Immersed microscope arrangement for sequencing systems with fast scanning

Abstract

An apparatus includes an imaging assembly and an actuation assembly. The imaging assembly includes an objective lens and an immersion fluid assembly. A first port of the immersion fluid assembly introduces an immersion fluid below a bottom surface of the objective lens. A second port of the immersion fluid assembly removes immersion fluid from below the bottom surface of the objective lens. The sidewalls of the immersion fluid assembly define an immersion fluid retention region below the objective lens. The immersion fluid retention region has a first horizontal dimension and a second horizontal dimension orthogonal to the first horizontal dimension. The first horizontal dimension is greater than the second horizontal dimension. The actuating assembly drives relative movement between the flow cell and the imaging assembly along the horizontal path in a direction parallel to the first horizontal dimension.

Description

Cross Reference to Related Applications

[0001] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 562,343, filed March 7, 2024, the contents of which are incorporated herein by reference in their entirety. BACKGROUND

[0002] Aspects of the present disclosure generally relate to devices, systems, and methods for providing biological or chemical analysis. Various protocols in biological or chemical research involve conducting a large number of controlled reactions on a local support surface or within a predefined reaction chamber. A specified reaction can then be observed or detected, and subsequent analysis can help identify or reveal properties of chemicals involved in the reaction. For example, in some multiplex assays, an unknown analyte with an identifiable marker (e.g., a fluorescent marker) can be exposed to thousands of known probes under controlled conditions. Each known probe can be placed into a corresponding well of a flow cell channel. Observing any chemical reactions that occur between the known probes and the unknown analyte within the well can help identify or reveal properties of the analyte. Other examples of such protocols include known DNA sequencing processes, such as sequencing by synthesis (SBS) or cyclic array sequencing.

[0003] While a variety of devices, systems, and methods have been made and used to provide biological or chemical analysis, it is believed that no one prior to the inventors has made or used the apparatus and techniques described herein. BRIEF DESCRIPTION OF DRAWINGS

[0004] Figure 1 A schematic diagram depicting an example of a system that can be used to provide biological or chemical analysis.

[0005] Figure 2 A schematic diagram depicting an example of a set of components of a fluidic path in a system that can work in concert to provide Figure 1 biological or chemical analysis.

[0006] Figure 3 A schematic diagram depicting another example of a system that can be used to provide biological or chemical analysis.

[0007] Figure 4 A cross-sectional view depicting an example of a flow cell that can be used in a system that can work in concert to provide Figure 1 biological or chemical analysis.

[0008] Figure 5 A cross-sectional view depicting another example of a flow cell that can be used in a system that can work in concert to provide Figure 1 biological or chemical analysis.

[0009] Figure 6 A top plan view of a flow cell that can work in concert to provide Figure 5 biological or chemical analysis, with an upper wafer omitted to reveal a lower wafer.

[0010] Figure 7 A schematic diagram depicting another example of a system that can be used to provide biological or chemical analysis.

[0011] Figure 8 A schematic diagram depicting an example of an imaging component that can be integrated into a system of Figure 7 .

[0012] Figure 9 A schematic diagram depicting another example of an imaging component that can be integrated into a system of Figure 7 .

[0013] Figure 10 A schematic diagram depicting another example of an imaging component that can be integrated into a system of Figure 7 .

[0014] Figure 11 A schematic diagram depicting another example of an imaging component that can be integrated into a system of Figure 7 .

[0015] Figure 12 A schematic diagram depicting an imaging component of Figure 11 , wherein a fluid line is coupled with a submersion fluid manifold.

[0016] Figure 13 A schematic diagram depicting an imaging component of Figure 11 , wherein a pressurized air assembly is coupled with an air curtain manifold and further coupled with an air blade manifold.

[0017] Figure 14 A perspective view of an imaging component of Figure 11 .

[0018] Figure 15 A side elevational view of an imaging component of Figure 11 .

[0019] Figure 16 A cross-sectional view of an imaging component of Figure 14 , taken along line 16-16 of Figure 11 .

[0020] Figure 17 Another side elevational view of an imaging component of Figure 11 .

[0021] Figure 18 A cross-sectional view of an imaging component of Figure 15 , taken along line 18-18 of Figure 11 .

[0022] Figure 19 A bottom plan view of an imaging component of Figure 11 .

[0023] Figure 20 depicts an exploded perspective view of an imaging component of Figure 11 .

[0024] Figure 21 depicts a cross-sectional side view of an objective lens assembly of an imaging component of Figure 11 .

[0025] Figure 22 is a bottom plan view of an objective lens assembly of Figure 22 .

[0026] Figure 23 depicts a cross-sectional side view of an example of an alternative objective lens element that can be incorporated into the objective lens assembly of Figure 22 .

[0027] Figure 24 depicts a perspective view of an air curtain manifold of an imaging component of Figure 11 .

[0028] Figure 25 depicts another perspective view of the air curtain manifold of Figure 24 .

[0029] Figure 26 depicts a cross-sectional view of the air curtain manifold of Figure 24 taken along line 26-26 of Figure 24 .

[0030] Figure 27 depicts a cross-sectional view of the air curtain manifold of Figure 26 taken along line 27-27 of Figure 24 .

[0031] Figure 28 depicts a magnified cross-sectional view of a portion of the air curtain manifold of Figure 24 .

[0032] Figure 29 depicts a perspective view of an immersion fluid manifold of an imaging component of Figure 11 .

[0033] Figure 30 depicts another perspective view of the immersion fluid manifold of Figure 29 .

[0034] Figure 31 depicts a bottom plan view of the immersion fluid manifold of Figure 29 .

[0035] Figure 32 depicts a cross-sectional view of the immersion fluid manifold of Figure 31 taken along line 32-32 of Figure 29 .

[0036] Figure 33 depicts a perspective view of an air blade manifold of an imaging component of Figure 11 .

[0037] Figure 34 depicts another perspective view of the air blade manifold of Figure 33 .

[0038] Figure 35 depicts a cross-sectional view of the air blade manifold of Figure 33 , taken along line 35-35 of Figure 33 .

[0039] Figure 36A depicts a schematic view of a flow cell with an immersion fluid footprint at a first location along the flow cell.

[0040] Figure 36B depicts a schematic view of the flow cell of Figure 36B , with the immersion fluid footprint at a second location along the flow cell.

[0041] Figure 37 depicts a flowchart illustrating an example of a set of steps that can be performed using the imaging component of Figure 11 .

[0042] Figure 38A depicts a cross-sectional side view of the imaging component of Figure 11 during an example of a first operational phase.

[0043] Figure 38B depicts a cross-sectional side view of the imaging component of Figure 11 during an example of a second operational phase.

[0044] Figure 38C depicts a cross-sectional side view of the imaging component of Figure 11 during an example of a third operational phase.

[0045] Figure 38D depicts a cross-sectional side view of the imaging component of Figure 11 during an example of a fourth operational phase.

[0046] Figure 38E depicts a cross-sectional side view of the imaging component of Figure 11 during an example of a fifth operational phase.

[0047] Figure 38F depicts a cross-sectional side view of the imaging component of Figure 11 during an example of a sixth operational phase.

[0048] Figure 38Gdepicted during an example of the seventh operational phase Figure 11 a cross-sectional side view of the imaging component. DETAILED DESCRIPTION

[0049] The following detailed description of certain examples will better be understood when read in connection with the following drawings. With respect to the figures illustrating functional blocks of various examples, the functional blocks do not necessarily indicate divisions between hardware components. Thus, for example, one or more of the functional blocks (e.g., a processor or memory) can be implemented in a single piece of hardware (e.g., a general purpose signal processor or random access memory, hard disk, etc.). Similarly, programs can be independent programs, can be incorporated as subroutines into an operating system, and can be functions in installed software packages, etc. It should be understood that the various examples are not limited to the arrangements and tools shown in the drawings. It should be understood that all combinations of the foregoing aspects and additional concepts discussed in greater detail below (provided such concepts are not mutually inconsistent) are contemplated as being part of the inventive subject matter and for realizing the benefits and advantages of the presently disclosed examples.

[0050] I. Overview of Systems for Biological or Chemical Analysis Examples described herein can be used in various biological or chemical processes and systems for academic, business, or other analysis. More specifically, examples described herein can be used in various processes and systems where detection of events, attributes, qualities, or characteristics indicative of a specified reaction is desired. Biological assay systems such as those described herein can be configured to perform multiple specified reactions that can be detected individually or collectively. For example, a biological assay system can be used to sequence a dense array of nucleic acid features through iterative cycles of enzyme manipulation and image capture. In some examples, the nucleic acids can be attached to a surface and amplified. Examples of such amplification are described in U.S. Patent No. 7,741,463, entitled "Method of Preparing Libraries of Template Polynucleotides," published June 22, 2010, the disclosure of which is incorporated by reference herein in its entirety; and / or U.S. Patent No. 7,270,981, entitled "Recombinase Polymerase Amplification," published September 18, 2007, the disclosure of which is incorporated by reference herein in its entirety.

[0051] Components used in a biological assay system can include one or more microfluidic channels that deliver reagents or other reaction components to reaction sites. Reaction sites can be randomly distributed across a substantially planar surface; or can be patterned across a substantially planar surface. Each of the reaction sites can be imaged to detect light from the reaction site. Signals indicative of photons emitted from a reaction site and detected by an image sensor can provide illumination values. These illumination values can be combined into images indicative of photons detected from reaction sites. These images can be further analyzed to identify compositions, reactions, conditions, etc. at each reaction site.

[0052] II. Examples of Fluidic Devices and Fluid Flow Paths A. Examples of Systems with Higher Volume Throughput Figure 1 A schematic diagram illustrating an example of a system (100) that can be used to analyze one or more samples of interest is shown. In some implementations, the samples can include one or more clusters of nucleotides (e.g., DNA) that are linearized to form single-stranded DNA (sstDNA). In the illustrated implementation, the system (100) is configured to receive a flow cell cartridge assembly (102) that includes a flow cell assembly (103) and a sample cartridge (104). The system (100) includes a flow cell receptacle (122) that receives the flow cell cartridge assembly (102), a vacuum chuck (124) that supports the flow cell assembly (103), and a flow cell interface (126) for establishing fluidic coupling between the system (100) and the flow cell assembly (103). The flow cell interface (126) can include one or more manifolds. The system (100) also includes an aspirator manifold assembly (106), a sample loading manifold assembly (108), and a pump manifold assembly (110). The system (100) also includes a drive assembly (112), a controller (114), an imaging system (116), and a waste reservoir (118). The controller (114) is electrically and / or communicatively coupled to the drive assembly (112) and the imaging system (116); and is configured to cause the drive assembly (112) and / or the imaging system (116) to perform various functions as disclosed herein.

[0053] In the present example, the flow cell assembly (103) includes a flow cell (128) having a channel (130) and defining a plurality of first openings (132) fluidically coupled to the channel (130) and arranged on a first side (134) of the channel (130). The flow cell (128) also includes a plurality of second openings (136) fluidically coupled to the channel (130) and arranged on a second side (138) of the channel (130). Thus, fluid can flow through the flow cell (128) via the channel. While the flow cell (128) is shown as including one channel (130), the flow cell (128) can include two or more channels (130). The flow cell assembly (103) also includes a flow cell manifold assembly (140) coupled to the flow cell (128) and having a first manifold fluid line (142) and a second manifold fluid line (144). The flow cell manifold assembly (140) can be in the form of a laminate including a plurality of layers, as discussed in greater detail below.

[0054] In the illustrated implementation, the first manifold fluid line (142) has first fluid line openings (146) and is fluidically coupled to each of the first openings (132) of the flow cell (128); and the second manifold fluid line (144) has second fluid line openings (148) and is fluidically coupled to each of the second openings (136). As shown, the flow cell assembly (103) includes a gasket (150) coupled to the flow cell manifold assembly (140) and fluidically coupled to the fluid line openings (146, 148). In some implementations in which the flow cell (128) includes a plurality of channels (130), the flow cell manifold assembly (140) can include an additional fluid line (152) coupling the first fluid line openings (146) to a single manifold port (154). In such implementations, a single gasket (150) can be coupled to the flow cell manifold assembly (140) that surrounds the manifold port (154) and is in fluid communication with the plurality of channels (130). In operation, the flow cell interface (126) engages with the corresponding gasket (150) to establish fluidic coupling between the system (100) and the flow cell (128). Engagement between the flow cell interface (126) and the gasket (150) reduces or eliminates fluid leakage between the flow cell interface (126) and the flow cell (128).

[0055] In the particular implementation shown, the first manifold fluid line (142) has a portion (156) that is substantially parallel to the longitudinal axis (158) of the channel (130); and the second manifold fluid line (144) has a portion (160) that is substantially parallel to the longitudinal axis (158) of the channel (130). Additionally, the first manifold fluid line (142) is shown as being at least partially adjacent to a first end (162) of the flow cell (128) and spaced apart from a second end (164) of the flow cell (128); and the second manifold fluid line (144) is shown as being at least partially adjacent to the second end (164) of the flow cell (128) and spaced apart from the first end (162). However, other arrangements of the manifold fluid lines (142, 144) can prove suitable.

[0056] In the particular implementation shown, the system (100) includes a sample cartridge receptacle (166) that receives a sample cartridge 104 that carries one or more samples of interest (e.g., analytes). The system (100) also includes a sample cartridge interface (168) that establishes a fluid connection with the sample cartridge (104). The sample loading manifold assembly (108) includes one or more sample valves (170). The pump manifold assembly (110) includes one or more pumps (172), one or more pump valves (174), and a cache (176). The valves (170, 174) and pumps (172) can take any suitable form. The cache (176) can include a serpentine cache, and can temporarily store one or more reaction components during bypass maneuvering of the system (100). While the cache (176) is shown as being included in the pump manifold assembly (110), the cache (176) can alternatively be located elsewhere (e.g., in the aspirator manifold assembly (106) or in another manifold downstream of the bypass fluid line (178), etc.).

[0057] The sample loading manifold assembly (108) and the pump manifold assembly (110) cause the one or more samples of interest to flow from the sample cartridge (104) to the flow cell cartridge assembly (102) through a fluid line (180). In some implementations, the sample loading manifold assembly (108) can individually load / address each channel (130) of the flow cell (128) with a respective sample of interest. The process of loading the channels 130 with samples of interest can occur automatically using the system (100). As Figure 1As shown, a sample cartridge (104) and a sample loading manifold assembly (108) are positioned downstream of the flow cell cartridge assembly (102). In the particular implementation shown, the sample loading manifold assembly (108) is coupled between the flow cell cartridge assembly (102) and a pump manifold assembly (110). To draw a sample of interest from the sample cartridge (104) and toward the pump manifold assembly (110), the sample valve (170), the pump valve (174), and / or the pump (172) can be selectively actuated to push the sample of interest toward the pump manifold assembly (110). The sample cartridge (104) can include a plurality of sample reservoirs that are fluidly accessible via corresponding sample valves (170). To individually flow the sample of interest toward the channels (130) of the flow cell (128) and away from the pump manifold assembly (110), the sample valve (170), the pump valve (174), and / or the pump (172) can be selectively actuated to push the sample of interest toward the flow cell cartridge assembly (102) and into the respective channels (130) of the flow cell (128).

[0058] The drive assembly (112) interfaces with the aspirator manifold assembly (106) and the pump manifold assembly (110) to flow one or more reagents that interact with a sample within the flow cell (128). In some cases, a reversible terminator is attached to a reagent to allow a single nucleotide to bind to a growing DNA strand. In some such implementations, one or more nucleotides have a unique fluorescent label that emits a color when excited. The color (or absence of color) is used to detect the corresponding nucleotide. In the implementation shown, the imaging system (116) excites one or more of the identifiable labels (e.g., fluorescent labels) and then obtains image data of the identifiable labels. The labels can be excited by incident light and / or laser light, and the image data can include one or more colors emitted by the respective labels in response to the excitation. The image data (e.g., detection data) can be analyzed by the system (100). Examples of features and functionality that can be incorporated into the imaging system (116) are described in more detail below.

