, 80 μm, 70 μm, 60 μm, 50 μm, 40 μm, 30 μm, 20 μm, 19 μm, 18 μm, 17 μm, 16 μm, 15 μm, 14 μm, 13 μm, 12 μm, 11 μm, 10 μm , 9 μm, 8 μm, 7 μm, 6 μm, 5 μm, 4 μm, 3 μm, 2 μm, 1 μm, 900 mm, 800 mm, 700 mm, 600 mm, 500 nm, 450 nm, 400 nm, 350 nm, 300 nm, 250 nm, 200 nm, 150 nm, 100 nm, 90 nm, 80 nm, 70 nm, 60 nm, 50 nm, 40 nm, 30 nm, 20 nm, 10 nm or less. In the alternative, or in addition, the thickness of the fluid layer may be between any two of the ranges of the aforesaid values.

[0138] Figure 2A illustrates a partial cross-sectional view of a barrier system 200 maintaining a fluid barrier 213. Figure 2B illustrates a close-up view of the barrier system 200. Figure 2C illustrates a perspective view of a chamber 215 of the barrier system 200. Barrier system 200 and / or its respective components may correspond to barrier system 100 and / or its respective components.

[0139] The barrier system 200 includes a sample environment 205 defined by a plate 203, a chamber 215, and a fluid barrier 213. The chamber 215 and the plate 203 may be separated by a physical gap. The sample environment 205 may be isolated (and / or blocked) from an external environment 207.

[0140] The fluid barrier 213 may act as a transition region between the sample environment 205 and the external environment 207. The fluid barrier 213 may contain fluid (e.g., air) from the sample environment 205, the external environment 207, or both. The fluid barrier 213 may be a low pressure region. The fluid barrier 213 may have a lower pressure than the sample environment, the external environment, or both. The fluid barrier 213 may be maintained via a fluid flow unit, such as via a pressure changer 211. The fluid barrier 213 may contain fluid in coherent or bulk motion.

[0141] The pressure change device 211 may be integrated with the chamber 215. For example, as illustrated in Figures 2A-2C, the pressure change device may be integrated as a flow path 220 in the wall of the chamber 215. For example, suction may be applied through the flow path 220 to draw fluid from the external environment 207 or the sample environment 205 or both to create a partial vacuum curtain (e.g., in coherent motion, in bulk motion, etc.), thereby creating a fluid barrier 213. Otherwise, the fluid may be exposed to negative pressure. Fluid exhaust may be exhausted at another end of the flow path. Alternatively, or in addition, the device may not be integrated with the chamber 215. The fluid flow unit and / or the pressure change device 211 may be operated via one or more compressors (e.g., to create negative pressure), via one or more pumps (e.g., to create positive pressure), via a suction device, and / or via other devices to create a lower pressure in the transition region. The chamber 215 may include one or more flow paths 220 for implementing the fluid barriers of the present disclosure.

[0142] Although two pressure altering devices 211 are illustrated in Figures 2A-2C, it will be understood that any number of such devices may be present. For example, there may be at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 or more such devices. Alternatively, or in addition, there may be at most about 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3 or 2 such devices. In some cases, one or more pressure altering devices 211 may be implemented as an annular flow path surrounding the sample environment region, or as other flow paths along the periphery or border of the sample environment region. In some cases, one or more additional fluid flow paths (e.g., 233) may be provided near the bottom of the chamber to draw excess fluid (e.g., liquid, gas) from the sample environment region.

[0143] Beneficially, the fluid barrier 213 may provide a low or no friction seal between the sample environment 205 and the external environment 207. In some cases, the fluid flow rate through the fluid barrier 213 may be at least about 5 liters per minute (L / min), 5.5 L / min, 6 L / min, 6.5 L / min, 7 L / min, 7.5 L / min, 8 L / min, 8.5 L / min, 9 L / min, 9.5 L / min, 10 L / min, 10.5 L / min, 11 L, 11.5 L / min, 12 L / min, 12.5 L / min, 13 L / min, 13.5 L / min, 14 L / min, 14.5 L / min, 15 L / min or more. In the alternative, or in addition, the fluid flow rate may be at most about 15 L / min, 14.5 L / min, 14 L / min, 13.5 L / min, 13 L / min, 12.5 L / min, 12 L / min, 11.5 L / min, 11 L / min, 10.5 L / min, 10 L / min, 9.5 L / min, 9 L / min, 8.5 L / min, 8 L / min, 7.5 L / min, 7 L / min, 6.5 L / min, 6 L / min, 5.5 L / min, 5 L / min or less. As will be appreciated, the fluid flow rate may vary depending on various parameters (e.g., minimum distance between the plate and the chamber, pressure, temperature, etc.). In some examples, when there is a gap of about 500 microns between the plate 203 and the chamber 215, the fluid flow rate can be about 10 L / min, or about 13 milliliters / min (mL / min) per millimeter (mm) along the circumference for a velocity of about 0.42 meters / second (m / s).

[0144] The systems of the present disclosure may be scaled, such as to have multiple sample environment regions defined by the same plate. Figure 3 illustrates a barrier system 300 having multiple sample environments. Barrier system 300 and / or its respective components may correspond to any other barrier system (e.g., 100 and / or 200) and / or their respective components described herein.

[0145] A single plate 303 may define at least two independent identical sample environments (305, 309) that are further defined by two independent chambers. Each sample environment may be controlled and maintained independent of the other sample environment. Each sample environment may be movable relative to the plate 303 independent of the other sample environments. A fluid barrier may be maintained between each sample environment and an external environment.

[0146] Although two sample environments are illustrated in FIG. 3, it will be understood that the system of the present disclosure can be implemented for any number of sample environments using only one plate. For example, at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 or more such sample environments may be present in a single plate system. Alternatively, or in addition, at most about 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3 or 2 such sample environments may be present. Any subset of the multiple sample environments, or all of the multiple sample environments, may be able to move independently of the other sample environments.

[0147] In some cases, a single detector in the plate 303 may be used to detect one or more sample environments. Alternatively, or in addition, a single plate 303 may allow at least two detectors to protrude through the single plate 303 and detect in parallel. For example, such detectors may protrude through the plate via one or more openings (321a, 321b) having a fluid-tight engagement with the detector. The detectors may be fixed relative to the plate. In some cases, multiple detectors may detect two different locations in the same sample environment in parallel. In some cases, multiple detectors may detect at least two different sample environments in parallel.

[0148] Although two detector openings are illustrated in Figure 3, it will be understood that the system of the present disclosure may be implemented for any number of detectors using only one plate. For example, there may be at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 or more detectors in a one plate system. In the alternative, or in addition, there may be at most about 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3 or 2 such detectors.

[0149] The sample environment (e.g., 105, 205, 305, 309) of the present disclosure may be controlled. For example, the environment may be maintained at a specified temperature or humidity. The environment (or any element thereof) may be maintained at at least about 20 degrees Celsius (°C), 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, 100°C or higher. In the alternative, the environment may be maintained below 20°C. In the alternative, or in addition, the environment (or whatever element thereof) may be maintained at a temperature that is at most about 100° C., 95° C., 90° C., 85° C., 80° C., 75° C., 70° C., 65° C., 60° C., 55° C., 50° C., 45° C., 40° C., 35° C., 30° C., 25° C., 20° C. or lower. The environment may be maintained at a temperature that is within a range defined by any two of the foregoing values.

[0150] Various different elements of the sample environment, such as the chamber in the sample environment, the protruding portion of the detector, the immersion fluid, the plate, the substrate, the solution and / or the sample, may be maintained at different temperatures or within different temperature ranges, such as the temperatures or temperature ranges described herein. Various elements of the system may be set at temperatures above the dew point to prevent condensation. Various elements of the system may be set at temperatures below the dew point to collect condensation.