[0059] After the image data is obtained, the drive assembly (112) interfaces with the aspirator manifold assembly (106) and the pump manifold assembly (110) to flow another reaction component (e.g., a reagent) through the flow cell (128), which is then received by the waste reservoir (118) via the main waste fluid line (182) and / or otherwise depleted by the system (100). Some reaction components can perform a wash operation that cleaves fluorescent labels and reversible terminators from the sstDNA. Subsequently, the sstDNA can be prepared for another cycle.

[0060] A main waste fluid line (182) is coupled between the pump manifold assembly (110) and the waste reservoir (118). In some implementations, the pump (172) and / or the pump valve (174) of the pump manifold assembly (110) selectively flow reaction components from the flow cell cartridge assembly (102) through the fluid line (180) and the sample loading manifold assembly (108) to the main waste fluid line (182). The flow cell cartridge assembly (102) is coupled to the central valve (184) via the flow cell interface (126). The central valve (184) is coupled to the flow cell interface (126) via the fluid line (185). An auxiliary waste fluid line (186) is coupled to the central valve (184) and the waste reservoir (118). In some implementations, the auxiliary waste fluid line (186) receives excess fluid from a sample of interest of the flow cell cartridge assembly (102) via the central valve (184) and flows the excess fluid of the sample of interest to the waste reservoir (118) when the sample of interest is being reverse loaded into the flow cell (128), as described herein.

[0061] The aspirator manifold assembly (106) includes a shared line valve (188) and a bypass valve (190). The shared line valve (188) can be referred to as a reagent selection valve. The central valve (184) and the valves (188, 190) of the aspirator manifold assembly (106) can be selectively actuated to control fluid flow through the fluid lines (192, 194, 196). The aspirator manifold assembly (106) can be coupled to a corresponding number of reagent reservoirs (198) via reagent aspirators (200). The reagent reservoirs (198) can hold fluid (e.g., reagents and / or another reaction component). In some implementations, the aspirator manifold assembly (106) includes a plurality of ports. Each port of the aspirator manifold assembly (106) can receive one of the reagent aspirators (200). The reagent aspirators (200) can be referred to as fluid lines. Some forms of the reagent aspirators (200) can include an array of aspirator tubes that extend downward along a z-dimension from ports in a main body of the aspirator manifold assembly (106). The reagent reservoirs (198) can be disposed in a cartridge, and the tubes of the reagent aspirators (200) can be configured to be inserted into corresponding reagent reservoirs (198) in the reagent cartridge such that liquid reagents can be aspirated from each reagent reservoir (198) into the aspirator manifold assembly (106).

[0062] The shared line valve (188) of the aspirator manifold assembly (106) is coupled to the central valve (184) via a shared reagent fluid line (196). Different reagents can flow through the shared reagent fluid line (196) at different times. In some versions, the pump manifold assembly (110) can draw a wash buffer through the shared reagent fluid line (196), the central valve (184), and the flow cell cartridge assembly (102) when a flush operation is performed prior to changing between one reagent and another reagent.

[0063] The bypass valve (190) of the aspirator manifold assembly (106) is coupled to the central valve (184) via a dedicated reagent fluid line (194, 196). Each of the dedicated reagent fluid lines (194, 196) can be associated with a single reagent. Fluids that can flow through the dedicated reagent fluid lines (194, 196) can be used during sequencing operations and can include cleavage reagents, integration reagents, scanning reagents, cleavage washes, and / or wash buffers.

[0064] The bypass valve (190) is also coupled to the cache (176) of the pump manifold assembly (110) via a bypass fluid line (178). One or more reagent priming operations, hydration operations, mixing operations, and / or transfer operations can be performed using the bypass fluid line (178). The priming operations, hydration operations, mixing operations, and / or transfer operations can be performed independent of the flow cell cartridge assembly (102). Thus, operations using the bypass fluid line (178) can occur during, for example, incubation of one or more samples of interest within the flow cell cartridge assembly (102). That is, the shared line valve (188) can be utilized independent of the bypass valve (190) such that the bypass valve (190) can utilize the bypass fluid line (178) and / or the cache (176) to perform one or more operations while the shared line valve (188) and / or the central valve (184) perform other operations simultaneously, substantially simultaneously, or offset synchronously.

[0065] The drive assembly (112) includes a pump drive assembly (202) and a valve drive assembly (204). The pump drive assembly (202) can be adapted to interface with one or more pumps (172) to pump fluid through a flow cell (128) and / or to load one or more samples of interest into the flow cell (128). The valve drive assembly (204) can be adapted to interface with one or more of the valves (170, 174, 184, 188, 190) to control positioning of the corresponding valve (170, 174, 184, 188, 190).

[0066] Figure 2An example of a fluidic arrangement (220) that can be incorporated into a variation of the system (100) is shown. The fluidic arrangement (220) of this example includes a pump manifold assembly (222) that can operate similarly to the pump manifold assembly (110) described above; a sample loading manifold assembly (228) that can operate similarly to the sample loading manifold assembly (108) described above; a flow cell interface (240) that can operate similarly to the flow cell interface (126) described above; an aspirator manifold assembly (250) that can operate similarly to the aspirator manifold assembly (106) described above; and a waste reservoir (270) that can operate similarly to the waste reservoir (118) described above. The pump manifold assembly (222) is coupled with a port assembly (258) of the aspirator manifold assembly (250) via a fluid line (224) that can be similar to the fluid line (178); and with the sample loading manifold assembly (228) via a fluid line (226). The sample loading manifold assembly (228) is coupled with the flow cell interface (240) via a fluid line (230) that can be similar to the fluid line (180); and with the port assembly (258) via fluid lines (232, 234). The flow cell interface (240) is coupled with the aspirator manifold assembly (250) via a fluid line (242) that can be similar to the fluid line (185). The aspirator manifold assembly (250) includes a manifold body (252) and a common output port (256) that provides fluid communication via a fluid line (185). A valve assembly (254) controls fluid flow through the common output port (256) and can operate similarly to the central valve (184). The port assembly (258) of the aspirator manifold assembly (250) is coupled with the waste reservoir (270) via a fluid line (272) that can be similar to the fluid line (186).

[0067] A plurality of reagent aspirators (260) extend from the manifold body (252) and are fluidically coupled with the valve assembly (254) via respective fluid channels (262) in the manifold body (252). The reagent aspirators (260) can operate similarly to the reagent aspirators (200). The valve assembly (254) is operable to selectively couple the fluid channels (262) with the flow cell interface (240) via the common output port (256) and the fluid line (230), thereby selectively providing various reagents to the flow cell interface (240). In other words, when each reagent aspirator (260) is disposed in a different respective reagent (e.g., in a respective reagent reservoir (198)), a flow cell (e.g., like the flow cell (128)) coupled with the flow cell interface (240) can selectively receive those different reagents based on control of the valve assembly (254).

[0068] The port assembly (258) can provide a fluidic interface between the pump manifold assembly (222) and the aspirator manifold assembly (250), thereby allowing the aspirator manifold assembly (250) to receive pressurized fluid from the pump manifold assembly (222). The port assembly (258) can also provide a fluidic interface between the sample loading manifold assembly (228) and the aspirator manifold assembly (250), thereby allowing the aspirator manifold assembly (250) to receive sample fluid from the sample loading manifold assembly (228). Further, the port assembly (258) can provide a fluidic interface between the waste reservoir (270) and the aspirator manifold assembly (250), thereby allowing the aspirator manifold assembly (250) to pass waste fluid to the waste reservoir (270). Fluid communication via the port assembly (258) can be at least partially regulated by the valve assembly (254).

[0069] Referring back to Figure 1 The controller (114) of the present example includes a user interface (206), a communication interface (208), one or more processors (210), and a memory (212) storing instructions executable by the one or more processors (210) to perform various functions including the disclosed implementations. The user interface (206), the communication interface (133), and the memory (212) are electrically and / or communicatively coupled to the one or more processors (210). The user interface (206) can be adapted to receive input from a user and to provide information associated with the operation of the system (100) and / or an ongoing analysis to the user. The user interface (206) can include a touchscreen, a display, a keyboard, a speaker, a mouse, a trackball, and / or a voice recognition system.

[0070] The communication interface (208) is adapted to enable communication between the system (100) and a remote system (e.g., a computer) via a network (e.g., the Internet, an intranet, a local area network (LAN), a wide area network (WAN), a coaxial cable network, a wireless network, a wired network, a satellite network, a digital subscriber line (DSL) network, a cellular network, a Bluetooth connection, a near field communication (NFC) connection, etc.). Some of the communications provided to the remote system can be associated with analysis results, imaging data, etc. generated or otherwise obtained by the system (100). Some of the communications provided to the system (100) can be associated with fluid analysis operations, patient records, and / or protocols to be performed by the system (100).

[0071] The one or more processors (210) and / or the system (100) can include one or more of a processor-based system or a microprocessor-based system. In some implementations, the one or more processors (210) and / or the system (100) include one or more of a programmable processor, a programmable controller, a microprocessor, a microcontroller, a graphics processing unit (GPU), a digital signal processor (DSP), a reduced instruction set computer (RISC), an application-specific integrated circuit (ASIC), a field-programmable gate array (FPGA), a field-programmable logic device (FPLD), a logic circuit, and / or another logic-based device that executes various functions including the functions described herein.

[0072] The memory (212) can include one or more of a semiconductor memory, a magnetically readable memory, an optically readable memory, a hard disk drive (HDD), an optical storage drive, a solid-state storage device, a solid state drive (SSD), a flash memory, a read-only memory (ROM), an erasable programmable read-only memory (EPROM), an electrically erasable programmable read-only memory (EEPROM), a random-access memory (RAM), a non-volatile RAM (NVRAM) memory, a compact disk (CD), a compact disk read-only memory (CD-ROM), a digital versatile disk (DVD), a Blu-ray disk, a redundant array of independent disks (RAID) system, a cache, and / or any other storage device or disk in which information is stored for any duration (e.g., for a permanent period, for a temporary period, for a long period, for a buffering, for a cache).

[0073] B. Examples of Systems with Lower Volume Throughput Figure 3 A schematic diagram illustrating another example of a system (300) that can be used to perform analysis on one or more samples of interest is shown. Unless otherwise described below, the system (300) of this example can be like the system (300) described above with reference to FIG. 2A. Figure 1The described system (100) is configured and operates as such. In some cases, the system (100) is used to provide a higher volume throughput; while the system (300) is used to provide a lower volume throughput. Alternatively, the systems (100, 300) can provide any other suitable amount or degree of throughput. The system (300) of the present example receives a cartridge (302), and partially comprises a gas source (304), a drive assembly (306), a controller (308), an imaging system (310), and a waste reservoir (312). The cartridge (302) can be referred to as a consumable, a reagent reservoir, or a reagent assembly. The controller (308) is electrically and / or communicatively coupled to the drive assembly (306) and the imaging system (310), and causes the drive assembly (306) and / or the imaging system (310) to perform various functions as disclosed herein.

[0074] The cartridge (302) in the illustrated implementation includes a well assembly (314) having a body (316). The body (316) has a first wall (318) defining a well (320) having a port (322). The first wall (318) has a distal end (324) defining an opening (326) having an opening perimeter (328). A second wall (330) surrounds the first wall (318) and has a distal end (332). The distal end (332) can be referred to as an edge or an outer edge. A cover (334) is coupled to the distal end (324) of the first wall (318) and covers the opening (326) along the opening perimeter (328) at a connected portion (336); and is separated from the distal end (324) of the first wall (318) at an unconnected portion (338). The connected portion (336) can be referred to as a connected segment or a connected segment, and the unconnected portion (338) can be referred to as an unconnected segment or an unconnected segment. The first wall (318) has a height, and the second wall (330) has a height that is greater than the height of the first wall (318). The first and second walls (318, 330) can alternatively have the same or similar heights. An impermeable barrier (340) is coupled to the distal end (332) of the second wall (330) and covers the well (320). The impermeable barrier (340) can be a metal foil, a plastic, or the like, and can prevent or inhibit moisture from permeating into the well (320) of the cartridge (302).

[0075] The unconnected portion (338) of the cover (334) forms a vent (342) that allows air to flow out of the well (320). A dry reagent (348) is contained within the well (320), and the vent (342) is sized to substantially retain the dry reagent (348) within the well (320). The body (316) can include a plurality of wells (320), while Figure 3A well (320) is shown. In practice, liquid (346) can flow into well (320) via port (322) to rehydrate dry reagent (348). Vent (342) can vent gas from well (320) as liquid (346) flows into well (320); and lid (334) prevents or inhibits reagent (348) and / or liquid (346) from escaping from well (320). In other words, vent (342) retains reagent (348) and / or liquid (346) within well (320); and prevents or inhibits reagent (348) and / or liquid (346) from migrating out of well (320). When cartridge (302) includes more than one well (320), vent (342) and lid (334) prevent or inhibit cross-contamination between reagents. Liquid (346) and dry reagent (348) can flow into and out of well (320) to mix liquid (346) and dry reagent (348) from liquid reservoir (362). In some implementations, system (300) and / or cartridge (302) can include a mixing chamber for mixing liquid (346) and dry reagent (348). Impermeable barrier (340) can be pierced prior to liquid (346) flowing into well (320).

[0076] Gas source (304) can be used to pressurize liquid reservoir (362) to cause liquid (346) to flow into well (320); and / or pump (350) can draw liquid (346) from liquid reservoir (362) and cause liquid (346) to flow into well (320) to rehydrate reagent (348). Gas source (304) can be provided by system (300) and / or can be carried by cartridge (302). Alternatively, gas source (304) can be omitted. Pump (350) can be implemented by a syringe pump, a peristaltic pump, a diaphragm pump, etc. While pump (350) can be positioned downstream of flow cell (368) as shown, pump (350) can be positioned upstream of flow cell (368) or omitted altogether.

[0077] Cartridge (302) and / or system (300) includes valve (352) that can be selectively actuatable to control fluid flow through fluid line (356). Such valve (352) can be implemented by a valve manifold, a rotary valve, a selector valve, a pinch valve, a flat valve, a solenoid valve, a check valve, a piezoelectric valve, etc. Regulator (354) can be positioned between gas source (304) and valve (352); and regulates the pressure of gas provided to valve (352). Regulator (354) can include a valve that controls the flow of gas from gas source (304).

[0078] The body (316) of the well assembly (314) has an edge (364); and the impermeable barrier (340) can be sealingly connected to the body (316) along the edge (364). The impermeable barrier (340) can comprise a metal foil, a plastic, and / or any other suitable material. The system (300) can pierce the impermeable barrier (340), the impermeable barrier (340) can be pierced by an individual prior to use, or the impermeable barrier (340) can be pierced by some other structure or method. In the particular implementation shown, the system (300) includes an actuator assembly (360) that interfaces with the impermeable barrier (340) to pierce the impermeable barrier (340). The system (300) can include a protrusion, such as a post having a blunt end or a sharp end, that can be moved by the actuator assembly (360) to pierce the impermeable barrier (340). The impermeable barrier (340) can alternatively be pierced by an operator prior to positioning the cartridge (302) in the system (300). The system (300) also includes a liquid reservoir (362) that contains a liquid (346). The liquid (346) can comprise a rehydration liquid, a wash buffer, and / or any other suitable type of liquid.

[0079] The system (300) also includes a flow cell receptacle (366) that receives a flow cell (368). The flow cell (368) can be configured and operated like the flow cell (128). In some variations, the flow cell (368) is carried by and / or integrated into the cartridge (302). The flow cell (368) can carry a sample of interest. The gas source (304) and / or the pump (350) can flow the liquid (346) to rehydrate the dry reagent (348) and to flow one or more liquid reagents that interact with the sample through the cartridge (302). The imaging system (310) can be configured and operated like the imaging system (116) such that the imaging system (310) can be used to obtain image data from the flow cell (368). After the image data is obtained, the drive assembly (306) can interface with the cartridge (302) to flow another reaction component (e.g., a reagent) through the flow cell (368) that is then received by the waste reservoir (312) and / or otherwise depleted by the cartridge (302). In the present example, the drive assembly (306) includes a pump drive assembly (370), a valve drive assembly (372), and the actuator assembly (360). The pump drive assembly (370) interfaces with the pump (350) to pump fluid through the cartridge (302) and / or the flow cell (368); and the valve drive assembly (372) interfaces with the valve (352) to control the positioning of the valve (352).