[0151] In some cases, the sample environment may be maintained at a higher humidity than the external environment. In some cases, the sample environment may be maintained at a relative humidity of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%. In the alternative, or in addition, the relative humidity may be maintained at a maximum of about 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25 20%, 15%, 10%, 5% or less. In the alternative, or in addition, the relative humidity may be maintained within a range defined by any two of the aforementioned values.

[0152] Environmental units (e.g., humidifiers, heaters, heat exchangers, compressors, etc.) may be configured to regulate one or more operating conditions in each sample environment. In some cases, each environment may be regulated by an independent environmental unit. In some cases, a single environmental unit may regulate multiple environments. In some cases, multiple environmental units may regulate different environments, individually or jointly. In the environmental units, active or passive methods may be used to regulate the operating conditions. For example, temperature may be controlled using heating or cooling elements. Humidity may be controlled using a humidifier or dehumidifier.

[0153] In some cases, the first portion of the sample environment may be further controlled from other portions of the sample environment. Different local portions may have different local temperatures, pressures and / or humidity. For example, the sample environment may include a first internal or local environment and a second internal or local environment, separated, for example, by a seal. In some cases, the seal may include an immersion objective as described elsewhere herein. For example, the immersion objective may be part of the seal that separates the sample environment into a first internal environment having 100% (or substantially 100%) relative humidity and a second environment having a different temperature, pressure or humidity. The second environment may be an ambient environment or may not be an ambient environment. The immersion objective may be in contact with a detector.

[0154] FIG. 4 illustrates an example barrier system 400 including different local environments (441, 442, 443), showing a cross-sectional side view. The example barrier system 400 and one or more components therein may correspond to the example barrier system 100 and one or more components therein. A fluid barrier 413 may be implemented between the sample environment 405 (e.g., a first region) and an external environment (e.g., a second region). The sample environment 405 may be a controlled environment that includes one or more samples therein. The external environment may be a closed or open environment. The region of the sample environment 405 may be defined by the chamber 415, the plate 403, and the fluid barrier 413. The fluid barrier 413 may be maintained between a physical gap between the chamber 415 and the plate 403. The chamber 415 and the plate 403 may be independent such that the chamber 145 and the region of the sample environment 405 defined by them are movable relative to the plate 403. The plate 403 and the chamber 415 may not be in direct mechanical contact such that a minimum distance exists between the plate and the chamber. The fluid barrier 413 may contain fluid from the sample environment, the external environment, or both, and may act as a transition region between the sample environment 405 and the external environment.

[0155] The sample environment 405 may include a substrate 417. One or more samples may be immobilized on or near the substrate 417. Alternatively, or in addition, one or more samples may be otherwise disposed on the substrate 417. In some cases, at least a portion of the chamber 415 may be or may include the substrate 417. In other examples, the chamber 415 may be coupled to the substrate 417. In some cases, the substrate 417 may be fixed relative to the chamber 415. In the alternative, the substrate 417 may be movable relative to the chamber 415, for example, in a linear and / or non-linear direction (e.g., rotational). For example, the substrate 417 may be fixed in XY coordinates (and / or Z coordinates) to the chamber 415, except that the substrate 417 may be rotatable relative to the chamber 415.

[0156] The detector 401 may protrude from the external environment into the sample environment 105 through the plate 403, such as through an opening in the plate 403. At least a portion of the detector 401 may be fixed relative to the plate 403. In some cases, the detector 401 may be capable of translating independently of the plate 403 along an axis substantially perpendicular to the plane of the plate 403 (e.g., through an opening). Within the sample environment 405, the detector 401 may be configured to detect one or more samples disposed on a substrate using an immersion optical system, such as the system described with respect to FIG. 1C. A portion of the detector 401 (e.g., an optical imaging objective, etc.) inside the sample environment 405 may be in optical communication with the substrate via a liquid fluid 431 medium. In some cases, the liquid fluid medium may be disposed in a localized region of the substrate 417. In the alternative, the detector 401 may be in optical communication with the substrate without a liquid fluid medium.

[0157] The sample environment 405 may include any number of different local environments (441, 442, 443) located in different portions of the sample environment. These different local environments may be regulated. The fluid barrier 413 may allow these different local environments to be maintained at different local environmental conditions. For example, the local environments may be maintained at a local temperature or local temperature range. For example, the local environments may be maintained at a local humidity or local humidity range. For example, the local environments may be maintained at a local pressure or local pressure range. The local temperatures may be at least about 20 degrees Celsius (°C), 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, 100°C or higher. In the alternative, the local temperatures may be maintained below 20°C. Alternatively, or in addition, the local temperature may be at most about 100° C., 95° C., 90° C., 85° C., 80° C., 75° C., 70° C., 65° C., 60° C., 55° C., 50° C., 45° C., 40° C., 35° C., 30° C., 25° C., 20° C. or less. The local environment may be maintained at a local temperature that is within a range defined by any two of the foregoing values. The local relative humidity may be at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%. Alternatively, or in addition, the local relative humidity may be at most about 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25 20%, 15%, 10%, 5% or less. Alternatively, or in addition, the relative humidity may be maintained within a range defined by any two of the aforementioned values. The local environment may be maintained using any of the environmental units described herein.

[0158] In the example illustrated in FIG. 4, a first localized environment 441 located at or near the top of the sample environment 405 (or chamber 415) is maintained as the highest localized temperature within the sample environment 405, such as to prevent condensation and unwanted dripping onto the substrate 417. A second localized environment 442 includes a humidity source 419, such as a body of liquid (e.g., water), at or near the bottom of the sample environment 405 (or chamber 415). The second localized environment 442 may be maintained at a second highest localized temperature within the sample environment 405, such as to generate steam from the humidity source 419. A third localized environment 443 is located at or near the surface of the substrate 417 and is maintained as the lowest localized temperature within the sample environment 405, such as to prevent the surface from drying out. Although three local environments are illustrated, it will be understood that the sample environment may have any number of different local environments maintained at different local environmental conditions, for example, at least one, two, three, four, five, six, seven, eight, nine, ten or more local environments, etc.

[0159] The objective lens of the detector 401 may be heated to prevent condensation and disturbance of the optical path between the objective lens and the substrate 417. Alternatively or additionally, another component or part of the detector in fluid contact with the substrate may be heated. The term "heating" as used herein may generally refer to subjecting an object to an elevated temperature compared to a baseline temperature before the heating operation. Heating may be performed by an environmental unit as described herein. Heating may be performed by heating or maintaining the temperature (or range) of a volume of immersion fluid in contact with the detector and substrate. A heating element may be coupled to or integrated with the objective lens (and / or other components of the detector).

[0160] A system for processing or analyzing an analyte is provided, the system including a chamber and a lid. The chamber may include an interior region including a substrate configured to immobilize the analyte immobilized on the substrate. The lid may be configured to be disposed proximate to the chamber. The system may include an environmental unit configured to maintain a first local environment, a second local environment, and a third local environment in the interior region. The environmental unit may be configured to maintain the first local environment at a first temperature or temperature range, the second local environment at a second temperature or temperature range, and the third local environment at a third temperature or temperature range. The first local environment may be disposed above the second local environment and the third local environment. The first local environment may be on or proximate to the lid. The second local environment may be disposed on or proximate to the surface of the substrate. The third local environment may be disposed below the first local environment and the second local environment. The first temperature or temperature range may be higher than the second temperature or temperature range and the third temperature or temperature range. The second temperature or temperature range may be lower than the third temperature or temperature range.

[0161] 5 illustrates a processing system 500 including an example barrier system. The processing system 500 may include one or more modular components.