[0080] The controller (308) of this example includes a user interface (374), a communication interface (376), a processor (378), and a memory (380). The user interface (374) can be configured and operated like the user interface (206) of the system (100). The communication interface (376) can be configured and operated like the communication interface (208) of the system (100). The processor (378) can be configured and operated like the processor (210) of the system (100). The memory (380) can be configured and operated like the memory (212) of the system (100).

[0081] Further examples and details of how various features of each system (100, 300) can be configured and operated will be described below. By way of further example only, various features of the system (100, 300) can be configured and operated in accordance with at least some of the teachings of International Publication No. WO 2023 / 055873, entitled “Flow Cells and Related Flow Cell Manifold Assemblies and Methods,” published April 6, 2023, the disclosure of which is incorporated by reference herein in its entirety; U.S. Patent No. 9,958,465, entitled “Detection Apparatus having a Microfluorometer, a Fluidic System, and a Flow Cell Latch Clamp Module,” issued May 1, 2018, the disclosure of which is incorporated by reference herein in its entirety; and / or U.S. Patent Application No. 63 / 325,462, entitled “Well Assemblies and Related Systems and Methods,” filed March 30, 2022, the disclosure of which is incorporated by reference herein in its entirety.

[0082] III. Examples of Flow Cell Structures As noted above, the system (100, 300) can perform reactions in a flow cell (128, 368) and / or perform analysis on one or more samples of interest in the flow cell (128, 368). Examples of forms that such a flow cell (128, 368) can take are described below, with the understanding that the flow cell (128, 368) can take various other forms and have various other features in addition to or instead of the features described below.

[0083] A. Examples of Single Surface Patterned Flow Cells Figure 4 An example of a flow cell (400) is shown that includes a patterned substrate (402) that includes recesses (404) separated by interstitial regions (406) and surface chemistry (410, 412) positioned in the recesses (404). The recesses (404) can be in the form of microwells or nanowells. The recesses (404) can be configured to hold nucleic acid strands or other oligonucleotides and thereby provide reaction sites for SBS and / or other types of processes. In some versions, each recess (404) has a cylindrical configuration with a generally circular cross-sectional profile. In some other versions, each recess (404) has a polygonal (e.g., hexagonal, octagonal, square, rectangular, elliptical, etc.) cross-sectional profile. Alternatively, the recesses (404) can have any other suitable configuration. It should also be understood that the recesses (404) can be arranged in any suitable pattern, including but not limited to a grid pattern.

[0084] The surface chemistry (410, 412) of the present example includes a functionalized coating layer (410) and a primer (412). Although not shown, it should be understood that the recesses (404) can also have a surface preparation or treatment chemistry (e.g., silane or silane derivative) positioned between the substrate (402) and the functionalized coating layer (410). This same surface preparation or treatment chemistry can also be positioned on the interstitial regions (406). In the present example, a hydrogel (440) is applied prior to bonding the cover (420) to the substrate (402). The hydrogel (440) covers the surface chemistry (410, 412) in the recesses (404) and at least a portion of the patterned substrate (402) (e.g., those interstitial regions (406) that are not also bonding regions (422)). By way of example only, the hydrogel (440) can include PAZAM, cross-linked polyacrylamide, agarose gel, etc.

[0085] The flow cell (400) of this example also includes a cover (420) bonded to the bonding regions (422) of the patterned substrate (402). In the present example, the cover (420) is bonded to the bonding regions (422) of the patterned substrate (402) prior to the application of the hydrogel (440). In other versions, the cover (420) is bonded to the bonding regions (422) of the patterned substrate (402) after the application of the hydrogel (440). In still other versions, the cover (420) is bonded to the bonding regions (422) of the patterned substrate (402) at the same time as the hydrogel (440) is applied. Figure 4In the illustrated example, the cover (420) includes a top portion (424) connected to a number of sidewalls (426), and these components (424, 426) define a portion of each of six flow channels (430A, 430B, 430C, 430D, 430E, 430F). A respective sidewall (426) isolates one flow channel (430A, 430B, 430C, 430D, 430E, 430F) from each adjacent flow channel (430A, 430B, 430C, 430D, 430E, 430F). Each flow channel (430A, 430B, 430C, 430D, 430E, 430F) is in selective fluid communication with a respective set of recesses (404).

[0086] The cover (420) can be bonded to the bonding regions (422) of the substrate (402) using any suitable technique, such as laser bonding, diffusion bonding, anodic bonding, eutectic bonding, plasma-activated bonding, glass frit bonding, or other methods known in the art. In some versions, a spacer layer (428) can be used to bond the cover (420) to the bonding regions (422). The spacer layer (428) can include any material that seals together at least some of the interstitial regions (404) (e.g., the bonding regions (422)) of the substrate (402) and the cover (420). Although not illustrated, the cover (420) or the patterned substrate (402) can include inlet and outlet ports that fluidly engage other ports (not shown), such as ports of a sample cartridge interface (168), for directing fluid (e.g., from a reagent cartridge or other fluid storage system) into respective flow channels (430A, 430B, 430C, 430D, 430E, 430F) and out of the flow channels (e.g., to a waste reservoir (118) or another waste removal system). The flow channels (430A, 430B, 430C, 430D, 430E, 430F) can be used, for example, to selectively introduce reaction components or reactants into the hydrogel (440) and the underlying surface chemistry (410, 412) in order to initiate a specified reaction in / at the recesses (404).

[0087] While flow cell (400) includes a pattern of recesses (404) to provide an array of reaction sites, other variations can provide reaction sites on or at various other types of structural features, including but not limited to continuous flat surfaces and / or protruding surfaces, etc. By way of further example only, flow cell (400) can be constructed and operated in accordance with at least some of the teachings of U.S. Patent No. 10,919,033, issued February 16, 2021, entitled “Flow Cells with Hydrogel Coating,” the disclosure of which is incorporated by reference herein in its entirety.

[0088] B. Examples of Double Surface Patterned Flow Cells While Figure 4 While one example of a flow cell (400) having a single surface patterned with reaction sites (i.e., recesses (404) formed in substrate (402)) is shown, in some cases it can be desirable to provide a variation of flow cell (400) that provides two surfaces patterned with reaction sites. Figures 5-6 An example of a dual-surface patterned flow cell (450) is shown. In this example, flow cell (450) includes a pair of wafers (452, 454) that are bonded together with a spacer layer (456) interposed between wafers (452, 454). Each wafer (452, 454) is patterned to provide a respective plurality of recesses (462, 464) such that, when flow cell (450) is assembled, recesses (462) of wafer (452) are aligned with recesses (464) of wafer (454). Recesses (462) are separated from one another by gap regions (466); and recesses (464) are separated from one another by gap regions (468). In this example, spacer layer (456) does not contact gap regions (466, 468).

[0089] Recesses (462, 464) of flow cell (450) can be configured and operated like recesses (404) of flow cell (400) described above. Each recess (462, 464) of the present example includes a grafting coating (470), which can be similar to functionalized coating layer (410); and a primer (472), which can be similar to primer (412) described above. Each recess (462, 464) can also include a hydrogel (like hydrogel (440)) and / or any other suitable features. As Figures 5-6As shown, recesses (462, 464) are provided within a plurality of flow channels (480A, 480B, 480C, 480D). The flow channels (480A, 480B, 480C, 480D) are separated from one another by walls (458) and ends (459) formed by the spacer layer (456). In Figure 6 In the example shown, the flow cell (450) provides four flow channels (480A, 480B, 480C, 480D), with each flow channel (480A, 480B, 480C, 480D) containing a number of rows and a number of columns of recesses (462, 464). When the flow cell (450) is used in a system (100, 300), the flow channels (480A, 480B, 480C, 480D) can be used, for example, to selectively introduce reaction components or reactant surface chemistries (470, 472) so as to initiate a specified reaction in / at the recesses (462, 464). In some cases, because each wafer (452, 454) has its own set of recesses (462, 464) that provide corresponding reaction sites, the flow cell (450) can provide twice as many reactions as a similarly sized flow cell (400) during a given time period.

[0090] Figure 6 The dashed lines in FIG. 45 indicate how the flow cell (450) can be diced to effectively form smaller flow cells (450A, 450A), with each smaller flow cell (450A, 450A) having its own respective pair of flow channels (480A, 480B, 480C, 480D). However, in the present example, the single flow cell (450) has more than two flow channels (480A, 480B, 480C, 480D). While the flow cell (450) includes a pattern of recesses (462, 464) to provide an array of reaction sites, other variations can provide reaction sites on or at various other types of structural features, including but not limited to continuous flat surfaces and / or protruding surfaces, etc. By way of further example only, the flow cell (450) can be constructed and operated in accordance with at least some of the teachings of U.S. Patent No. 10,955,332, issued March 23, 2021, entitled “Flow Cell Package and Method for Making the Same,” the disclosure of which is incorporated by reference herein in its entirety.

[0091] IV. Examples of Imaging System Features As noted above, the system (100, 300) includes an imaging system (116, 310) that excites one or more identifiable markers (e.g., fluorescent markers) in a sample in a reaction site provided by a recess (404, 462, 464) of a flow cell (128, 368, 400, 450); image data of the identifiable markers is then obtained. The image data is used to identify nucleotides as part of a nucleic acid sequencing process. Alternatively, the image data can be used for various other purposes. The following description provides details on how some versions of the imaging system (116, 310) can be configured and operated.

[0092] Figure 7 A schematic diagram illustrating another example of a system (500) that can be used to perform analysis on one or more samples of interest is shown. Unless otherwise described below, the system (500) of this example can be configured and operated like the systems (100, 300) described above. The system (500) is configured to perform a large number of parallel reactions within a flow cell (510). The flow cell (510) can be configured and operated like the flow cells (400, 450) described above, or can have any other suitable configuration. Thus, the flow cell (510) can include one or more flow channels that receive a solution from the system (500) and direct the solution toward reaction sites of the flow cell (510).

[0093] The system (500) includes a system controller (520) that can communicate with various components, assemblies, and subsystems of the system (500). The controller (520) can be configured and operated like the controllers (114, 308) described above. The imaging assembly (522) of the system (500) includes a light emitting assembly (550) that emits light that reaches reaction sites on the flow cell (510). The light emitting assembly (550) can include a non-coherent light emitter (e.g., that emits a light beam output by one or more excitation diodes) or a coherent light emitter (such as an emitter of light output by one or more lasers or laser diodes). In some implementations, the light emitting assembly (550) can include a plurality of different light sources (not shown) that each emit light of a different wavelength range. Some versions of the light emitting assembly (550) can also include one or more collimating lenses (not shown), light structuring optical assemblies (not shown), projection lenses (not shown) that are operable to adjust the shape and path of a structured light beam, epi-fluorescence microscope components, and / or other components. Although the system (500) is illustrated as having a single light emitting assembly (550), multiple light emitting assemblies (550) can be included in some other implementations.

[0094] In this example, light from the light-emitting assembly (550) is directed by the dichroic mirror assembly (546) through the objective lens assembly (542) onto a sample on the flow cell (510) positioned on the motion stage (570). In the case of fluorescence microscopy of the sample, fluorescent elements associated with the sample of interest fluoresce in response to the excitation light, and the resulting light is collected by the objective lens assembly (542) and directed to the image sensor of the camera system (540) to detect the emitted fluorescence. In some implementations, a lens tube assembly can be positioned between the objective lens assembly (542) and the dichroic mirror assembly (546) or between the dichroic mirror (546) and the image sensor of the camera system (540). A movable lens element can be able to translate along a longitudinal axis of this lens tube assembly to address focusing on the upper or lower interior surface of the flow cell (510) and / or spherical aberrations introduced by movement of the objective lens assembly (542).

[0095] In this example, a filter switching assembly (544) is interposed between the dichroic mirror assembly (546) and the camera system (540). The filter switching assembly (544) includes one or more emission filters that can be used to pass a particular range of emission wavelengths and block (or reflect) other ranges of emission wavelengths. For example, the emission filters can be used to direct emission light of different wavelength ranges to different image sensors of the camera system (540) of the imaging assembly (522). For example, the emission filters can be implemented as dichroic mirrors that direct emission light of different wavelengths from the flow cell (510) to different image sensors of the camera system (540). In some variations, a projection lens is interposed between the filter switching assembly (544) and the camera system (540). In some versions, the filter switching assembly (544) can be omitted.

[0096] The system (500) can include a fluid delivery assembly (590) that can direct reagents (e.g., fluorescently labeled nucleotides, buffers, enzymes, lysis reagents, etc.) to flow to (and through) the flow cell (510) and a waste valve (580). The fluid delivery assembly (590) can be configured and operated like the various fluid delivery components described above in the context of Figures 1-3 The system (500) of this example also includes a temperature station actuator (530) and a heater / cooler (532) that can optionally adjust the conditioned temperature of the fluid within the flow cell (510). In some implementations, the heater / cooler (532) can be affixed to the sample stage (570) on which the flow cell (510) is placed and / or can be integrated into the sample stage (570).

[0097] The flow cell (510) can be removably mounted on a sample stage (570), which can provide movement and alignment of the flow cell (510) relative to the objective lens assembly (542). The sample stage (570) can have one or more actuators to allow the sample stage (570) to move in any of three dimensions. For example, actuators can be provided to allow the sample stage (570) to move in an x-direction, a y-direction, and a z-direction relative to the objective lens assembly (542), to tilt relative to the objective lens assembly (542), and / or to otherwise move relative to the objective lens assembly (542). Movement of the sample stage (570) can allow one or more sample locations on the flow cell (510) to be positioned in optical alignment with the objective lens assembly (542). Movement of the sample stage (570) relative to the objective lens assembly (542) can be achieved by moving the sample stage (570) itself, by moving the objective lens assembly (542), by moving some other component of the imaging assembly (522), by moving some other component of the system (500), or any combination of the foregoing. For example, in some implementations, the sample stage (570) can be capable of actuation in the x- and y-directions relative to the objective lens assembly (542), while a focus component (562) or z-stage can move the objective lens assembly (542) along the z-direction relative to the sample stage (570).

[0098] In some implementations, a focus component (562) can be included to control positioning of one or more elements of the objective lens assembly (542) in a focusing direction (e.g., along a z-axis or z-dimension) relative to the flow cell (510). The focus component (562) can include one or more actuators that are physically coupled to the objective lens assembly (542), an optical stage, the sample stage (570), or a combination thereof, to move the flow cell (510) on the sample stage (570) relative to the objective lens assembly (542) to provide proper focus for imaging operations. In the present example, the focus component (562) utilizes a focus tracking module (560) that is configured to detect a displacement of the objective lens assembly (542) relative to a portion of the flow cell (510) and output data indicative of a focus position to the focus component (562) or a component thereof, or is operable to control the focus component (562) (such as the controller (520)) to move the objective lens assembly (542) to position a corresponding portion of the flow cell (510) at a focal point of the objective lens assembly (542).

[0099] In some implementations, the focus component (562) or an actuator for the sample stage (570) can be physically coupled to the objective lens assembly (542), the optical stage, the sample stage (570), or a combination thereof, such as by being directly or indirectly attached or in contact to the stage or component thereof, e.g., mechanically, magnetically, fluidically, or otherwise. The actuator of the focus component (562) can be configured to move the objective lens assembly (542) in the z-direction while maintaining the sample stage (570) in the same plane (e.g., maintaining a horizontal or level attitude perpendicular to the optical axis). In some implementations, the sample stage (570) includes x-direction and y-direction actuators to form an x-y stage. The sample stage (570) can also be configured to include one or more flip or tilt actuators to flip or tilt the sample stage (570) and / or a portion thereof to account for any slope on its surface.

[0100] The camera system (540) can include one or more image sensors to monitor and track imaging (e.g., sequencing) of the flow cell (510). The camera system (540) can be implemented as, e.g., a CCD or CMOS image sensor camera, although other image sensor technologies (e.g., active pixel sensors) can be used. By way of further example only, the camera system (540) can include a dual-sensor time delay integration (TDI) camera, a single-sensor camera, a camera with one or more two-dimensional image sensors, and / or other types of camera technology. While the camera system (540) and associated optical components are shown positioned above the flow cell (510) in Figure 7 the system (500) as will be apparent to those of skill in the art in view of the teachings herein. For example, one or more image sensors can be positioned below the flow cell (510), such as within or below the sample stage (570); or even integrated into the flow cell (510).

[0101] Figure 8 Examples of various components that can be integrated into the imaging assembly (522) of the system (500) are shown. In particular, Figure 8 The arrangement shown can represent a variation of the illumination assembly (550). Figure 8 The arrangement shown can be particularly useful in the case where the camera system (540) includes a TDI camera. In Figure 8In the illustrated arrangement, a line generation module (LGM) (602) and an emission optics module (EOM) (604) are aligned and mechanically coupled to a precision mounting plate (610) and to each other. The EOM (604) includes an objective lens assembly (606) that is aligned with a tube lens (620) via a mirror (608), which in turn is optically coupled to the LGM (602). The LGM (602) can include one or more light sources (e.g., a coherent light source such as a laser diode). In some examples, the LGM (602) can include a first light source configured to emit light of a red wavelength and a second light source configured to emit light of a green wavelength. The LGM (602) can also include optical components such as focusing surfaces, lenses, reflective surfaces, or mirrors. The optical components can be positioned within a housing of the LGM (602) to direct and focus light emitted from the one or more light sources into adjacent module subassemblies. One or more of the optical components of the LGM (602) can also be configured to shape the light emitted from the one or more light sources into a desired pattern. For example, in some implementations, the optical components can shape the light into a line pattern (e.g., by using one or more Powell lenses or other beam shaping lenses, diffractive components, or scattering components). In some variations, the LGM (602) can include one or more laser modules that can be individually removed and replaced from the LGM (602).

[0102] The light beam produced by the LGM (602) transmits through an interface baffle between the LGM (602) and the EOM (604), through the objective lens assembly (606), and impinges on an optical target (e.g., a flow cell (510)). In some versions, the interface baffle includes an aperture shaped to enable light to pass through its center while obscuring interference from external light sources. Responsive optical radiation from the target can return through the objective lens assembly (606) and into a tube lens (622). The lens element (622), which can form part of the tube lens (620), is configured to articulate along an axis (e.g., a z-axis) to correct for spherical aberration artifacts introduced by the objective lens assembly (606) imaging through different thicknesses of the flow cell (510) component. As shown, the lens element (622) can articulate closer or further away from the objective lens assembly (606) to adjust the beam shape and path. The objective lens assembly (606) can emit excitation light toward the optical target (e.g., the flow cell (510)) and receive fluorescent emissions from the optical target. An actuator can be configured to position the objective lens assembly (606) to a region of interest proximate to the optical target. Subsequently, a processor of the controller (520) can execute program instructions for detecting the fluorescent emissions from the optical target.

[0103] Figure 9 An example of another configuration that can be provided in an imaging assembly (522) is shown. Specifically, Figure 9 An imaging assembly (650) is shown positioned relative to a flow cell (670). The flow cell (670) can represent any of the various variants of the flow cells (128, 368, 400, 450, 510) described herein. The flow cell (670) has an upper layer (671) and a lower layer (673) separated by a fluid-filled channel (675). In the configuration shown, the upper layer (671) is optically transparent, and the imaging assembly (650) is focused to an area (676) on an inner surface (672) of the upper layer (671). In other variants, the imaging assembly (650) can be focused on an inner surface (674) of the lower layer (673). One or both of the surfaces (672, 674) can include an array feature that will be detected by the imaging assembly (650).

[0104] The imaging assembly (650) includes an objective lens assembly (666) configured to direct excitation radiation from a light emitting assembly (652) to the flow cell (670); and to direct emissions from the flow cell (670) to a detector (664). In the arrangement shown, excitation radiation from the light emitting assembly (652) passes through a lens (658), through a beam splitter (660), and through the objective lens assembly (666) to the flow cell (670). In this example, the light emitting assembly (652) includes two light emitting diodes (LEDs) (656, 654) that produce radiation at different wavelengths from one another. Emission radiation from the flow cell (670) is captured by the objective lens assembly (666) and reflected by the beam splitter (660) through conditioning optics (662) and to the detector (664) (e.g., a CMOS sensor). The beam splitter (660) is used to direct the emission radiation in a direction orthogonal to the path of the excitation radiation. The positioning of the objective lens assembly (666) can be moved in the z-dimension to alter the focal point of the imaging assembly (650). The imaging assembly (650) can be moved back and forth in the y-direction to capture images of several areas of at least one inner surface (672, 674) of the flow cell (670).

[0105] In this example, a single imaging assembly (650) includes two LEDs (656, 654) that emit light at two different respective wavelengths, with a single detector (664) detecting light emitted by a fluorophore in the flow cell (670) in response to illumination at the two different wavelengths. In some other versions, there are two or more imaging assemblies (650), with each imaging assembly (650) including a single LED (656, 654) and a single detector (664), such that each imaging assembly (650) provides illumination at only one single respective wavelength. As another variation, two or more detectors (664) can receive excitation radiation from a common light-emitting assembly (652).

[0106] Figure 10 An example of another configuration that can be provided in an imaging assembly (522) is shown. Specifically, Figure 10 An imaging assembly (700) is shown positioned relative to a flow cell (774). The flow cell (774) can represent any of the various variations of the flow cells (128, 368, 400, 450, 510) described herein. The flow cell (770) has a translucent cover plate (772), a base (774), and a liquid layer (776) interposed between the cover plate (772) and the base (774). A biological sample can be located on an interior surface of the cover plate (772) (above the liquid layer (776)) and / or on an interior surface of the base (774) (below the liquid layer (776)).

[0107] The imaging assembly (700) of this example includes an LGM (710) in which two light sources (712, 714) are provided. The light sources (712, 714) can include laser diodes, diode-pumped solid state lasers, or other light sources known in the art that output a laser beam at different wavelengths (e.g., red or green light). The beams output from the light sources (712, 714) are directed through one or more beam shaping lenses 716. In some implementations, one or more beam shaping lenses can be used to shape the beams output from each or both light sources. The LGM (710) can use one or more Powell lenses to expand and / or shape the laser beams from single mode or near single mode laser light sources. Other beam shaping optics such as active beam expanders, attenuators, one relay lens, cylindrical lenses, actuated mirrors, diffractive elements, and scattering components can be used to control uniformity and increase tolerance. The laser beams can intersect at the back focal point of the objective lens to provide better tolerance on the surface of the flow cell (770).

[0108] The LGM (710) of this example also includes mirrors (718, 720). The beam produced by light source (712) is reflected by mirror (718) so as to be directed through an aperture or semi-reflective surface of mirror (720) and into EOM (740) through a single interface port. Similarly, the beam produced by light source (714) is reflected by mirror (720) so as to be directed into EOM (740) through the single interface port. In some examples, an additional set of articulating mirrors can be incorporated adjacent to mirrors (718, 720) to provide additional tuning surfaces. A dichroic mirror (720) can be used to combine the two beams. Mirrors (718, 720) can each be configured to be articulated using manual controls or automated controls to align the beams from light sources (712, 714). In this example, the beams also pass through a shutter element (722).

[0109] EOM (740) includes an objective lens assembly (756) and a z-stage (758) that moves objective lens assembly (756) longitudinally closer to or further from flow cell (770). LGM (710) is configured to produce uniform line illumination through objective lens assembly (756). Subsequently, z-stage (758) can move objective lens assembly (756) to focus the beam onto any of the interior surfaces of flow cell (770) (e.g., to focus on a biological sample). In some implementations, objective lens assembly (756) can be configured to focus the beam at a focal point outside of flow cell (770), such as to increase the line width of the beam at the surface of flow cell (770).

[0110] EOM (740) of this example also includes a semi-reflective mirror (754) to direct light through objective lens assembly (756) while allowing light returning from flow cell (774) to pass through. EOM (740) also includes a tube lens (744) and a correction lens (748). Correction lens (748) can be articulated longitudinally closer to or further from objective lens assembly (756) by z-stage (746) to ensure accurate imaging (e.g., to correct for spherical aberration caused by moving objective lens assembly (756); and / or spherical aberration caused by imaging through a thicker base, etc.). Light transmitted through correction lens (748) and tube lens (744) passes through a filter element (742) and into camera system (730). Camera system (730) includes one or more optical sensors (732) to detect light emitted from a biological sample in response to an incident beam of light.

[0111] In this example, the EOM (740) also includes a half-mirror (752) for reflecting a focus tracking beam emitted from a focus tracking module (FTM) (760) onto a flow cell (774) and subsequently reflecting light returned from the flow cell (774) back into the FTM (760). The FTM (760) can include a focus tracking optical sensor for detecting a characteristic of the returned focus tracking beam and generating a feedback signal to optimize the focusing of the objective lens assembly (756) on the flow cell (774).

[0112] The direction, size, and / or polarization of the laser beam can be adjusted by using lenses, mirrors, and / or polarizers. Optical lenses (e.g., cylindrical, spherical, or aspherical) can be used to actively adjust the illumination focal point on the double surface of the flow cell (770) target. The LGM (710) can also include multiple units, where each unit is designed for a specific / different wavelength and polarization. Stacking multiple units can be used to increase the laser power and increase the wavelength options. Two or more laser wavelengths can be combined with dichroics and polarizers.

[0113] By way of example only, the focus tracking module (560) and / or other components of the imaging assembly (522) can be constructed and operated in accordance with at least some of the teachings of U.S. Patent No. 10,416,428, issued September 17, 2019, entitled “Systems and Methods for Improved Focus Tracking Using a Light Source Configuration,” the disclosure of which is incorporated by reference herein in its entirety; U.S. Publication No. 2023 / 0228984, published July 20, 2023, entitled “Dynamic Detilt Focus Tracking,” the disclosure of which is incorporated by reference herein in its entirety; and / or U.S. Patent Application No. 63 / 410,961, filed September 28, 2022, entitled “Spot Error Handling for Focus Tracking,” the disclosure of which is incorporated by reference herein in its entirety. By way of further example only, components of the imaging assembly (522) can be configured and operated in accordance with at least some of the teachings of U.S. Patent No. 10,774,371, issued September 15, 2020, entitled “Laser Line Illuminator for High Throughput Sequencing,” the disclosure of which is incorporated by reference herein in its entirety; and / or U.S. Patent No. 9,958,465, issued May 1, 2018, entitled “Detection Apparatus having a Microfluorometer, a Fluidic System, and a FlowCell Latch Clamp Module,” the disclosure of which is incorporated by reference herein in its entirety.

[0114] VI. Examples of Immersion Microscope Features for Sequencing Systems As noted above, some versions of the imaging assembly (522, 650, 700) in the sequencing system (500) can include an objective lens assembly (542, 606, 666, 756) positioned above the flow cell (128, 368, 400, 450, 450A, 510, 670, 770) for capturing images of fluorescent emissions having fluorophore labels from nucleotides in the flow cell (128, 368, 400, 450, 450A, 510, 670, 770). In some cases, it can be desirable to provide an immersion fluid (e.g., water, oil, etc.) between the objective lens assembly (542, 606, 666, 756) and an upper surface of the flow cell (128, 368, 400, 450, 450A, 510, 670, 770), thereby providing an immersion microscope arrangement. Such an immersion fluid can enhance the resolution and clarity of images captured via the objective lens assembly (542, 606, 666, 756) by effectively increasing the numerical aperture of the objective lens assembly by increasing the refractive index that would otherwise be provided in an air gap between the objective lens assembly (542, 606, 666, 756) positioned above the flow cell (128, 368, 400, 450, 450A, 510, 670, 770). Examples of how to integrate an immersion fluid feature into an imaging assembly of a sequencing system, such as any of the imaging assemblies (522, 650, 700) described herein, are provided below.

[0115] A. Overview Figure 11 An example of an arrangement (1000) that can incorporate any of the various sequencing systems described herein is shown. The arrangement (1000) of this example includes a frame (1010), a sample stage (1020), a flow cell (1030), an imaging assembly (1040), an air curtain manifold (2020), and an air blade manifold (2060). The frame (1010) is shown schematically and takes various suitable forms. For example, the frame (1010) can include a fixed superstructure within a sequencing instrument or system housing, thereby providing a mechanical ground for other features of the arrangement (1000).

[0116] The sample stage (1020) can be configured and operated like the sample stage (570) described above, such that the sample stage (1020) can provide structural support for the flow cell (1030); and further provide movement and alignment of the flow cell (1030) relative to the objective lens assembly (2000). The sample stage (1020) can have one or more actuators to allow the sample stage (1020) to move in any of three dimensions. For example, actuators can be provided to allow the sample stage (1020) to move in an x-direction, a y-direction, and a z-direction relative to the objective lens assembly (2000), to tilt relative to the objective lens assembly (2000), and / or to otherwise move relative to the objective lens assembly (2000). The flow cell (1030) can be configured and operated like any of the other flow cells (128, 368, 400, 450, 450A, 510, 670, 770) described herein.

[0117] The imaging assembly (1040) of this example includes an objective lens assembly (2000), an immersion fluidic manifold (2040), and Figure 11 A set of additional imaging components (1044) are schematically shown in FIG. 104. These additional imaging components (1044) can include any of the various imaging components of any of the various imaging assemblies (522, 650, 700) described herein. The specific features and functionality of the objective lens assembly (2000) and the immersion fluidic manifold (2040) will be described in greater detail below. In this example, the imaging assembly (1040) of this example is coupled with the frame (1010) via a mount (1042). In some versions, the mount (1042) provides a static fixed relationship between the imaging assembly (1040) and the frame (1010), such that the imaging assembly (1040) does not move relative to the frame (1010). In some other versions, at least a portion of the imaging assembly 1040 moves relative to the frame 1010. For example, the mount (1042) can include one or more motors, servo systems, or other actuators, etc. that provide controlled movement of the imaging assembly (1040) relative to the frame (1010) in one or more of the x-dimension, the y-dimension, or the z-dimension. In addition or in the alternative, one or more features within the imaging assembly can provide movement of the objective lens assembly (2000) and / or one or more focusing lens elements relative to the frame (1010), such as to provide controlled focusing of an imaging region on or in the flow cell (1030), etc.

[0118] As Figures 11-20As shown, the air curtain manifold (2020) of this example includes an annular body (2022) positioned to encircle a distal portion of the objective lens assembly (2000). The specific features and functionality of the air curtain manifold (2020) will be described in greater detail below. In this example, the air curtain manifold (2020) is coupled with the frame (1010) via mounts (1012) and corresponding beams (1014). Thus, in this example, the mounts (1012) and beams (1014) support the air curtain manifold (2020) relative to the objective lens assembly (2000) with a gap (G) maintained between the air curtain manifold (2020) and the objective lens assembly (2000), as Figure 16 , Figure 18 and Figures 38A-38G best seen. In some versions, the mounts (1012) and beams (1014) provide a static fixed relationship between the air curtain manifold (2020) and the frame (1010). In some other versions, each mount (1012) can include one or more motors, servos, or other actuators, etc. that provide controlled movement of the air curtain manifold (2020) relative to the frame (1010) in one or more of the x, y, or z dimensions.