[0162] The processing system 500 may include one or more barrier systems, such as a first barrier system 505a and a second barrier system 505b. The barrier systems (e.g., 505a, 505b) of the processing system 500 and one or more components thereof may correspond to any barrier system and one or more components thereof described herein. Although two barrier systems are illustrated in Figure 5, it will be understood that the processing system may have any number of barrier systems.

[0163] In some cases, any of the barrier systems of the present disclosure may be used to handle tasks instead of or in addition to detection.

[0164] For example, any of the barrier systems of the present disclosure may have one or more operational units instead of or in addition to a detector (e.g., 501). An operational unit may include one or more devices or a collection thereof and may be configured to facilitate an operation on a sample or a sample environment (or its local environment(s)). For example, an operational unit may include one or more detectors configured to facilitate detection of a signal or signal change from a sample. In another example, an operational unit may include a fluid dispenser (e.g., 509a, 509b) configured to facilitate dispensing of a reagent or fluid to the sample. In another example, an operational unit may include an environmental unit configured to facilitate environmental conditioning of the sample environment. In another example, an operational unit may include a light source, a heat source, or a humidity source. In another example, an operational unit may include one or more sensors of any type. A barrier system may have multiple operational units of the same or different types.

[0165] An operating unit (e.g., 509a) may protrude from the external environment into the sample environment of the barrier system through a plate (e.g., 503), such as through an opening in the plate. The engagement between the operating unit and the opening may be fluid-tight, such that there is no fluid communication through the opening when the operating unit engages through the opening. In the alternative, or in addition, the opening may be sealed. In the alternative, the plate may be integral with the operating unit, or the operating unit may be integral with the plate. In the alternative, the operating unit may be fully contained in the sample environment, for example, by attaching an end that does not face the sample to the plate. In some cases, at least a portion of the operating unit may be fixed relative to the plate. In some cases, the operating unit may be capable of translating independently of the plate along an axis substantially orthogonal to the plane of the plate (e.g., through the opening). In some cases, at least a portion of the operating unit (e.g., the portion of the operating unit that is inside the sample environment region) may be capable of moving independently of the plate (e.g., linearly or non-linearly, e.g., rotating, etc.).

[0166] In some cases, the processing system 500 may include multiple modular plates (e.g., 503a, 503b, 503c) that may be interconnected or otherwise fastened to provide an uninterrupted plate 503. The fit between adjacent modular plates may be fluid-tight such that there is no fluid communication between the modular plates. Alternatively, or in addition, the fit may include an airtight seal. Adjacent modular plates (e.g., a first modular plate and a second modular plate) may be coupled via one or more fastening mechanisms. Examples of fastening mechanisms may include, but are not limited to, complementary threads, form-fitting pairs, hooks and loops, latches, threads, screws, staples, clips, clamps, prongs, rings, brads, rubber bands, rivets, grommets, pins, ties, snaps, Velcro, adhesives (e.g., glue), tapes, vacuums, seals, magnets, magnetic seals, combinations thereof, or any other type of fastening mechanism.

[0167] In some cases, the first and second modular plates can be fastened to each other via complementary fastening units. For example, the first and second modular plates can complete a form-fitting pair. The first modular plate can include a form-fitting male component and the second modular plate can include a form-fitting female component, and / or conversely, the first modular plate can include a form-fitting female component and the second modular plate can include a form-fitting male component. In some cases, the outer diameter of the protruding type fastening unit of the first modular plate can be substantially equal to the inner diameter of the recessed type fastening unit of the second modular plate to form an interference fit, or vice versa. Alternatively or in addition, the two modular plates can include other types of complementary units or structures (e.g., hooks and loops, latches, snap-ons, buttons, nuts and bolts, magnets, etc.) that can be fastened together. Alternatively, or in addition, the two modular plates can be fastened using other fastening mechanisms, such as, but not limited to, staples, clips, clamps, prongs, rings, brads, rubber bands, rivets, grommets, pins, ties, snaps, Velcro, adhesives (e.g., glue), magnets or magnetic fields, tape, combinations thereof, or any other type of fastening mechanism.

[0168] In some cases, the first modular plate and the second modular plate can be fastened to each other via an intermediate structure. The intermediate structure may be a linker or connector between the first modular plate and the second modular plate. In some cases, the intermediate structure may be fastened to one or both of the first modular plate and the second modular plate via any one or more of the fastening mechanisms described herein. The intermediate structure may be a solid. The intermediate structure may be a liquid or a gas. The intermediate structure may be a gel. In some cases, the intermediate structure may be applied as one phase (e.g., a liquid) and change to another phase (e.g., a solid) after a period of time, such as to achieve fastening. For example, the intermediate structure may include a fluid adhesive that solidifies to achieve fastening. In some cases, the intermediate structure may be capable of changing from a first phase to a second phase, e.g., from liquid to solid or from solid to liquid, upon application of a stimulus (e.g., heat change, pH change, pressure change, magnetic field, electric field, etc.) to achieve fastening or unfastening (or both). In some cases, the first modular plate and / or the second modular plate may include an intermediate structure. For example, the intermediate structure may be integral with the first modular plate and / or the second modular plate. In some cases, the first modular plate and / or the second modular plate may be capable of changing in part or in whole from a first phase to a second phase, e.g., from liquid to solid or from solid to liquid, upon application of a stimulus (e.g., heat change, pH change, pressure change, magnetic field, electric field, etc.) to achieve fastening or unfastening (or both) to the other plate. In some cases, when the two modular plates are fastened together, it is possible for one or both of the two modular plates to be cut or pierced by the other.

[0169] The fastening between the first and second modular plates can be temporary, such as to allow for subsequent unfastening of the two modular plates with no or minimal damage to the two modular plates (e.g., permanent deformation, cosmetic changes, etc.). In some cases, the first modular plate may be repeatedly and easily unfastened from the second modular plate and / or the remainder of plates 503.

[0170] In some cases, a modular plate may be removable from another modular plate or the remainder of the plate, such as during operation (e.g., reagent distribution, washing, detection, etc.) by one or more operational units of one or more barrier systems, without disturbing one or more sample environments of the respective one or more barrier systems, including at least a portion of the remainder of the plate. Beneficially, removal of a modular plate may allow access to the chambers, such as for loading or removing the chambers in the processing system 500. Removal of a modular plate may also allow access to the interior of the chambers of the barrier system, such as for loading or removing a substrate from the chamber. Removal of a modular plate may also allow access to one or more operational units coupled to or otherwise associated with the removed modular plate, such as for maintenance, repair, and / or replacement of the one or more operational units. Such removal may occur while another barrier system continues normal operation (e.g., chemical processing operations, detection operations, etc.). In some cases, removal of a modular plate may be along the Z axis or substantially along the Z axis, or along any other axis (e.g., XY plane, etc.). In some cases, any of the modular plates may be removable from another modular plate. In some cases, any of the modular plates may be movable relative to another modular plate. In some cases, any of the modular plates may be movable with respect to a reference coordinate at the time of removal. In some cases, any of the modular plates may be substantially stationary with respect to a reference coordinate at the time of removal. In some cases, a first modular plate (e.g., 520a, 520c, etc.) may be movable with respect to a reference coordinate and a second modular plate (e.g., 520b) may be stationary with respect to a reference coordinate.

[0171] The processing system 500 may include a variety of different operation stations (e.g., 520a, 502b, 520c). The operation stations may be positioned relative to a given area of ​​the plate 503. In some cases, only one modular plate may include one or more operation units for one operation station. In some cases, multiple modular plates may include one or more operation units for one operation station. In some cases, only one modular plate may include one or more operation units for multiple operation stations. In some cases, multiple modular plates may include one or more operation units for multiple operation stations. The operation stations may include chemical operation stations (e.g., 520a, 520c), such as for reagent dispensing and / or washing. The operation stations may include detection stations (e.g., 520b), such as for detecting a signal or a signal change. Any of the barrier systems (e.g., 505a, 505b) of the processing system may be capable of moving between the different operation stations. Alternatively, or in addition, the plate 503 may be capable of traversing relative to either barrier system in order to position the barrier system relative to an operating station (e.g., located relative to a given area of ​​the plate). In some cases, the barrier system may be provided with rails or tracks 507 or other motion paths to enable traversal between the different operating stations. In some cases, different barrier systems may share the same rails or tracks or other motion paths (e.g., as illustrated in FIG. 5) for traversal between the different operating systems. In such cases, the different barrier systems may be configured to move independently of one another on the same rails or tracks or other motion paths, or to move in unison. In some cases, the different barrier systems may move on dedicated separate rails or tracks or other motion paths.In some cases, the fluid barrier of the barrier system may be maintained during relative motion between the plate 503 and the barrier system, such as during switching of operating stations. In some cases, one or more operating units may be capable of movement relative to the plate 503 (such as movement along an axis perpendicular to the plate) or removal from the plate 503 to allow the barrier system to be positioned with respect to the operating stations.

[0172] The external environment (e.g., 107, 207) of the present disclosure may be any environment outside the sample environment. For example, the external environment may be an indoor environment. The external environment may be an ambient environment. The external environment may be self-regulated, such as, for example, via one or more environmental units described elsewhere herein. The external environment may be open or closed. In some cases, the external environment may be at indoor temperature, pressure, and / or humidity. In some cases, the external environment may be at ambient temperature, pressure, and / or humidity.

[0173] The chambers (e.g., 115, 215, 415) of the present disclosure may include a base and a sidewall to define an opening that is in close contact with the plate (or lid). The sidewall may be a closed continuous surface or a plurality of adjacent (and / or contiguous) surfaces. For example, the base may include or be a substrate. In some cases, the base may be coupled to the substrate. The substrate may be translationally fixed to the substrate. The substrate may be rotatable relative to the substrate. The translational movement may include a movement of an object from a first coordinate to a second coordinate. The translational movement may include a movement of a reference point of the object from a first coordinate to a second coordinate. In some cases, at least a portion of the sidewall of the chamber may have a thickness dimension large enough to allow integration of one or more flow paths to allow operation of the pressure altering device. In some cases, the sidewall of the chamber may have a thickness dimension large enough to maintain a low pressure fluid barrier. The chamber may be made in whole or in part of glass, silicon, metals, such as aluminum, copper, titanium, chromium or steel, ceramics, such as titanium dioxide or silicon nitride, plastics, such as polyethylene (PE), low density polyethylene (LDPE), high density polyethylene (HDPE), polypropylene (PP), polystyrene (PS), high impact polystyrene (HIPS), polyvinyl chloride (PVC), polyvinylidene chloride (PVDC), acrylonitrile butadiene styrene (ABS), polyacetylene, polyamide, polycarbonate, etc. The polymeric material may include one or more of polyvinyl chloride, polyether, polyester, polyurethane, polyepoxide, polymethyl methacrylate (PMMA), polytetrafluoroethylene (PTFE), phenol formaldehyde (PF), melamine formaldehyde (MF), urea formaldehyde (UF), polyether ether ketone (PEEK), polyetherimide (PEI), polyimide, polylactic acid (PLA), furans, silicone, polysulfone, any mixture of any of the foregoing materials, or any other suitable material.

[0174] The substrate (e.g., 417) of the present disclosure may be an open substrate. The substrate may be a solid substrate. The substrate may be made in whole or in part of glass, silicon, metal, such as aluminum, copper, titanium, chromium, or steel, ceramic, such as titanium dioxide or silicon nitride, plastic, such as polyethylene (PE), low density polyethylene (LDPE), high density polyethylene (HDPE), polypropylene (PP), polystyrene (PS), high impact polystyrene (HIPS), polyvinyl chloride (PVC), polyvinylidene chloride (PVDC), acrylonitrile butadiene styrene (ABS), polyacetylene, polyamide, polycarbonate, or the like. The substrate may comprise one or more of the following: ethylene, polyester, polyurethane, polyepoxide, polymethylmethacrylate (PMMA), polytetrafluoroethylene (PTFE), phenol formaldehyde (PF), melamine formaldehyde (MF), urea formaldehyde (UF), polyetheretherketone (PEEK), polyetherimide (PEI), polyimide, polylactic acid (PLA), furans, silicones, polysulfone, any mixture of any of the foregoing materials, or any other suitable material. The substrate may be made in whole or in part of a metal, such as aluminum, copper, silver, or gold, an oxide, such as silicon oxide (Si x O yThe substrate may be coated with one or more layers of a coating material such as a photoresist, e.g., SU8, a surface coating such as an aminosilane or a hydrogel, polyacrylic acid, polyacrylamide dextran, polyethylene glycol (PEG), or any combination of any of the foregoing materials, or any other suitable coating material. The one or more layers may be at least 1 nanometer (nm), at least 2 nm, at least 5 nm, at least 10 nm, at least 20 nm, at least 50 nm, at least 100 nm, at least 200 nm, at least 500 nm, at least 1 micrometer (μm), at least 2 μm, at least 5 μm, at least 10 μm, at least 20 μm, at least 50 μm, at least 100 μm, at least 200 μm, at least 500 μm, or at least 1 millimeter (mm) in thickness. The one or more layers may have a thickness that is within a range defined by any two of the foregoing values.

[0175] The substrate and / or chamber may have any shape, form or dimension. In some cases, for example, the substrate may have an overall form of a cylinder, a cylindrical shell or disk, a rectangular prism, or any other geometric form. The substrate may have a thickness (e.g., smallest dimension) of at least about 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 1 cm, 2 cm, 3 cm, 4 cm, 5 cm, or more. The substrate may have a thickness that is within a range defined by any two of the aforementioned values. The substrate may have a first lateral dimension (e.g., width for a substrate whose overall form is a rectangular prism, or radius for a substrate whose overall form is a cylinder) of at least about 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 1 cm, 2 cm, 3 cm, 4 cm, 5 cm, 6 cm, 7 cm, 8 cm, 9 cm, 10 cm, 20 cm, 30 cm, 40 cm, 50 cm, 60 cm, 70 cm, 80 cm, 90 cm, 1 meter (m) or more. The substrate may have a first lateral dimension that is within a range defined by any two of the foregoing values. The substrate may have a second lateral dimension (e.g., length for a substrate whose overall form is a rectangular prism) of at least about 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 1 cm, 2 cm, 3 cm, 4 cm, 5 cm, 6 cm, 7 cm, 8 cm, 9 cm, 10 cm, 20 cm, 30 cm, 40 cm, 50 cm, 60 cm, 70 cm, 80 cm, 90 cm, 1 meter (m) or more. The substrate may have a second lateral dimension that is within a range defined by any two of the aforementioned values. The substrate surface may be flat or substantially flat. Alternatively, or in addition, the substrate surface may be textured or patterned. For example, the substrate may include grooves, troughs, hills, and / or pillars. In some cases, the substrate may include wells. In some cases, the substrate may define one or more cavities (e.g., microscale cavities or nanoscale cavities). The substrate may have a regular texture and / or pattern across the surface of the substrate.For example, the substrate may have regular geometric structures (e.g., wedges, cubes, cylinders, spheres, hemispheres, etc.) that are above or below the reference level of the surface. Alternatively, the substrate may have irregular surface textures and / or patterns across the substrate's surface. For example, the substrate may have any arbitrary structure above or below the reference level of the substrate. In some cases, the substrate's surface texture may include structures having a maximum dimension that is at most about 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.1%, 0.01%, 0.001%, 0.0001%, 0.00001% of the total thickness of the substrate or a given layer of the substrate. In some cases, the surface texture and / or pattern of the substrate may define at least a portion of the individually addressable locations on the substrate. The surface textured and / or patterned substrate may be substantially flat.