[0119] Each mount (1012) and / or the frame (1010) can also provide a substantial mass to absorb any vibrations that can be transmitted by the air curtain manifold (2020) along the beams (1014) during operation of the air curtain manifold (2020) as described below. This can effectively isolate the objective lens assembly (2000) from such vibrations, preventing such vibrations from otherwise adversely affecting the quality of images captured via the objective lens assembly (2000). Additionally or in the alternative, foam, rubber, or other elastomeric material, etc. can be interposed between the air curtain manifold (2020) and the objective lens assembly (2000) to prevent vibrations of the air curtain manifold (2020) from reaching the objective lens assembly (2000).

[0120] As Figures 11-20As shown, the air vane manifold (2060) of this example includes a body (2062) positioned laterally offset from the field of view of the objective lens assembly (2000). Specific features and functionality of the air vane manifold (2060) will be described in more detail below. In this example, the air vane manifold (2060) is connected to the frame (1010) via a mounting (1016) and a corresponding beam (1018). Thus, in this example, the mounting (1016) and beam (1016) support the air vane manifold (2060) relative to the objective lens assembly (2000). In some forms, the mounting (1016) and beam (1018) provide a static fixing relationship between the air vane manifold (2060) and the frame (1010). In some other forms, the mounting (1016) may include one or more motors, servo systems, or other actuators that provide controlled movement of the air vane manifold (2060) relative to the frame (1010) in one or more of the x, y, or z dimensions. The mounting (1016) and / or the frame (1010) may also provide considerable mass to absorb any vibrations that may be transmitted by the air vane manifold (2060) along the beam (1018) during operation of the air vane manifold (2060) as described below.

[0121] like Figure 12 As shown, an immersion fluid source (3000) is fluidly connected to an immersion fluid manifold (2040) via a fluid conduit (3002). The immersion fluid source (3000) may include any suitable type of immersion fluid, including but not limited to water (e.g., HPLC double-distilled water), oil, etc. A suction source (3010) is also fluidly connected to the immersion fluid manifold (2040) via a fluid conduit (3012). By way of example only, each fluid conduit (3002, 3012) may include flexible tubing and / or any other suitable components. In some configurations, the suction source (3010) is also fluidly connected to the immersion fluid source (3000) via a third conduit (not shown), such that fluid transferred from the immersion fluid source (3000) to the immersion fluid manifold (2040) can be recirculated back to the immersion fluid source (3000) via the suction source (3010) and the third conduit. In some other configurations, the suction source (3010) is fluidly connected to a tank or other fluid container such that fluid drawn from the immersion fluid manifold (2040) by the suction source (3010) is deposited in the tank or other fluid container for disposal or other treatment.

[0122] like Figure 13As shown, a pressurized air source (3020) is fluidically coupled with the valve (3030) via a fluid conduit (3022). By way of example only, the pressurized air source (3020) can comprise a pump or a cartridge containing pressurized air. The valve (3030) is fluidically coupled with the air curtain manifold (2020) via a fluid conduit (3032); and with the air vane manifold (2060) via another fluid conduit (3034). The valve (3030) is operable to switch between at least two different states, including a first state in which pressurized air from the pressurized air source (3020) is delivered to the air curtain manifold (2020) and a second state in which pressurized air from the pressurized air source (3020) is delivered to the air vane manifold (2020). The valve (3030) can take any suitable form that can be apparent to those of skill in the art in view of the teachings herein. The operation of the pressurized air source (3020) and the valve (3030) can be automated under the control of features similar to the controller (114, 308) in view of the teachings herein. By way of further example only, each fluid conduit (3022, 3032, 3034) can comprise a flexible tube and / or any other suitable component.

[0123] B. Examples of Objective Lens Assemblies Figures 21-22 Features of the objective lens assembly (2000) are shown in further detail. As shown, the objective lens assembly (2000) of this example includes an objective lens element (2002) supported by a housing (2006). The housing (2006) includes a tapered distal portion (2008) that terminates at an annular distal face (2010). By way of example only, the annular distal face (2010) can comprise polytetrafluoroethylene, nickel, and / or any other suitable material. The annular distal face (2010) encloses a distal face (2004) of the objective lens element (2002). In some versions, the distal face (2004) of the objective lens element (2002) is substantially flush with the annular distal face (2010). In some other versions, the objective lens element (2002) is recessed relative to the annular distal face (2010). In some such versions, the objective lens element (2002) is recessed relative to the annular distal face (2010) at a depth in a range of about 100 pm to about 50 pm. Alternatively, the objective lens element (2002) can be recessed relative to the annular distal face (2010) at any other suitable depth.

[0124] In the present example, an annular recess (2012) is formed in the annular distal face (2010). In this example, the annular recess (2012) has a cross-sectional profile similar to a truncated pyramid. Alternatively, the annular recess (2012) can have any other suitable kind of cross-sectional profile, including but not limited to circular / concave, triangular, rectangular, etc. While only one annular recess (2012) is provided in this example, other versions can include two or more annular recesses (2012) (e.g., a concentric arrangement along the annular distal face (2010)). The annular recess (2012) of the present example is configured to facilitate the retention of a volume of immersion fluid (IF) between the objective lens assembly (2000) and the upper surface (1032) of the flow cell (1030), even during movement of the flow cell (1030) relative to the objective lens assembly (2000), as described in greater detail below. By way of example only, the annular recess (2012) can have a depth ranging from approximately 12.5 pm to approximately 50 pm. Alternatively, the annular recess (2012) can have any other suitable depth.

[0125] Figure 23 An example of an alternative objective lens element (2003) is shown that can be incorporated into the objective lens assembly (2000) in place of the objective lens element (2002). While the objective lens element (2002) has a flat distal face (2004), the objective lens element (2003) of this example has a concave distal face (2005). In some cases, the concave configuration of the distal face (2005) can facilitate the retention of a volume of immersion fluid (IF) between the objective lens assembly (2000) and the upper surface (1032) of the flow cell (1030), even during movement of the flow cell (1030) relative to the objective lens assembly (2000), as described in greater detail below. By way of example only, some versions of the objective lens assembly (2000) can include a combination of a concave distal face (2005) and one or more annular recesses (2012) to facilitate the retention of a volume of immersion fluid (IF) between the objective lens assembly (2000) and the upper surface (1032) of the flow cell (1030). Additionally or in the alternative, the distal faces (2004, 2005) of the objective lens elements (2002, 2003) and / or the distal face (2010) of the housing (2006) can incorporate a hydrophilic material (e.g., polyetherimide, acrylic, inconel, etc.) and / or other treatments to facilitate the retention of a volume of immersion fluid (IF) between the objective lens assembly (2000) and the upper surface (1032) of the flow cell (1030).

[0126] C. Examples of Air Curtain Manifolds Figures 24-28 Features of the air curtain manifold (2020) are shown in greater detail. As shown, the air curtain manifold (2020) of this example includes an annular body (2022) having an outer surface (2024) that is angled to taper inwardly toward a bottom of the body (2022). A plurality of openings (2026) are formed through the outer surface (2024). In this example, the openings (2026) are equally spaced apart from one another around an entire circumference of the annular body (2022).

[0127] As Figures 26-28 As best seen, an annular channel (2032) is formed in the body (2022) and is in fluid communication with the openings (2026). The air curtain manifold (2020) also includes a pair of ports (2028) that extend upwardly from the body (2022). Each port (2028) defines a passageway (2034) that is in fluid communication with the channel (2032). Each port (2028) is configured to fluidly couple with a respective fluid conduit (3032). As described above with reference to Figure 13 As described above with reference to

[0128] Accordingly, the ports (2028) allow the air curtain manifold (2020) to receive the pressurized air from the pressurized air source (3020). The pressurized air passes through the passageways (2034) and the channel (2032) to reach the openings (2026) through which the pressurized air is expelled from the air curtain manifold (2020). As described in greater detail below, with the openings (2026) positioned on the angled surface (2026) of the air curtain manifold (2020), and with the air curtain manifold (2020) coaxially positioned about the objective lens assembly (2000), the pressurized air creates a truncated conical shaped air curtain that is oriented downwardly and outwardly relative to the central longitudinal axis (LA) of the imaging assembly (1040) as the pressurized air exits the openings (2026). As will also be described in greater detail below, this air curtain provided by the air curtain manifold (2020) can substantially prevent any fluid, debris, etc. on the upper surface (1032) of the flow cell (1030) from reaching the field of view below the objective lens assembly (2000). In other words, the air curtain can tend to blow or sweep such fluid, debris, etc. away from the upper surface (1032) before it would otherwise reach the field of view below the objective lens assembly (2000).

[0129] The air curtain manifold (2020) of the present example also includes a pair of openings (2030) on an area above the surface (2024) of the body (2022). The openings (2030) are configured to receive fasteners (e.g., pins, screws, etc.) to secure the air curtain manifold (2020) to the beam (1014). As noted above with reference to Figure 11 As noted, the beam (1014) is secured to the frame (1010) via respective mounts (1012). In this example, the openings (2030) are not in fluid communication with the channels (2032), the passageways (2034), or the openings (2026). In other versions, the air curtain manifold (2020) is secured to the frame (1010) (or otherwise secured relative to the objective lens assembly (2000)) using other structures or arrangements. In the present example, the air curtain manifold (2020) is secured at a position along the central longitudinal axis (LA) that is slightly higher than a position of the immersion fluid manifold (2040). In some other versions, the air curtain manifold (2020) can be positioned at the position of the immersion fluid manifold (2040) or slightly lower than the position of the immersion fluid manifold along the central longitudinal axis (LA).

[0130] D. Examples of Immersion Fluid Manifolds Figures 29-32 Features of the immersion fluid manifold (2040) are shown in greater detail. As shown, the immersion fluid manifold (2040) of the present example includes an annular body (2042) having an inner surface (2043) that is angled to taper inwardly toward the bottom of the body (2022). The angle of the inner surface (2043) is configured to be complementary to the angle of the tapered distal portion (2008) of the housing (2006) of the objective lens assembly (2000). Thus, when the immersion fluid manifold (2040) is secured at the bottom of the objective lens assembly (2000), the inner surface (2043) fits together in juxtaposition with the tapered distal portion (2008), as Figure 16 and Figure 18 best seen. In some versions, the fit between the inner surface (2043) and the tapered distal portion (2008) is substantially fluid-tight, thereby preventing the undesired migration of immersion fluid during operation into a space that might otherwise be defined between the inner surface (2043) and the tapered distal portion (2008), as described in greater detail below.

[0131] The central opening (2045) of the immersion fluidic manifold (2040) is sized to accommodate the annular distal face (2010) of the housing (2006) of the objective lens assembly (2000) such that the immersion fluidic manifold (2040) does not obstruct the field of view through the objective lens element (2002). Similarly, the immersion fluidic manifold (2040) does not cover any area of the annular recess (2012) such that the immersion fluidic manifold (2040) does not impede the fluid retention capability of the annular recess (2012) as described herein.

[0132] In some versions, the immersion fluidic manifold (2040) is secured to the housing (2006) of the objective lens assembly (2000) via an adhesive or epoxy. In some other versions, the immersion fluidic manifold (2040) is secured to the housing (2006) of the objective lens assembly (2000) via complementary threads and / or some other mechanical feature. Alternatively, the immersion fluidic manifold (2040) can be secured to the housing (2006) of the objective lens assembly (2000) in any other suitable manner. As yet another example of an alternative arrangement, the immersion fluidic manifold (2040) can be secured relative to the housing (2006) without being directly secured to the housing (2006). For example, the immersion fluidic manifold can be secured relative to the housing (2006) via a structure such as the beam (1014) and mount (1012).

[0133] Also as shown Figures 29-32 The immersion fluidic manifold (2040) of the present example also includes a recess (2044) below and around the central opening (2045). The recess (2044) is surrounded by an edge (2046) at the bottom of the body (2042). As Figure 31 As best seen, the recess (2044) has an elliptical shape with a major axis (Al) and a minor axis (A2). By way of example only, the major axis (Al) can be about 18 mm, while the minor axis (A2) can be about 14 mm. Alternatively, any other suitable length can be used for the major axis (Al) or the minor axis (A2). During the image capture process, and as will be described in greater detail below, the immersion fluidic manifold (2040) is oriented such that the major axis (Al) of the recess (2044) is oriented along the path of relative motion between the flow cell (1030) and the objective lens assembly (2000); while the minor axis (A2) of the recess (2044) is oriented transversely relative to the path of relative motion between the flow cell (1030) and the objective lens assembly (2000). In Figure 31 and Figures 38A-38GIn the reference axis provided, the relative motion path during the image capture process is along the x-axis. As will be described in more detail below, the elliptical shape and orientation of the recess (2044) can facilitate the retention of a certain volume of immersion fluid (IF) in the space between the objective lens element (2002) and the upper surface (1032) of the flow cell (1030), including when there is relative motion between the flow cell (1030) and the objective lens assembly (2000) during the image capture process.

[0134] like Figure 32 Best viewed in this example, the immersion fluid manifold (2040) also includes a first port (2050), a first channel (2052), a second port (2056), and a second channel (2058). The first port (2050) is located in a laterally facing region of the body (2042) and is configured to connect to an immersion fluid source (3000) via a fluid conduit (3002), as referenced above. Figure 12 As described. Therefore, the immersion fluid manifold (2040) receives immersion fluid via a fluid conduit (3002) and a first port (2050). In some configurations, the first port (2050) includes outwardly extending rigid fittings and / or other structural features to facilitate connection with the fluid conduit (3002). A first channel (2052) extends from the first port (2050) to a recess (2044), thereby providing a path for conveying immersion fluid from the immersion fluid source (3000) to the recess (2044). Thus, a given volume of immersion fluid from the immersion fluid source (3000) can be conveyed via the fluid conduit (3002), the first port (2050), and the first channel (2052) to the space between the objective lens element (2002) and the upper surface (1032) of the flow cell (1030).

[0135] The second port (2056) is also located on the laterally facing area of ​​the main body (2042) and is configured to connect to the suction source (3010) via a fluid conduit (3012), as referenced above. Figure 12As described above, the immersion fluid is drawn from the immersion fluid manifold (2040) via the fluid conduit (3012) and the second port (2056). In some versions, the second port (2056) includes outwardly extending rigid fittings and / or other structural features to facilitate coupling with the fluid conduit (3012). The second channel (2058) extends from the second port (2056) to the recess (2044), thereby providing a path for the transfer of immersion fluid from the recess (2044). Thus, the suction source (3010) can be activated to draw / remove a volume of immersion fluid from the space between the objective lens element (2002) and the upper surface (1032) of the flow cell (1030) via the second channel (2058), the second port (2056), and the fluid conduit (3012). This drawing / removal of immersion fluid can be provided during a de-priming step of an operation as described in greater detail below. Additionally or in the alternative, this drawing of immersion fluid by the suction source (3010) can be provided concurrently with the transfer of immersion fluid from the immersion fluid source (3000), such as during circulation of immersion fluid within the space between the objective lens element (2002) and the upper surface (1032) of the flow cell (1030).

[0136] E. Examples of Air Blade Manifolds Figures 33-35 Features of the air blade manifold (2060) are shown in greater detail. As shown, the air blade manifold (2060) of this example includes a body (2062) having a mounting portion (2064) and an arcuate portion (2066). The mounting portion (2064) is configured to couple with the beam (1018) via one or more fasteners (e.g., pins, screws, etc.). As noted above with reference to Figure 11 As noted above, the beam (1018) is secured to the frame (1010) via the mount (1016). In other versions, the air blade manifold (2060) is secured to the frame (1010) (or otherwise relative to the objective lens assembly (2000)) using other structures or arrangements.

[0137] As Figure 35 As best seen, a main channel (2068) is formed in the body (2062). The main channel (2068) is configured to fluidly couple with the fluid conduit (3034). As described above with reference to Figure 13 As described above, the fluid conduit (3034) is configured to receive pressurized air from the pressurized air source (3020) via the fluid conduit (3022) and the valve (3030). In some versions, the body (2062) includes outwardly extending rigid fittings and / or other structural features to facilitate coupling of the main channel (2068) with the fluid conduit (3022). Also as Figure 35As best seen, a plurality of passages (2072) are also in fluid communication with the main passage (2068). The passages (2072) are oriented along respective radii emanating from a single center point.

[0138] Each passage (2072) terminates at a respective opening (2070) such that pressurized air delivered to the main passage (2068) via the fluid conduit (3022) will pass through the passages (2072) and exit the air blade manifold (2060) via the openings (2070). The openings (2070) are positioned along the arcuate portion (2066). In this example, the openings (2070) are all positioned along the same x-y plane as one another, but the openings (2070) can alternatively have any other suitable arrangement. In the present example, the angular range of the array of openings (2070) is less than 180 degrees. The arcuate portion (2066) is positioned to face the space between the objective lens element (2002) and the upper surface (1032) of the flow cell (1030). When pressurized air is delivered from the pressurized air source (3020) to the air blade manifold (2060) as described above, the pressurized air is expelled via the openings (2070) into the space between the objective lens element (2002) and the upper surface (1032) of the flow cell (1030). This pressurized air creates a substantially flat air blade that can tend to clear any residual immersion fluid on the objective lens assembly (2000) and / or the upper surface (1032) of the flow cell (1030).

[0139] As noted above, the channels (2072) are oriented along respective radii emanating from a single center point; and the openings (2070) are all oriented toward the single center point. In some versions, the single center point is positioned along an optical axis of the objective lens assembly (2000) such that the channels (2072) and the openings (2070) are all oriented toward a central region of a space between the objective lens element (2002) and the upper surface (1032) of the flow cell (1030). Thus, air expelled via the openings (2070) is substantially directed toward the central region of the space between the objective lens element (2002) and the upper surface (1032) of the flow cell (1030). This inwardly oriented air flow pattern can enhance the ability of the air blades formed by the air blade manifold (2060) to remove residual immersion fluid from the objective lens assembly (2000) and / or the upper surface (1032) of the flow cell (1030), as described in greater detail below. In some other versions, the channels (2072) and the openings (2070) are positioned along straight lines rather than arcs such that the air flow pattern of the air blades formed by the air blade manifold (2060) is not focused centrally on the central region of the space between the objective lens element (2002) and the upper surface (1032) of the flow cell (1030). In some cases, this non-centrally focused air flow pattern can be less efficient or effective than a centrally focused air flow pattern; but is still acceptable.

[0140] While an air blade manifold (2060) is used in this example to remove residual immersion fluid from the objective lens assembly (2000) and / or the upper surface (1032) of the flow cell (1030), other structures or techniques can be used to remove residual immersion fluid from the objective lens assembly (2000) and / or the upper surface (1032) of the flow cell (1030). Such other structures or techniques can be used in addition to or instead of using the air blade manifold (2060). Such other structures or techniques can include absorbent features, non-absorbent wiping features, and / or any other suitable structure or technique. In some other versions, the air blade manifold (2060) is omitted and no other additional features are provided to remove residual immersion fluid from the objective lens assembly (2000) and / or the upper surface (1032) of the flow cell (1030). In some such versions, depriming provided via the suction source (3010) and the immersion fluid manifold (2040) can sufficiently remove immersion fluid from the objective lens assembly (2000) and / or the upper surface (1032) of the flow cell (1030).

[0141] F. Examples of Use of Imaging Assemblies with Immersion Microscope Features Figures 36A-36BAn example of an imaging process in which the imaging assembly (1040) is used to capture images of fluorescent emissions having fluorophore labels from nucleotides in the flow cell (1030) is schematically depicted. As shown, the flow cell (1030) includes eight channels (1034). By way of example only, each channel (1034) of the flow cell (1030) can be constructed and operated like each channel (130) of the flow cell (128) described above, like each channel (430) of the flow cell (400) described above, like each channel (480) of the flow cell (450) described above, or otherwise. At the start of the imaging process, the imaging assembly (1040) is positioned at a first end of a first channel (1034a), as shown. Figure 36A Figure 36A The ellipse (1036) in

[0142] Through the imaging process, the flow cell (1030) is moved relative to the imaging assembly (1040) along the x-y plane, allowing the imaging assembly (1040) to capture multiple images along the length and width of the first channel (1034a). As noted above, this relative movement is provided by one or more actuators of the sample stage (1020); while the imaging assembly (1040) remains stationary along the x-y plane. Also as noted above, some other variations can provide for driven movement of the imaging assembly (1040) along the x-y plane; while the flow cell (1030) remains stationary along the x-y plane. In either case, after the imaging assembly (1040) has captured sufficient images along the first channel (1034a), the relative positioning between the imaging assembly (1040) and the flow cell (1030) is changed to allow the imaging assembly (1040) to capture images along a second channel (1034b), along a third channel (1034c), along a fourth channel (1034d), along a fifth channel (1034e), along a sixth channel (1034f), along a seventh channel (1034g), and along an eighth channel (1034h). At the end of this imaging process, the imaging assembly (1040) is positioned at a second end of the eighth channel (1034h), as shown. Figure 36B

[0143] Figure 37 Figures 38A-38G Examples of how the air curtain manifold (2020), the immersion fluid manifold (2040), and the air blade manifold (2060) can be utilized during an imaging process, such as the process described above with reference to Figures 36A-36B Figure 38A ​​​​As shown, the process can begin by activating the air curtain via the air curtain manifold (2020). Figure 37 (Box 3050). As noted above, this can be achieved via activation by a pressurized air source (3020), wherein the valve (3030) is in an operating state in which the valve (3030) directs pressurized air to the fluid conduit (3032). In some types, the air curtain remains continuously activated throughout the process until the air blades are activated, as referenced below. Figure 38F To describe in more detail.

[0144] After the air blades have been initially activated, and as Figure 38B As shown, the process can continue to position the flow cell (1030) ( Figure 37 The frame 3052 is located below the objective lens assembly (2000). In some configurations, the flow cell (1030) is driven to this position via the sample stage (1020), while the imaging assembly (1040) remains stationary. In some other configurations, the imaging assembly (1040) is driven to position (e.g., via the mounting element (1042)), while the flow cell (1030) remains stationary. In either case, once the flow cell (1030) is properly positioned below the objective lens assembly (2000), the truncated conical air curtain exiting from the air curtain manifold (2020) is oriented downward and outward relative to the central longitudinal axis of the objective lens assembly (2000) as described above, such that any fluid, debris, etc., on the upper surface (1032) of the flow cell (1030) would otherwise reach the field of view below the objective lens assembly (2000). When the air curtain reaches the upper surface (1032), the pressurized air is deflected outward by the upper surface (1032). Figure 38B The operation phases shown can be related to Figure 36A This corresponds to the initial part of the operation phase shown.

[0145] With the flow cell (1030) properly positioned below the objective lens assembly (2000), and as Figure 38C As shown, the submersible fluid manifold (2040) is filled with ( Figure 37 (Box 3054). Although the immersion fluid source (3000) was not in Figure 38CAs shown, however, the infusion step includes transferring immersion fluid from the immersion fluid source (3000) to the recess (2044) via a conduit (3002), a first port (2050), and a first channel (2052). This infusion step generates a volume of immersion fluid (IF) in the space between the objective lens element (2002) and the upper surface (1032) of the flow cell (1030). By way of example only, the volume of the immersion fluid (IF) can be approximately 125 μL. Alternatively, the volume of the immersion fluid (IF) can have any other suitable size.

[0146] In some configurations, the immersion fluid is continuously circulated to the space by continuous deposition of immersion fluid from an immersion fluid source (3000) and continuous extraction of immersion fluid from a suction source (3010). In some of these configurations, the immersion fluid is provided at a specific temperature or within a specific temperature range to regulate the temperature of the objective lens element (2002) and / or the flow cell (1030). Furthermore, or in alternative configurations, one or more thermal elements (e.g., within the objective lens assembly (2000), within the immersion fluid manifold (2040), and / or elsewhere) may be used to regulate the temperature of the objective lens element (2002), the volume of the immersion fluid (IF), and / or the flow cell (1030).

[0147] For example Figure 38C As shown, some configurations may include a machine vision sensor (1038) (e.g., a camera or other optical sensor, etc.) oriented to observe the space between the objective lens assembly (2000) and the upper surface (1032) of the flow cell (1030). Therefore, data from such a machine vision sensor (1038) can indicate whether / when a sufficient volume of immersion fluid (IF) has been deposited in the space between the objective lens assembly (2000) and the upper surface (1032) of the flow cell (1030). In some of these types, the machine vision sensor (1038) and the immersion fluid source (3000) may be connected to the same controller (e.g., controller (114, 308)) such that the controller may issue a command signal to cause the immersion fluid source (3000) to continue depositing immersion fluid into the space between the objective lens assembly (2000) and the upper surface (1032) of the flow cell (1030) until feedback data from the machine vision sensor (1038) indicates that a sufficient volume of immersion fluid (IF) has reached the space between the objective lens assembly (2000) and the upper surface (1032) of the flow cell (1030).

[0148] Once sufficient volume of immersion fluid (IF) has been provided in the space between the objective lens assembly (2000) and the upper surface (1032) of the flow cell (1030), and as Figure 38DAs shown, this process can proceed to scanning the flow cell (1030). Figure 37 (See box 3056). During this scanning process, relative movement can be provided between the flow cell (1030) and the imaging assembly (1040) such that the imaging assembly (1040) captures images along the length and width of each channel (1034) of the flow cell (1030), as referenced above. Figures 36A-36B As described above, this relative movement is provided by one or more actuators of the sample stage (1020) such that the flow cell (1030) moves along the xy plane while the imaging assembly (1040) remains stationary along the xy plane. Also as noted above, some other variations can provide driven movement of the imaging assembly (1040) along the xy plane while the flow cell (1030) remains stationary along the xy plane.

[0149] like Figure 38D As shown, during the relative movement between the flow cell (1030) and the imaging assembly (1040), the volume of immersion fluid (IF) can be retained in the space between the objective lens element (2002) and the upper surface (1032), although the volume of immersion fluid (IF) may experience some degree of lateral deformation due to shear forces. Even when the relative movement between the flow cell (1030) and the imaging assembly (1040) is substantially rapid (e.g., between approximately 20 mm / s and approximately 100 mm / s, or faster than approximately 100 mm / s), the volume of immersion fluid (IF) can be adequately maintained between the objective lens element (2002) and the upper surface (1032).

[0150] As noted above, various features can facilitate maintaining the volume of immersion fluid (IF) in the space between the objective lens element (2002) and the upper surface (1032) during the relative movement between the flow cell (1030) and the imaging assembly (1040). Such features can include an annular recess (2012) in the annular distal face (2010) of the housing (2006) of the objective lens assembly (2000). Such features can also include a hydrophilic coating on the distal face (2004) of the objective lens element (2002) and / or on the distal face (2010) of the housing (2006). Such features can also include a hydrophobic coating (e.g., fluoropolymer, diamond-like nanocomposite, diamond-like nanostructure, amorphous carbon, fluorinated diamond-like nanocomposite, etc.) on the upper surface (1032) of the flow cell (1030). Such features can also include an elliptical-shaped recess (2044) on the underside of the immersion fluid manifold (2040), which can tend to minimize the surface tension of the immersion fluid. Such features can also include a recessed spacing of the distal faces (2004, 2005) of the objective lens elements (2002, 2003) relative to the annular distal face (2010) of the housing (2006). Such features can also include a concave recess formed in the distal face (2005) of the objective lens element (2003).

[0151] As noted above, some versions can include a machine vision sensor (1038) oriented to view the space between the objective lens assembly (2000) and the upper surface (1032) of the flow cell (1030). In some such versions, the machine vision sensor (1038) can continue to optically monitor the space between the objective lens assembly (2000) and the upper surface (1032) to confirm that a sufficient volume of immersion fluid (IF) remains in the space between the objective lens assembly (2000) and the upper surface (1032) during the scanning process. If feedback from the machine vision sensor (1038) indicates that there is no longer a sufficient volume of immersion fluid (IF) in the space between the objective lens assembly (2000) and the upper surface (1032) (or that the volume of immersion fluid (IF) is approaching an insufficient level), then a controller (e.g., controller (114, 308)) can issue a command signal to cause the immersion fluid source (3000) to deposit additional immersion fluid into the space between the objective lens assembly (2000) and the upper surface (1032) until a suitable volume of immersion fluid (IF) is again achieved. This feedback loop can be maintained throughout the scanning process. It should also be noted that the air curtain remains activated throughout the scanning process to substantially prevent any other fluids, debris, etc. on the upper surface (1032) of the flow cell (1030) from reaching the field of view beneath the objective lens assembly (2000) during the scanning process.

[0152] After the flow cell (1030) has been sufficiently scanned by the imaging assembly (1040), in the event that a sufficient number of images have been captured, the process can proceed to a de-priming step (block 3058) as shown in Figure 38E Figure 37 In some versions, when the de-priming step is initiated, the relative movement between the flow cell (1030) and the imaging assembly (1040) has ceased. To provide de-priming, the suction source (3010) is activated to draw a volume of immersion fluid (IF) from the space between the objective lens assembly (2000) and the upper surface (1032).

[0153] In versions in which the machine vision sensor (1038) is positioned to optically monitor the space between the objective lens assembly (2000) and the upper surface (1032), feedback from the machine vision sensor (1038) can be used to determine when the suction source (3010) has removed as much of the immersion fluid as possible from the space between the objective lens assembly (2000) and the upper surface (1032) that can be removed via the immersion fluid manifold (2040). In some other versions (e.g., in the event that the machine vision sensor (1038) is omitted), the suction source (3010) can be activated for a predetermined duration without regard to any machine vision feedback; then deactivated after the predetermined duration has expired. In some cases, and as shown in Figure 38E

[0154] After de-priming by the suction source (3010) and the immersion fluid manifold (2040), the air blade (block 3060) of Figure 37 may be activated, as shown in Figure 38F As noted above, this can be accomplished via sustained activation of the pressurized air source (3020) with the valve (3030) switched to an operational state in which the valve (3030) directs pressurized air to the fluid conduit (3034). This switching of the valve (3030) will stop the flow of pressurized air to the air curtain manifold (2020) to redirect the pressurized air to the air blade manifold (2060). As described above, the air blade provided via the air blade manifold (2060) is oriented toward the space between the objective lens assembly (2000) and the upper surface (1032) such that the air blade effectively blows away residual immersion fluid (RF) on the objective lens assembly (2000) and / or the upper surface (1032). In Figure 38F ​​In the example shown, the suction source (3010) is not activated, while the air blades are activated. In some other types, the suction source (3010) remains activated when the air blades are activated.

[0155] In a configuration where a machine vision sensor (1038) is positioned to optically monitor the space between the objective lens assembly (2000) and the upper surface (1032), feedback from the machine vision sensor (1038) can be used to determine when the air blade has removed residual immersion fluid (RF) from the objective lens assembly (2000) and / or the upper surface (1032). In some of these configurations, a controller (e.g., controller (114, 308)) can maintain the air blade active until feedback from the machine vision sensor (1038) indicates that the air blade has removed residual immersion fluid (RF) from the objective lens assembly (2000) and / or the upper surface (1032). In other configurations (e.g., where the machine vision sensor (1038) is omitted), the air blade can be activated for a predetermined duration without any machine vision feedback; it can then be deactivated after the predetermined duration has expired.

[0156] As noted above, the removal of residual submerged fluid (RF) from the objective lens assembly (2000) and / or the upper surface (1032) using air blades can be supplemented or replaced by removing such residual submerged fluid (RF) using absorption features and / or wiping away such residual submerged fluid (RF) using wiping features and / or any other suitable features.

[0157] After the residual immersion fluid (RF) has been removed from the objective lens assembly (2000) and / or the upper surface (1032), and as Figure 38G As shown, flow cell (1030) can be removed. Figure 37 (Box 3062). In some cases, there is no additional flow cell (1030) to scan, so the process can then be terminated ( Figure 37 (Box 3066). In some other cases, there are at least one more flow pool (1030) to scan. In such cases, the process can return to, for example... Figure 38A The activated air curtain shown Figure 37 (e.g., box 3050) Figure 38B The positioning of the next flow cell is shown. Figure 37 The starting steps of box 3052, etc., are looped through. Figure 37 and Figures 38A-38G The above steps are repeated until all flow cells (1030) have been scanned.