[0176] The substrate may include an array. For example, the array may be located on a side of the substrate. The array may be a planar array. The array may have an overall shape of a circle, ring, rectangle, or any other shape. The array may include linear and / or non-linear rows. The array may be equally spaced or evenly distributed. The array may be arbitrarily spaced or arbitrarily distributed. The array may have regular spacing. The array may have irregular spacing. The array may be a textured array. The array may be a patterned array. FIG. 6 illustrates various examples of arrays of individually addressable locations 601 on a substrate (e.g., from a top view), where panel A shows a substantially rectangular substrate with a regular linear array, panel B shows a substantially circular substrate with a regular linear array, and panel C shows an arbitrarily shaped substrate with an irregular array.

[0177] An array may include a plurality of individually addressable locations (e.g., 501). In some cases, these locations may correspond to individually addressable coordinates on a substrate. Alternatively, or in addition, these locations may correspond to physical structures (e.g., wells) on a substrate. Analytes to be processed and / or detected by a detector may be immobilized on the array. The array may include one or more binders as described herein, such as one or more physical or chemical linkers or adapters that are linked to or configured to be linked to the analytes. For example, the array may include linkers or adapters that are linked to nucleic acid molecules. Alternatively, or in addition, the analytes may be linked to beads, and the beads may be immobilized on the array.

[0178] An individually addressable location may include a location of an analyte or group of analytes that is accessible for manipulation. Manipulation may include placement, extraction, reagent dispensing, seeding, heating, cooling or stirring. Extraction may include extracting individual analytes or groups of analytes. For example, extraction may include extracting at least 2, at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 500, or at least 1,000 analytes or groups of analytes. Alternatively or in addition, extraction may include extracting at most 1,000, at most 500, at most 200, at most 100, at most 50, at most 20, at most 10, at most 5, or at most 2 analytes or groups of analytes. Manipulation may be accomplished, for example, via localized microfluidic, pipette, optical, laser, acoustic, magnetic and / or electromagnetic interactions with the analyte or its surroundings.

[0179] The array may be coated with a binder. For example, the array may be coated with a binder randomly. Alternatively, the array may be coated with a binder arranged in a regular pattern (e.g., in a linear array, a radial array, a hexagonal array). The array may be coated with a binder on at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the number of individually addressable locations or the surface area of ​​the substrate. The array may be coated with a binder with a percentage of the individually addressable locations within a range defined by any two of the aforementioned values ​​or a percentage of the surface area of ​​the substrate within a range defined by any two of the aforementioned values. The binder may be integral with the array. The binder may be added to the array, for example, the binder may be added to the array as one or more coating layers on the array.

[0180] The binder may immobilize the analyte through non-specific interactions, such as one or more of hydrophilic interactions, hydrophobic interactions, electrostatic interactions, and physical interactions (e.g., adhesion to pillars or sedimentation in wells). In some cases, the binder may immobilize the biological analyte through specific interactions. For example, when the biological analyte is a nucleic acid molecule, the binder may include an oligonucleotide adaptor configured to bind to the nucleic acid molecule. Alternatively, or in addition, the binder may include one or more of antibodies, oligonucleotides, aptamers, affinity binding proteins, lipids, carbohydrates, and the like, such as for binding to other types of analytes. The binder may immobilize the biological analyte through any possible combination of interactions. For example, the binder may immobilize the nucleic acid molecule through a combination of physical interactions and chemical interactions, a combination of protein interactions and nucleic acid interactions, and the like. An array may include at least about 10, 100, 1000, 10,000, 100,000, 1,000,000, 10,000,000, 100,000,000 or more binders. Alternatively, or in addition, an array may include at most about 100,000,000, 10,000,000, 1,000,000, 100,000, 10,000, 1000, 10 or less binders. An array may have a number of binders that is within a range defined by any two of the aforementioned values. In some cases, a single binder may bind to a single analyte (e.g., a nucleic acid molecule). In some cases, a single binder may bind to multiple analytes (e.g., multiple nucleic acid molecules). In some cases, multiple binders may bind to a single analyte.Although some examples herein describe the interaction of the binders with nucleic acid molecules, the binders may also immobilize other molecules (e.g., proteins, etc.), other particles, cells, viruses, or other organisms, and non-biological analytes.

[0181] In some cases, each location, or a subset of such locations, may have an analyte (e.g., a nucleic acid molecule, a protein molecule, a carbohydrate molecule, etc.) immobilized thereto. In other cases, a portion of the plurality of individually addressable locations may have an analyte immobilized thereto. The plurality of analytes immobilized to the substrate may be copies of a template analyte. For example, the plurality of analytes (e.g., nucleic acid molecules) may have sequence homology. In other examples, the plurality of analytes immobilized to the substrate may not be copies. The plurality of analytes may be the same type of analyte (e.g., nucleic acid molecules) or may be a combination of different types of analytes (e.g., nucleic acid molecules, protein molecules, etc.).

[0182] In some cases, an array may include multiple types of binders, such as for binding with different types of analytes. For example, an array may include a first type of binder (e.g., oligonucleotides) configured to bind with a first type of analyte (e.g., nucleic acid molecules), and a second type of binder (e.g., antibodies) configured to bind with a second type of analyte (e.g., proteins), and so forth. In another example, an array may include a first type of binder (e.g., a first type of oligonucleotide molecule) for binding with a first type of nucleic acid molecule, and a second type of binder (e.g., a second type of oligonucleotide molecule) for binding with a second type of nucleic acid molecule, and so forth. For example, a substrate may be configured to bind with different types of analytes at specific portions or specific locations of a substrate surface by having different types of binders at specific portions or specific locations of the substrate surface.

[0183] Analytes may be immobilized on the array at defined individually addressable locations of a plurality of individually addressable locations. The array may have any number of individually addressable locations. For example, the array may have at least 1, at least 2, at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, at least 10,000, at least 20,000, at least 50,000, at least 100,000, at least 200,000, at least 500,000, at least 1,000,000, at least 2,000,000, at least 5,000,000, at least 10,000,000. An array may have at least 20,000,000, at least 50,000,000, at least 100,000,000, at least 200,000,000, at least 500,000,000, at least 1,000,000,000, at least 2,000,000,000, at least 5,000,000,000, at least 10,000,000,000, at least 20,000,000,000, at least 50,000,000,000, or at least 100,000,000,000 individually addressable locations. An array may have a number of individually addressable locations that is within a range defined by any two of the foregoing values. Each individually addressable location may be individually digitally and / or physically accessible (from a plurality of individually addressable locations). For example, each individually addressable location may be electronically or digitally located, identified, and / or accessed for mapping, sensing, association with a device (e.g., a detector, processor, dispenser, etc.), or otherwise processing. Alternatively, or in addition, each individually addressable location may be physically located, identified, and / or accessed, such as for physical manipulation or extraction of an analyte, reagent, particle, or other component located at the individually addressable location.

[0184] Each individually addressable location may have an overall shape or form of a circle, a rectangle, a pit, a bump, or any other shape or form. Each individually addressable location may have a first lateral dimension (e.g., a radius for an individually addressable location whose overall shape is a circle, or a width for an individually addressable location whose overall shape is a rectangle, etc.). The first lateral dimension may be at least 1 nanometer (nm), at least 2 nm, at least 5 nm, at least 10 nm, at least 20 nm, at least 50 nm, at least 100 nm, at least 200 nm, at least 500 nm, at least 1,000 nm, at least 2,000 nm, at least 5,000 nm, or at least 10,000 nm. The first lateral dimension may be within a range defined by any two of the aforementioned values. Each individually addressable location may have a second lateral dimension (e.g., a length for an individually addressable location whose overall shape is a rectangle, etc.). The second lateral dimension may be at least 1 nanometer (nm), at least 2 nm, at least 5 nm, at least 10 nm, at least 20 nm, at least 50 nm, at least 100 nm, at least 200 nm, at least 500 nm, at least 1,000 nm, at least 2,000 nm, at least 5,000 nm, or at least 10,000 nm. The second lateral dimension may be within a range defined by any two of the aforementioned values. In some cases, each individually addressable location may have or be linked to a binder as described herein to immobilize an analyte at the location. In some cases, only a portion of the individually addressable locations may have or be linked to a binder. In some cases, an individually addressable location may have or be linked to multiple binders to immobilize an analyte at the location.

[0185] Analytes that are bound to individually addressable locations may include, but are not limited to, molecules, cells, organisms, nucleic acid molecules, nucleic acid colonies, beads, clusters, polonies, or DNA nanoballs. The bound analytes may be immobilized on the array in a regular, patterned, periodic, random or pseudorandom arrangement, or any other spatial arrangement.

[0186] Although the examples of the present disclosure describe the processing and / or detection of samples and analytes immobilized at individually addressable locations on a substrate surface, the systems, devices and methods described herein also allow for detection of the substrate itself (without any samples and / or analytes disposed thereon).

[0187] The substrate may be configured to move relative to the plate. Such motion may be facilitated by one or more actuators or other devices (e.g., gears, stages, actuators, disks, pulleys, motors, etc.). Such actuators and devices may be mechanically connected to the substrate directly or indirectly through intermediate components. Such actuators and devices may be automated. Alternatively, or in addition, the actuators and devices may receive manual input. The substrate may be configured to move at any speed that allows for detection. In some cases, or in rotational motion, the axis of rotation may be an axis that passes through the center of the substrate. The axis may be an off-center axis. For example, the substrate may be attached to a chuck (e.g., a vacuum chuck, etc.). The substrate may be configured to rotate at a rotational speed of at least 1 revolution per minute (rpm), at least 2 rpm, at least 5 rpm, at least 10 rpm, at least 20 rpm, at least 50 rpm, at least 100 rpm, at least 200 rpm, at least 500 rpm, at least 1,000 rpm, at least 2,000 rpm, at least 5,000 rpm, or at least 10,000 rpm. The substrate may be configured to rotate at a rotational speed that is within a range defined by any two of the aforementioned values. The substrate may be configured to rotate at different rotational speeds during the various different operations described herein. The substrate may be configured to rotate at a rotational speed that varies according to a time-dependent function, such as, for example, a ramp function, a sinusoidal function, a pulse function, or other function or combination of functions. The time-varying function may be periodic or non-periodic.

[0188] The fluid barriers provided herein may provide for frictionless or low-friction relative motion between the substrate and the detector. There may be no mechanical contact between the plate (coupled to the detector) and the chamber (coupled to the substrate).

[0189] The detectors (e.g., 101, 1110) of the present disclosure may include devices capable of detecting signals. For example, the signals can be signals indicative of the presence or absence of one or more components (e.g., incorporated nucleotides, fluorescent labels, electronic signals, etc.) and / or signals indicative of a change in state in one or more components. The detectors may detect multiple signals. The signal or multiple signals may be detected in real time before, during (or substantially during), or after a reaction, such as a sequencing reaction. In some cases, the detectors may include optical and / or electronic components capable of detecting signals. The detectors may perform one or more detection methods. Non-limiting examples of detection methods include optical detection, spectroscopic detection, electrostatic detection, electrochemical detection, acoustic detection, and magnetic detection, etc. Optical detection methods include, but are not limited to, light absorption, ultraviolet-visible (UV-vis) light absorption, infrared light absorption, light scattering, Rayleigh scattering, Raman scattering, surface-enhanced Raman scattering, Mie scattering, fluorescence, luminescence, and phosphorescence. Spectroscopic detection methods include, but are not limited to, mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy and infrared spectroscopy. Electrostatic detection methods include, but are not limited to, gel-based techniques, such as gel electrophoresis. Electrochemical detection methods include, but are not limited to, electrochemical detection of amplification products after high performance liquid chromatography separation of the amplification products.

[0190] A detectable signal, such as an optical signal (e.g., a fluorescent signal), may be generated when an analyte reacts with another component (e.g., a probe). For example, the signal may originate from the probe and / or the analyte. The detectable signal may indicate a reaction or interaction between the probe and the analyte. The detectable signal may be a non-optical signal. For example, the detectable signal may be an electronic signal. The detectable signal may be detected by one or more sensors. For example, an optical signal may be detected via one or more optical detectors in an optical detection scheme described elsewhere herein. The signal may be detected during the period of motion of the substrate. The signal may be detected after the motion has ceased. In some cases, after detection, the signal may weaken, such as by cleaving a label from the probe and / or analyte and / or modifying the probe and / or analyte. Such cleavage and / or modification may be caused by one or more stimuli, such as by exposure to a chemical, an enzyme, light (e.g., ultraviolet light) or a temperature change (e.g., heat). In some cases, the signal may be rendered undetectable, in other cases, by stopping or changing the mode (e.g., detection wavelength) of one or more sensors, or terminating or reversing the excitation of the signal. In some cases, detection of the signal may include capturing an image or producing a digital output (e.g., between different images).

[0191] The detector may be capable of continuous area scanning during continuous linear motion and / or continuous non-linear motion (e.g., rotational motion) between the sample and the substrate. For example, the detector may scan the substrate or array along a linear or substantially linear path. Alternatively, or in addition, the detector may scan along a non-linear path, including a loop, spiral, or arc path on a rotating substrate. The detector may be a continuous area scanning detector. The continuous area scanning detector may include an imaging array sensor capable of continuous integration over a scan area. The scanning may be electronically synchronized to the image of the object in relative motion. The continuous area scanning detector may include a time-delay integration (TDI) charge-coupled device (CCD), a hybrid TDI, and / or a complementary metal-oxide semiconductor (CMOS) pseudo-TDI.

[0192] For a rotational scan path, the scan direction may be substantially θ in a (R, θ) coordinate system where the rotational motion of the object is in the θ direction. Over any field of view at the surface of the object (substrate) imaged by the scanning system, the apparent velocity is

number

[0193] The tangential velocity blur may vary with the radial position (R) of the field point on the object surface as tangential velocity blur. A continuous surface scanning detector may scan at the same speed for all image locations, and therefore may not be able to operate at the correct scan speed for all imaged points in a curved (or arcuate or non-linear scan) scan. Thus, the scan may be corrupted by velocity blur for imaged field points that move at a speed different from the scan speed. A continuous rotational surface scan may include an optical detection system or method that provides algorithmic, optical and / or electronic correction to substantially compensate for this tangential velocity blur, thereby reducing this scanning aberration. For example, this compensation is achieved algorithmically by using an image processing algorithm that deconvolves differential velocity blur at various image locations corresponding to different radii at the rotating substrate to compensate for the differential velocity blur. In another example, this compensation is achieved by using an anamorphic magnification gradient. This may be useful for magnifying the substrate by different amounts in one axis at two or more substrate locations across the scan direction (anamorphic magnification). The anamorphic magnification gradient may change the imaging speed of two or more such locations to be substantially equal, thereby compensating for the tangential velocity difference of the two locations on the substrate. This compensation may be adjustable to account for different velocity gradients across the field of view at different radii of the substrate surface. In some cases, the imaging field of view may be segmented into two or more regions, each of which may be electronically controlled to scan at different speeds. These speeds may be adjusted to match the average projected object speed within each region. These regions may be optically defined using one or more beam splitters or one or more mirrors. These two or more regions may be directed to two or more detectors. These regions may be defined as segments of only one detector.