[0158] Although this example is Figure 37 and Figures 38A-38GThe entire process provides a fixed position for the air blade manifold (2060), but some variations are possible, in which the air blade manifold (2060) is in Figure 37 and Figures 38A-38G The movement occurs during at least a portion of the process. This movement of the air blade manifold (2060) can be provided by a mounting (1016), which may include one or more motors, servo systems, or other actuators, as noted above. In some of this type, the initial positioning of the flow pool (1030)... Figure 37 During the infusion step (frame 3052) Figure 37 During the scanning step (frame 3054) Figure 37 During the frame 3056) and in the de-infusion step ( Figure 37 During frame 3058, the air vane manifold (2060) can be positioned substantially away from the imaging assembly (1040) and the flow cell (1030). In some cases, positioning the air vane manifold (2060) substantially away from the imaging assembly (1040) and the flow cell (1030) allows the objective lens assembly (2000) to be closer to the upper surface (1032) of the flow cell (1030), which in turn can facilitate the use of a smaller volume of immersion fluid (IF). The air vane manifold (2060) can be moved to... Figure 38F The positioning shown is to provide activation of the air blades as described above. Figure 37 The box is 3060); then you can remove it ( Figure 37 The frame 3062) flow cell (1030) is moved back to an unobstructed position before or after it.

[0159] Some sequencing systems can perform a preliminary scan of the flow cell (1030) to map its tilt and morphology, thereby collecting calibration data for subsequent scans. In such preliminary scans, even if images are captured during the preliminary scan, these images may tend not to be used for collecting nucleotide sequencing data—only for calibration purposes. In some such systems, an air curtain can be continuously activated throughout the preliminary scan to provide initial cleaning of debris, etc., from the upper surface (1032) of the flow cell; during the execution Figure 37 and Figures 38A-38G Before the process, images are obtained that will be used for collecting nucleotides for sequencing, etc. In some of these preliminary scans, immersion fluids may also be used as described above.

[0160] G. Examples of Additional Immersion Microscope Features, Aspects, and Variations As noted above, the temperature of the immersion fluid can be adjusted, thereby adjusting the temperature of the objective lens element (2002) and / or the temperature of the flow cell (1030). For example, in some cases, the objective lens assembly (2000) can tend to reach an operating temperature that is significantly higher than the desired operating temperature in the flow cell (1030). In some such cases, the volume of immersion fluid (IF) between the objective lens element (2002) and the flow cell (1030) can tend to undesirably conduct this heat from the objective lens assembly (2000) to the flow cell (1030). In some cases, the heat transferred from the objective lens assembly (2000) to the flow cell (1030) can tend to adversely affect the nucleotide sequencing processes occurring in the flow cell (1030). In some other cases (e.g., where the desired operating temperature of the flow cell (1030) is higher than the desired operating temperature of the objective lens assembly (2000)), the combination of the objective lens assembly (2000) and the volume of immersion fluid (IF) between the objective lens element (2002) and the flow cell (1030) can tend to form a heat sink that undesirably draws heat away from the flow cell (1030), which can also adversely affect the nucleotide sequencing processes occurring in the flow cell (1030). Additionally or in the alternative, the heat transferred from the flow cell (1030) to the objective lens element (2002) via the immersion fluid can tend to induce thermal aberration / warping effects in the objective lens element (2002), which can adversely affect the quality of the images obtained through the objective lens element (2002).

[0161] By appropriately controlling the temperature of the immersion fluid before it reaches the space between the objective lens element (2002) and the flow cell (1030), the immersion fluid can mitigate the heat transfer that would otherwise occur between the objective lens assembly (2000) and the flow cell (1030). Additionally, in versions in which the immersion fluid is circulated within the space between the objective lens element (2002) and the flow cell (1030) during the scanning process, the circulation of the immersion fluid can further facilitate the use of the immersion fluid to mitigate the heat transfer that would otherwise occur between the objective lens assembly (2000) and the flow cell (1030). Some variations of the immersion fluid manifold (2040) can provide one or more temperature sensors in fluid communication with the immersion fluid. Such temperature sensors can be in electrical communication with a controller (e.g., the controller (114, 308)), and the controller can drive the heating and / or cooling elements to maintain the immersion fluid at a desired temperature; or within a desired temperature range.

[0162] In the examples provided above with reference to Figure 37 and Figures 38A-38G In the examples provided above with reference to Figure 37of the volume of immersion fluid (ID) from the space between the objective lens element (2002) and the upper surface (1032) of the flow cell (1030) by applying suction via the immersion fluid manifold (2040), as shown in FIG. 30B, block 3058. In some other variations, rather than providing this de-priming step in this manner, relative movement is provided along the x-y plane between the flow cell (1030) and the imaging assembly (1040) until the recess (2044) of the immersion fluid manifold (2040) passes over the edge of the flow cell (1030) and is no longer positioned over the upper surface (1032) of the flow cell (1030). In some cases, a substantial amount of immersion fluid can remain in place in the recess (2044). Such an amount of immersion fluid can remain in place due to the combination of surface tension and hydrophobic properties of the immersion fluid manifold (2040) and the objective lens element (2002), among others. To the extent that any residual immersion fluid is present on the upper surface (1032) of the flow cell (1030), such residual immersion fluid can then be removed using air blades from the air blade manifold (2060), using an absorbent member, using a wiping member, and / or using any other suitable structure or technique. The process can then proceed to removal of the flow cell (1030) (FIG. 30B, block 3062); and, if applicable, ultimately to positioning of the next flow cell (1030) (FIG. 30B, block 3052). At this stage, the substantial amount of immersion fluid remaining in place in the recess (2044) from the previous scanning process can effectively pre-prime the immersion fluid manifold (2040) during the priming step (FIG. 30B, block 3054) for the next flow cell (1030). If needed, additional immersion fluid can be delivered to the space between the objective lens element (2002) and the upper surface (1032) of the flow cell (1030) to complete the priming process, but the amount of such additional immersion fluid can be less than would otherwise be needed in the absence of the pre-priming provided by the substantial amount of immersion fluid remaining in place in the recess (2044) from the previous scanning process. The machine vision sensor (1038) can be used to detect the amount of immersion fluid; and provide feedback, informing of any supplementation of immersion fluid that can be needed in the space between the objective lens element (2002) and the upper surface (1032). Figure 38E Figure 37 Figure 37 Figure 37

[0163] ​​​​As another example, relative movement along the z-axis between the flow cell (1030) and the imaging assembly (1040) can be provided to further separate the objective lens assembly (2000) from the upper surface (1032) of the flow cell (1030). In some cases, a significant amount of immersion fluid can be retained in place in the recess (2044) due to the combination of surface tension and hydrophobic properties of the immersion fluid manifold (2040) and the objective lens element (2002), etc. To the extent that any residual immersion fluid is present on the upper surface (1032) of the flow cell (1030), such residual immersion fluid can then be removed using air blades from the air blade manifold (2060), by using an absorption member, by using a wiping member, and / or by using any other suitable structure or technique. The process can then proceed to the removal of the flow cell (1030). Figure 37 (box 3062); and if applicable, finally proceed to positioning the next flow pool (1030) ( Figure 37 (Box 3052). As noted above, the large amount of submerged fluid retained in place in the recess (2044) from the previous scanning process can be used in the infusion step for the next flow cell (1030). Figure 37 During the pre-filling of the submerged fluid manifold (2040) in frame 3054.

[0164] While the example described above utilizes a machine vision sensor (1038) to detect the amount of immersion fluid in the space between the objective lens assembly (2000) and the upper surface (1032) of the flow cell (1030), other features besides or instead of the machine vision sensor (1038) can be used to detect the amount of immersion fluid in the space between the objective lens assembly (2000) and the upper surface (1032) of the flow cell (1030). For example, an immersion fluid manifold (2040) may include electrodes exposed within a recess (2044) to contact the immersion fluid in the recess (2044). Such electrodes may be positioned at or near opposite ends of the long axis (A1) of the recess (2044). When the immersion fluid fills the recess (2044) to the point where the immersion fluid simultaneously contacts both electrodes, the immersion fluid can complete the circuit between the electrodes. In the type in which the electrodes form a capacitor, the electrodes can be covered in an additional insulator to prevent electrolysis by the immersion fluid, which could otherwise lead to contamination of the surfaces in contact with the immersion fluid.

[0165] The aforementioned example with two electrodes provides an indication of whether the immersion fluid level has reached a certain threshold (i.e., the point where the immersion fluid contacts both electrodes). In some other variations, more than two electrodes may be provided at different locations (e.g., along the edge (2046) surrounding the recess (2044) and / or elsewhere on the immersion fluid manifold (2040). Such multi-electrode variations can provide multi-point immersion fluid level measurements, thereby generating signals indicating different volumes of immersion fluid in the space between the objective lens assembly (2000) and the upper surface (1032) of the flow cell (1030). Regardless of the number of electrodes provided, in some cases it may be desirable to position one electrode between the first channel (2052) and the edge (2046), and another electrode between the second channel (2058) and the edge (2046). This positioning allows those electrodes to provide immersion fluid level sensing before the channels (2052, 2058) dry out, which may tend to undesirably introduce air bubbles into the immersion fluid.

[0166] As another example, force sensing can be used to measure the deformation of the submerged fluid (IF) relative to the upper surface (1032) of the flow cell (1030). When deformation occurs upon contact, an additional force can be applied in the z-direction to compensate for the deformation. In some types, the additional force is applied in the z-direction via a sample stage (1020). In some other types, the additional force is applied in the z-direction via a mounting (1042). In some cases, once the infusion step ( Figure 37 An immersion fluid is provided in box 3054, which can then exert a sudden increase in force on the imaging component (1040) along the z-axis. This initial force along the z-axis provides a baseline against which subsequent force data can be tracked. A decrease in the force induced by the immersion fluid below this baseline indicates a necessary reduction in the deformation of the immersion fluid (IF) volume. In other words, if the force exerted by the immersion fluid along the z-axis on the imaging component (1040) during scanning decreases below a certain threshold (…), the immersion fluid will induce a sudden increase in force. Figure 37 If the force is applied along the z-axis to the imaging assembly (1040) or the flow cell (1030) (in box 3056), an additional force can be applied to the imaging assembly (1040) or the flow cell (1030) until the force applied to the imaging assembly (1040) along the z-axis by the immersion fluid returns to the baseline.

[0167] As yet another example, a laser beam can pass through the objective lens element (2002), reflect off a sample in the flow cell (1030), and be passed back into the imaging assembly (1040) for image capture. The laser beam can intercept the surface at different positions along the z-dimension. The angle of the incident beam results in a lateral shift of the reflected beam as it passes back through the optical path, depending on the height of the surface intercept. The reflected beam is imaged as a spot on a sensor in the imaging assembly (1040), and different surfaces have spots that are laterally shifted on the sensor. In the case where the immersion fluid is disposed as a droplet on the objective lens element (2002) before the immersion fluid also contacts the upper surface (1032) of the flow cell (1030), the reflected spots can tend to form an odd pattern, as the first surface (the immersion fluid droplet) is curved, and subsequent surfaces will observe a beam focused by the curvature of the immersion fluid droplet. In any case, once the droplet contacts the upper surface (1032) of the flow cell (1030), the spot profile on the sensor will change significantly (to the expected number of spots). This threshold can be used to measure the height of the immersion fluid droplet.

[0168] As yet another example, a collimated beam can be output via the objective lens element (2002). The collimated beam will then be focused by a droplet of immersion fluid disposed on the objective lens element (2002) before the immersion fluid also contacts the upper surface (1032) of the flow cell (1030). When relative movement is provided along the z-dimension between the imaging assembly (1004) and the flow cell (1030), the focused collimated beam will be incident on the upper surface (1032) of the flow cell (1030), producing a cat's eye reflection (e.g., in a similar manner to a collimator), resulting in a spot on an image sensor in the imaging assembly (1004). The droplet of immersion fluid on the objective lens element (2002) can act as a lens, such that the characteristics of the cat's eye reflection can be indicative of the radius of curvature of the droplet of immersion fluid on the objective lens element (2002). Thus, as the radius of curvature of the droplet changes, the focal length of the lens effectively formed by the droplet will change. Similarly, when the droplet engages the upper surface (1032) of the flow cell (1030), the reflection, radius of curvature, and focal length associated with the droplet will change. The reflection profile before engagement between the droplet and the upper surface (1032) of the flow cell (1030) can provide a baseline indicative of the droplet height, such that a change in the reflection profile after engagement between the droplet and the upper surface (1032) of the flow cell (1030) can be indicative of a change in the droplet height.

[0169] VII. Embodiments of Combinations The following examples illustrate various non-exhaustive ways in which the teachings herein can be combined or applied. The following examples are not intended to limit the scope of any claims that may be provided at any time in this application or in subsequent filings thereof. No disclaimer is intended. The following examples are provided merely for illustrative purposes. It is contemplated that the various teachings herein may be arranged and applied in a variety of other ways. Some variations are also expected to omit certain features mentioned in the following examples. Therefore, none of the aspects or features mentioned below should be considered definitive unless otherwise expressly indicated, for example, by the inventor or a successor of the inventor of interest, at a later date. If any claim set forth in this application or in a subsequent filing related to this application includes additional features beyond those mentioned below, such additional features should not be presumed to have been added for any reason related to patentability.

[0170] Embodiment 1 An apparatus comprising: an imaging assembly including: an objective lens having a bottom surface; and an immersion fluid assembly including: a first port for introducing immersion fluid below the bottom surface of the objective lens; a second port for removing immersion fluid from below the bottom surface of the objective lens; and a sidewall defining an immersion fluid holding region below the objective lens having a first horizontal dimension and a second horizontal dimension orthogonal to the first horizontal dimension, the first horizontal dimension being greater than the second horizontal dimension; and an actuation assembly for driving relative movement between a flow cell and the imaging assembly along a horizontal path in a direction parallel to the first horizontal dimension.

[0171] Embodiment 2 According to the apparatus of embodiment 1, the apparatus further includes a flow cell having an upper surface below the fluid holding region, and the immersion fluid assembly is used to maintain immersion fluid between the bottom surface of the objective lens and the upper surface of the flow cell.

[0172] Embodiment 3 According to the device of Embodiment 2, the upper surface comprises a hydrophobic material.

[0173] Embodiment 4 According to any one of Embodiments 2 to 3, the flow cell further includes a plurality of channels, each of the plurality of channels including a plurality of reaction sites.

[0174] Example 5 The apparatus of embodiment 4, the plurality of reaction sites comprises a plurality of nucleotides.

[0175] Example 6 The apparatus of any one of embodiments 4-5, at least some of the plurality of channels are oriented along the first horizontal dimension.

[0176] Example 7 The apparatus of embodiment 6, each of the plurality of channels is spaced apart from another of the plurality of channels along the second horizontal dimension.

[0177] Example 8 The apparatus of any one of embodiments 4-7, the actuation assembly is to drive relative movement between the flow cell and the imaging assembly to position the imaging assembly successively over each of the plurality of channels.

[0178] Example 9 The apparatus of any one of embodiments 1-8, the bottom surface of the objective lens is flat.

[0179] Example 10 The apparatus of any one of embodiments 1-9, the bottom surface of the objective lens comprises a concave surface.

[0180] Example 11 The apparatus of any one of embodiments 1-10, further comprising an air curtain manifold to expel pressurized air outward relative to the objective lens.

[0181] Example 12 The apparatus of embodiment 11, the air curtain manifold is positioned to surround the objective lens.

[0182] Example 13 The apparatus of embodiment 12, the objective lens is centered along a longitudinal axis, the air curtain manifold is centered along the longitudinal axis.

[0183] Example 14 The apparatus of any one of embodiments 11-13, the air curtain manifold defines a central opening.

[0184] Example 15 The apparatus of embodiment 14, a portion of the imaging assembly is positioned within the central opening of the air curtain manifold.