[0194] The systems, devices and methods described herein may have specific biological applications. In some examples, the fluid barrier system may be used in nucleic acid sequencing applications. The sample environment may be provided inside a chamber having a substrate that includes an array. A plurality of nucleic acid molecules may be immobilized at individually addressable locations in the array. A solution of labeled nucleotides may be dispensed onto the substrate under conditions sufficient to allow at least a subset of the labeled nucleotides to be incorporated into at least a subset of the plurality of nucleic acid molecules, if appropriate (e.g., if the labeled nucleotides are complementary to vacant positions in the nucleic acid molecules), and unincorporated nucleotides may be washed away with a wash solution. The sample environment, including temperature, pressure and / or humidity, may be maintained consistent with the particular sample (e.g., nucleic acid molecules) used in the sample environment and / or the process (e.g., the incorporation reaction) to be performed. Thereafter, while implementing the fluid barrier and thereby maintaining the sample environment conditions, a detector configured as described elsewhere herein, projecting through the plate into the sample environment, may detect one or more detectable signals from the incorporated labeled nucleotides from the individually addressable locations in the array during the relative motion between the detector and the substrate. For example, the substrate may be moved relative to the detector, such as to allow the detector to detect all individually addressable locations in the substrate (or a desired sub-area of ​​the substrate). In some cases, the substrate may undergo repeated cycles of rotational motion followed by linear motion, such that after each rotational motion the detector can scan an annular ring, and after each linear motion the detector is positioned to scan another annular ring at a different radius from the center of the substrate. In the alternative, or in addition, the substrate may undergo only rotational motion. In the alternative, or in addition, the substrate may undergo only linear motion.

[0195] A fluid barrier maintained during detection may provide a barrier between the controlled sample environment and the external environment and allow low or no friction relative motion between the detector and the sample while maintaining the controlled sample environment. Advantageously, such a barrier may allow continuous scanning in a 100% or substantially 100% relative humidity environment. The barrier may prevent humidity from escaping the sample environment, which may condense if leaked and affect (e.g., cause corrosion, contamination, etc.) sensitive instruments, such as optical instruments. Additionally, the barrier may prevent contaminants from the external environment from entering the sample environment, which may affect fluidics and / or detection (e.g., imaging).

[0196] As will be understood, the systems, devices and methods described herein may also have non-biological applications, such as, for example, for analyzing non-biological samples.

[0197] Computer Systems The present disclosure provides a computer control system programmed to carry out the methods of the present disclosure. Figure 7 shows a computer system 701 programmed or otherwise configured to process and / or detect samples. The computer system 701 can coordinate various aspects of the methods and systems of the present disclosure. The computer system may be configured to coordinate or communicate with any barrier system or components thereof and / or any processing system or components thereof described herein. For example, the computer system 701 may comprise or be a controller configured to communicate with the fluid flow units, actuators and / or detectors of the systems described herein.

[0198] The computer system 701 includes a central processing unit (CPU, also referred to herein as "processor" and "computer processor") 705, which can be a single-core processor or a multi-core processor, or multiple processors for parallel processing. The computer system 701 also includes memory or memory locations 710 (e.g., random access memory, read-only memory, flash memory), electronic storage 715 (e.g., hard disk), communication interface 720 (e.g., network adapter) for communicating with one or more other systems, and peripherals 725, such as cache, other memory, data storage, and / or electronic display adapters. The memory 710, storage 715, interface 720, and peripherals 725 are in communication with the CPU 705 via a communication bus (solid lines), such as a motherboard. The storage 715 can be a data storage device (or data repository) for storing data. The computer system 701 can be operatively coupled to a computer network ("network") 730 with the aid of the communication interface 720. The network 730 can be the Internet, an Internet and / or an extranet, or an intranet and / or an extranet in communication with the Internet. The network 730 is in some cases a telecommunications network and / or a data network. The network 730 can include one or more computer servers that can enable distributed computing, such as cloud computing. The network 730 can implement a peer-to-peer network, which in some cases can enable devices coupled to the computer system 701 to act as clients or servers, with the help of the computer system 701.

[0199] CPU 705 can execute a series of machine-readable instructions, which may be embodied in a program or software. The instructions may be stored in a memory location, such as memory 710. The instructions may be directed to CPU 705, which may then program or otherwise configure CPU 705 to perform the methods of the present disclosure. Examples of operations performed by CPU 705 may include fetch, decode, execute, and writeback.

[0200] The CPU 705 may be part of a circuit, such as an integrated circuit. One or more other components of the system 701 may be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).

[0201] The storage device 715 can store files such as drivers, libraries, and saved programs. The storage device 715 can store user data, such as user preferences and user programs. The computer system 701 can include one or more additional data storage devices in some cases that are external to the computer system 701, such as located on a remote server in communication with the computer system 701 via an intranet or the Internet.

[0202] Computer system 701 can communicate with one or more remote computer systems via network 730. For example, computer system 701 can communicate with a user's remote computer system. Examples of remote computer systems include a personal computer (e.g., a portable PC), a slate or tablet PC (e.g., Apple® iPad, Samsung® Galaxy Tab), a phone, a smartphone (e.g., Apple® iPhone, Android-enabled device, Blackberry®), or a personal digital assistant. A user can access computer system 701 via network 730.

[0203] Methods as described herein may be implemented as machine (e.g., computer processor) executable code stored in an electronic storage location of the computer system 701, such as in memory 710 or electronic storage 715. The machine executable or machine readable code may be provided in the form of software. During use, the code may be executed by the processor 705. In some cases, the code may be retrieved from storage 715 and stored in memory 710 for easy access by the processor 705. In some situations, electronic storage 715 may not be included and machine executable instructions are stored in memory 710.

[0204] The code can be precompiled and configured for use with a machine having a processor adapted to execute the code, or can be compiled during run-time. The code can be provided in a programming language that can be selected to allow the code to be executed in a precompiled or compiled manner.

[0205] Various aspects of the systems and methods provided herein, such as the computer system 701, can be embodied in a program. Various aspects of the technology may be considered to be "products" or "articles of manufacture" that are typically in the form of machine (or processor) executable code and / or associated data conveyed on or embodied in a machine-readable medium of a given type. The machine executable code can be stored in electronic storage, such as memory (e.g., read-only memory, random access memory, flash memory) or hard disk. "Storage" type media can include any or all of the tangible memory of a computer, processor or the like, or their associated modules, such as various semiconductor memories, tape drives, disk drives, and the like, which may provide non-transitory storage at any time for software programming. All or a portion of the software may be communicated from time to time via the Internet or various other telecommunications networks. Such communication may, for example, allow software to be loaded from one computer or processor to another, for example, from a management server or host computer to a computer platform of an application server. Thus, other types of media that may carry software elements include light waves, radio waves, and electromagnetic waves, such as those used between physical interfaces between local devices, over wired and optical land-line networks, and by various air links. Physical elements that transmit such waves, such as wired or wireless links, optical links, or the like, may also be considered to be media that carry software. As used herein, except when limited to non-transitory tangible "storage" media, terms such as computer or machine "readable media" refer to any media that participates in providing instructions to a processor for execution.

[0206] Thus, machine-readable media, such as computer executable code, may take many forms, including but not limited to tangible storage media, carrier wave media, or physical transmission media. Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or similar, such as the storage devices shown in the figures that may be used to implement, for example, databases, etc. Volatile storage media include dynamic memory, such as the main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that make up a bus in a computer system. Carrier wave transmission media may take the form of electric or electromagnetic signals, or sound or light waves, such as those generated during radio frequency (RF) and infrared (IR) data communications. Thus, common forms of computer readable media include, for example, a floppy disk, a flexible disk, a hard disk, a magnetic tape, any other magnetic medium, a CD-ROM, a DVD or a DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium having a pattern of holes, RAM, ROM, PROM and EPROM, FLASH-EPROM, any other memory chip or memory cartridge, a carrier wave carrying data or instructions, a cable or link carrying such a carrier wave, or any other medium from which a computer may read programming code and / or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0207] The computer system 701 can, for example, include or be in communication with an electronic display 735 with a user interface (UI) 740 for providing detection results to a user. The UI may also provide a console for configuring the fluid barrier system of the present disclosure and / or its components (e.g., pressure altering device, environmental unit, detector, immersion housing, detector movement, plate movement, vessel movement, substrate movement, sample processing, etc.). Examples of UIs include, but are not limited to, graphical user interfaces (GUIs) and web-based user interfaces.

[0208] The methods and systems of the present disclosure may be implemented as one or more algorithms. The algorithms may be implemented as software when executed by the central processing unit 705.

[0209] While various preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. The present invention is not intended to be limited by the specific examples provided within the scope of this specification. Although the present invention has been described with reference to the foregoing specification, the description and illustration of the embodiments herein are not meant to be construed in a limiting sense. Numerous changes, modifications and substitutions will now occur to those skilled in the art without departing from the present invention. Furthermore, it should be understood that all aspects of the present invention are not limited to the specific depictions, configurations or relative proportions shown herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the present invention described herein may be used in carrying out the present invention. Thus, the present invention is also intended to encompass any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the present invention, and that methods and structures within the scope of these claims and their equivalents are covered by such claims.

[0210] [Example] [Example 1] Imaging for sequencing of nucleic acid molecules FIG. 8 shows an example of an image generated by imaging a substrate on which biological analytes are immobilized in the sample environment of the barrier system of the present disclosure. A substrate 810 including a substantially planar array has immobilized thereon a plurality of biological analytes, such as a plurality of nucleic acid molecules, at a plurality of individually addressable locations 820. The individually addressable locations may be arranged randomly or in an ordered pattern. The biological analytes may be bound to beads, in which case the beads are immobilized in the array. A single bead may contain multiple analytes. The beads may be associated with the individually addressable locations. A plurality of fluorescent probes (e.g., a plurality of fluorescently labeled A, T, C, or G-containing nucleotides or analogs thereof) are dispensed onto the substrate 810 with the aid of one or more operational units (e.g., reagent dispensers) in a chemical processing operational station. In some embodiments, the substrate is configured to rotate about a given axis. The substrate 810 is then subjected to conditions sufficient to allow a reaction between at least one of the multiple probes and the biological analyte to occur, linking the at least one probe to the biological analyte. Unlinked probes are washed away with the aid of one or more operational units. At the detection operational station, while the fluid barrier is maintained, the binding of the at least one probe to the biological analyte is detected using photometry, which includes imaging at least a portion of the substrate 810 (e.g., via scanning or fixed field imaging) and measuring a signal at each individually addressable location 820. Nucleic acid molecules that include nucleotides that are complementary to the fluorescent probes have fluorescence at the individually addressable locations 820. These operations may then be repeated at each operational station, and signals from the images are matched with signals from previous images of the same substrate to obtain a signal trajectory in time for each biological analyte at each individually addressable location 820.The sequences of the multiple fluorescent probes are known for each iteration of the run, resulting in a known sequence for the analyte at each of the individually addressable locations 820.

[0211] [Example 2] Signal Processing FIG. 9 shows signal data processed by imaging a substrate on which biological analytes are immobilized in the sample environment of the barrier system of the present disclosure. A substrate containing a substantially planar array has nucleic acid molecules from E. coli immobilized thereon. Sequencing by synthesis was performed using flow-type chemistry using the processing system described herein. Imaging was performed while the fluid barrier of the barrier system was maintained as described elsewhere herein. Panel (A) shows the signal distribution for a set of several hundred colonies, each one being a replica of only one synthetic mono-template. The x-axis shows the length of sequencing after each cycle (e.g., each chemical flow run). In panel (B), the same data is processed by a parametric model, in which the causes of different template counts (amplitudes) and background levels are accounted for for each colony. The signals are deconvoluted by a model of lead and lag phasing, which also accounts for the causes of the overall signal loss per cycle. In the example shown here, the nominal phasing was 0.54% delay, 0.41% advance, and 0.45% signal loss. Residual systematic variation may be due to signal variation due to sequence context and can be further corrected using other algorithms (not shown).

Claims

1. 1. A method for processing a biological analyte, comprising: (a) providing a barrier between a first region and a second region, wherein the first region comprises a substrate adjacent which the biological analyte is immobilized, wherein the barrier maintains the first region at a first atmosphere that is different from a second atmosphere of the second region, wherein the barrier comprises a chamber, a plate, and a physical gap between the chamber and the plate, and wherein a portion of the barrier comprises fluid in bulk movement in the physical gap; and (b) detecting one or more signals from the biological analyte or changes therein using a detector contained at least in part in the first region while (i) the detector is undergoing motion relative to the substrate and there is no direct mechanical contact between the substrate and the detector, and (ii) the first region is maintained at the first atmosphere that is different from the second atmosphere of the second region. The method includes:

2. The method of claim 1 , wherein the portion of the barrier comprises a partial vacuum.

3. The method of claim 1 , wherein the portion of the barrier contains fluid from the first region, the second region, or both.

4. The method of claim 1 , wherein the first atmosphere is maintained at a first humidity that is different from a second humidity of the second atmosphere.

5. The method of claim 4 , wherein the first atmosphere has a relative humidity of greater than 90%.

6. The method of claim 1 , wherein the detector is an optical detector and the one or more signals are one or more optical signals or optical signal changes.

7. The method of claim 1 , wherein there is no direct mechanical contact between the plate and the chamber, and the plate is moveable relative to the chamber.

8. The method of claim 7 , wherein the detector is fixed relative to the plate and the substrate is translationally fixed relative to the chamber.

9. 1. A system for processing an analyte, comprising: A first region configured to contain (i) a substrate having the analyte immobilized therein and (ii) at least a portion of a detector; and a barrier disposed between the first region and the second region; the barrier comprises a chamber, a plate, and a physical gap between the chamber and the plate; a portion of the barrier includes fluid in bulk motion in the physical gap; the barrier is configured to maintain the first region at a first atmosphere different from a second atmosphere of the second region while the detector and the substrate undergo relative motion with respect to one another to detect one or more signals or changes therein from the analyte; system.

10. The system of claim 9 , wherein the portion of the barrier is configured to be under a vacuum.

11. The system of claim 9 , wherein the portion of the barrier is configured to contain fluid from the first region, the second region, or both the first region and the second region.

12. The system of claim 9 , wherein a portion of the barrier is configured to contain air.

13. 10. The system of claim 9, wherein the barrier is configured to maintain the first region at a first humidity or a first humidity range, the first humidity or first humidity range being different from a second humidity or second humidity range of the second region.

14. The system of claim 13 , wherein the first atmosphere has a relative humidity of greater than 90%.

15. 10. The system of claim 9, wherein the barrier is configured to maintain the first region at a first temperature or first temperature range, the first temperature or first temperature range being different from a second temperature or second temperature range of the second region.

16. 10. The system of claim 9, wherein the first region includes a first portion and a second portion, and the barrier is configured to maintain the first portion at a first local atmosphere and to maintain the second portion at a second local atmosphere different from the first local atmosphere.

17. 17. The system of claim 16, wherein the barrier is configured to maintain the first localized atmosphere at a first localized temperature or first localized temperature range that is different from a second localized temperature or second localized temperature range of the second localized atmosphere.

Images