[0185] Example 16 The apparatus of embodiment 15, the central opening is sized to define a gap between the air curtain manifold and the portion of the imaging assembly positioned within the central opening.

[0186] Example 17 The apparatus of any one of embodiments 11-16, the air curtain manifold has an annular shape with a circumferentially extending surface with openings positioned along the circumferentially extending surface, the air curtain manifold for expelling pressurized air outward relative to the objective lens via the openings positioned along the circumferentially extending surface.

[0187] Example 18 The apparatus of embodiment 17, the circumferentially extending surface is angled along a vertical plane.

[0188] Example 19 The apparatus of any one of embodiments 17-18, the air curtain manifold further comprises an annular channel in fluid communication with the openings positioned along the circumferentially extending surface.

[0189] Example 20 The apparatus of any one of embodiments 11-19, the apparatus further comprising a frame, the imaging assembly is fixed to the frame, the air curtain manifold is fixed to the frame independently of the imaging assembly.

[0190] Example 21 The apparatus of any one of embodiments 11-20, the air curtain manifold is for expelling pressurized air outward relative to the objective lens in the form of a frustoconical air curtain.

[0191] Example 22 The apparatus of embodiment 21, the frustoconical air curtain is centered along a longitudinal axis, the objective lens is centered along the longitudinal axis.

[0192] Example 23 The apparatus of any one of embodiments 11-22, the air curtain manifold is positioned to expel pressurized air downward toward the flow cell.

[0193] Example 24 The apparatus of any of embodiments 1-23, the imaging assembly further comprising an objective lens housing supporting the objective lens, the objective lens housing having a distal face that encompasses an outer periphery of the objective lens.

[0194] Example 25 The apparatus of embodiment 24, the distal face comprising a hydrophilic material.

[0195] Example 26 The apparatus of any of embodiments 24-25, the bottom surface of the objective lens comprising a hydrophilic material.

[0196] Example 27 The apparatus of any of embodiments 24-26, the distal face further comprising at least one annular recess spaced outwardly from the outer periphery of the objective lens.

[0197] Example 28 The apparatus of any of embodiments 1-27, the apparatus further comprising an air blade manifold for expelling pressurized air toward a space between the bottom surface of the objective lens and an upper surface of the flow cell.

[0198] Example 29 The apparatus of embodiment 28, the air blade manifold comprising a plurality of openings for expelling pressurized air toward the space between the bottom surface of the objective lens and an upper surface of the flow cell.

[0199] Example 30 The apparatus of embodiment 29, the plurality of openings being positioned along an arc.

[0200] Example 31 The apparatus of any of embodiments 29-30, the air blade manifold further comprising a plurality of channels in fluid communication with the plurality of openings.

[0201] Example 32 The apparatus of embodiment 31, each channel in fluid communication with the plurality of openings being oriented along a respective radius extending toward a center point, such that the plurality of openings all extend along the radii of their orientations toward the center point.

[0202] Example 33 The apparatus of any one of embodiments 32 to 35, wherein the center point is positioned to correspond to a center region of the space between the bottom surface of the objective lens and an upper surface of the flow cell.

[0203] Example 34 The apparatus of any one of embodiments 28 to 33, wherein the air blade manifold is to expel pressurized air in the form of a substantially flat air blade toward the space between the bottom surface of the objective lens and an upper surface of the flow cell.

[0204] Example 35 The apparatus of any one of embodiments 28 to 34, further comprising a frame to which the imaging assembly is fixed, the air blade manifold being fixed to the frame independently of the imaging assembly.

[0205] Example 36 The apparatus of any one of embodiments 28 to 35, further comprising an air curtain manifold to expel pressurized air outward relative to the objective lens.

[0206] Example 37 The apparatus of embodiment 36, further comprising a source of pressurized air to provide pressurized air to the air blade manifold, the source of pressurized air also to provide pressurized air to the air curtain manifold.

[0207] Example 38 The apparatus of embodiment 37, further comprising a valve to direct pressurized air to a selected one of the air blade manifold or the air curtain manifold.

[0208] Example 39 The apparatus of any one of embodiments 1 to 38, further comprising a sensor to monitor a presence of an immersion fluid below the bottom surface of the objective lens.

[0209] Example 40 The apparatus of embodiment 39, the sensor comprising an optical sensor.

[0210] Example 41 The apparatus of embodiment 40, the optical sensor being positioned and oriented to view the immersion fluid below the bottom surface of the objective lens along a horizontal dimension.

[0211] Example 42 The apparatus of any one of embodiments 40 to 42, wherein the optical sensor is positioned and oriented to view light transmitted through the immersion fluid and reflected back through the objective lens.

[0212] Example 43 The apparatus of any one of embodiments 39 to 42, wherein the sensor comprises a set of electrodes.

[0213] Example 44 The apparatus of any one of embodiments 39 to 43, wherein the sensor comprises a force sensor.

[0214] Example 45 An apparatus comprising: an imaging assembly comprising: an objective lens assembly comprising: an objective lens element having a bottom surface; and a housing having a bottom surface adjacent to the bottom surface of the objective lens element, the bottom surface of the housing comprising a fluid retention feature; and an immersion fluid assembly comprising: a first port for introducing immersion fluid beneath the objective lens assembly; a second port for removing immersion fluid from beneath the objective lens assembly; and a sidewall defining an immersion fluid retention region beneath the objective lens assembly; the fluid retention feature of the housing of the objective lens assembly being interposed laterally between the objective lens element of the objective lens assembly and the sidewall of the immersion fluid assembly.

[0215] Example 46 The apparatus of embodiment 45, wherein the fluid retention feature comprises an annular recess surrounding the objective lens element.

[0216] Example 47 The apparatus of embodiment 46, wherein the fluid retention feature is positioned on the bottom surface of the housing.

[0217] Example 48 The apparatus of any one of embodiments 45 to 47, wherein the bottom surface of the housing comprises a hydrophilic material.

[0218] Example 49 The apparatus of any one of embodiments 45 to 48, wherein the bottom surface of the objective lens element comprises a hydrophilic material.

[0219] Example 50 The apparatus of any one of embodiments 45-49, the immersion fluid retention region has a first horizontal dimension and a second horizontal dimension orthogonal to the first horizontal dimension, the first horizontal dimension being greater than the second horizontal dimension.

[0220] Example 51 The apparatus of embodiment 50, further comprising an actuation assembly for driving relative movement between a flow cell and the imaging assembly along a path.

[0221] Example 52 The apparatus of embodiment 51, the path comprises a horizontal path in a direction parallel to the first horizontal dimension.

[0222] Example 53 The apparatus of any one of embodiments 45-52, further comprising a flow cell having an upper surface below the fluid retention region, the immersion fluid assembly for maintaining immersion fluid between the bottom surface of the objective lens and the upper surface of the flow cell.

[0223] Example 54 The apparatus of embodiment 53, the upper surface comprises a hydrophobic material.

[0224] Example 55 The apparatus of any one of embodiments 53-54, the flow cell further comprising a plurality of channels, each channel of the plurality of channels comprising a plurality of reaction sites.

[0225] Example 56 The apparatus of embodiment 55, the plurality of reaction sites comprises a plurality of nucleotides.

[0226] Example 57 The apparatus of any one of embodiments 55-56, the immersion fluid retention region has a first horizontal dimension and a second horizontal dimension orthogonal to the first horizontal dimension, the first horizontal dimension being greater than the second horizontal dimension, at least some channels of the plurality of channels are oriented along the first horizontal dimension.

[0227] Example 58 The apparatus of embodiment 57, each channel of the plurality of channels is spaced apart from another channel of the plurality of channels along the second horizontal dimension.

[0228] Example 59 The apparatus of any one of embodiments 55-58, further comprising an actuation assembly for driving relative movement between the flow cell and the imaging assembly to position the imaging assembly successively over each of the plurality of channels.

[0229] Example 60 The apparatus of any one of embodiments 45-59, the bottom surface of the objective lens element is flat.

[0230] Example 61 The apparatus of any one of embodiments 45-60, the bottom surface of the objective lens element comprises a concave surface.

[0231] Example 62 The apparatus of any one of embodiments 45-61, further comprising an air curtain manifold for expelling pressurized air outward relative to the objective lens element.

[0232] Example 63 The apparatus of embodiment 62, the air curtain manifold is positioned to surround the objective lens element.

[0233] Example 64 The apparatus of embodiment 63, the objective lens element is centered along a longitudinal axis, the air curtain manifold is centered along the longitudinal axis.

[0234] Example 65 The apparatus of any one of embodiments 62-64, the air curtain manifold defines a central opening.

[0235] Example 66 The apparatus of embodiment 65, a portion of the imaging assembly is positioned within the central opening of the air curtain manifold.

[0236] Example 67 The apparatus of embodiment 66, the central opening is sized to define a gap between the air curtain manifold and the portion of the imaging assembly positioned within the central opening.

[0237] Example 68 The apparatus of any one of embodiments 62-67, the air curtain manifold having a ring shape with a circumferentially extending surface with openings positioned along the circumferentially extending surface, the air curtain manifold for expelling pressurized air outward relative to the objective lens via the openings positioned along the circumferentially extending surface.

[0238] Example 69 The apparatus of embodiment 68, the circumferentially extending surface is angled along a vertical plane.

[0239] Example 70 The apparatus of any one of embodiments 68-69, the air curtain manifold further comprising an annular channel in fluid communication with the openings positioned along the circumferentially extending surface.

[0240] Example 71 The apparatus of any one of embodiments 62-70, the apparatus further comprising a frame to which the imaging assembly is fixed, the air curtain manifold being fixed to the frame independently of the imaging assembly.

[0241] Example 72 The apparatus of any one of embodiments 62-71, the air curtain manifold for expelling pressurized air outward relative to the objective lens element in the form of a frustoconical air curtain.

[0242] Example 73 The apparatus of embodiment 72, the frustoconical air curtain being centered along a longitudinal axis along which the objective lens element is centered.

[0243] Example 74 The apparatus of any one of embodiments 62-73, the apparatus further comprising an actuation assembly for driving relative movement along a path between a flow cell and the imaging assembly, the air curtain manifold being positioned to expel pressurized air downward toward the flow cell.

[0244] Example 75 The apparatus of any one of embodiments 45-74, the apparatus further comprising an air vane manifold for expelling pressurized air toward a space between the bottom surface of the objective lens element and an upper surface of the flow cell.

[0245] Example 76 The apparatus of embodiment 75, the air vane manifold comprising a plurality of openings for expelling pressurized air toward the space between the bottom surface of the objective lens element and an upper surface of the flow cell.

[0246] Example 77 The apparatus of embodiment 76, the plurality of openings being positioned along an arc.

[0247] Example 78 The apparatus of any one of embodiments 76-77, the air vane manifold further comprising a plurality of channels in fluid communication with the plurality of openings.

[0248] Example 79 The apparatus of embodiment 78, each channel in fluid communication with the plurality of openings being oriented along a respective radius extending toward a center point, such that the plurality of openings all extend along the radius of their orientation toward the center point.

[0249] Example 80 The apparatus of embodiment 79, the center point being positioned to correspond with a central region of the space between the bottom surface of the objective lens element and an upper surface of the flow cell.

[0250] Example 81 The apparatus of any one of embodiments 75-80, the air vane manifold for expelling pressurized air toward the space between the bottom surface of the objective lens element and an upper surface of the flow cell in the form of a substantially flat air vane.

[0251] Example 82 The apparatus of any one of embodiments 75-81, the apparatus further comprising a frame to which the imaging assembly is fixed, the air vane manifold being fixed to the frame independently of the imaging assembly.

[0252] Example 83 The apparatus of any one of embodiments 75-82, the apparatus further comprising an air curtain manifold for expelling pressurized air outward relative to the objective lens element.

[0253] Example 84 The apparatus of embodiment 83, the apparatus further comprising a source of pressurized air for providing pressurized air to the air vane manifold, the source of pressurized air also for providing pressurized air to the air curtain manifold.

[0254] Example 85 The apparatus of example 84, further comprising a valve for directing pressurized air to a selected one of the air vane manifold or the air curtain manifold.

[0255] Example 86 The apparatus of any of examples 45-85, further comprising a sensor for monitoring a presence of immersion fluid below the bottom surface of the objective lens element.

[0256] Example 87 The apparatus of example 86, the sensor comprising an optical sensor.

[0257] Example 88 The apparatus of example 87, the optical sensor positioned and oriented to view the immersion fluid below the bottom surface of the objective lens element along a horizontal dimension.

[0258] Example 89 The apparatus of example 88, the optical sensor positioned and oriented to view light transmitted through the immersion fluid and reflected back through the objective lens element.

[0259] Example 90 The apparatus of any of examples 86-89, the sensor comprising a set of electrodes.

[0260] Example 91 The apparatus of any of examples 86-90, the sensor comprising a force sensor.

[0261] Example 92 An apparatus comprising: an imaging assembly comprising: an objective lens having a bottom surface; an immersion fluid assembly comprising: a first port for introducing immersion fluid beneath the bottom surface of the objective lens; a second port for removing immersion fluid from beneath the bottom surface of the objective lens; and a sidewall defining an immersion fluid retention region beneath the objective lens; and an air delivery assembly coupled with the objective lens assembly for delivering pressurized air into one or both of the objective lens or an upper surface of a flow cell, thereby dislodging one or both of debris or immersion fluid relative to one or both of the objective lens or an upper surface of a flow cell.

[0262] Example 93 The apparatus of embodiment 92, the immersion fluid retention region having a first horizontal dimension and a second horizontal dimension orthogonal to the first horizontal dimension, the first horizontal dimension being greater than the second horizontal dimension.

[0263] Example 94 The apparatus of embodiment 93, further comprising an actuation assembly for driving relative movement between a flow cell and the imaging assembly along a horizontal path in a direction parallel to the first horizontal dimension.

[0264] Example 95 The apparatus of any one of embodiments 92-94, further comprising a flow cell having an upper surface beneath the fluid retention region, the immersion fluid assembly for maintaining immersion fluid between the bottom surface of the objective lens and the upper surface of the flow cell.

[0265] Example 96 The apparatus of embodiment 95, the upper surface comprising a hydrophobic material.

[0266] Example 97 The apparatus of any one of embodiments 95-96, the flow cell further comprising a plurality of channels, each channel of the plurality of channels comprising a plurality of reaction sites.

[0267] Example 98 The apparatus of embodiment 97, the plurality of reaction sites comprising a plurality of nucleotides.

[0268] Example 99 The apparatus of any one of embodiments 97-98, at least some of the plurality of channels are oriented along the first horizontal dimension.

[0269] Example 100 The apparatus of embodiment 99, each of the plurality of channels is spaced apart from another of the plurality of channels along the second horizontal dimension.

[0270] Example 101 The apparatus of any one of embodiments 97-100, the actuation assembly is to drive relative movement between the flow cell and the imaging assembly to position the imaging assembly successively over each of the plurality of channels.

[0271] Example 102 The apparatus of any one of embodiments 92-101, the bottom surface of the objective lens is flat.

[0272] Example 103 The apparatus of any one of embodiments 92-102, the bottom surface of the objective lens comprises a concave surface.

[0273] Example 104 The apparatus of any one of embodiments 92-103, the air delivery assembly comprises an air curtain manifold to expel pressurized air outward relative to the objective lens.

[0274] Example 105 The apparatus of embodiment 104, the air curtain manifold is positioned to surround the objective lens.

[0275] Example 106 The apparatus of embodiment 105, the objective lens is centered along a longitudinal axis, the air curtain manifold is centered along the longitudinal axis.

[0276] Example 107 The apparatus of any one of embodiments 104-20+, the air curtain manifold defines a central opening.

[0277] Example 108 The apparatus of embodiment 107, a portion of the imaging assembly is positioned within the central opening of the air curtain manifold.

[0278] Example 109 The apparatus of embodiment 108, a size of the central opening is set to define a gap between the air curtain manifold and the portion of the imaging assembly positioned within the central opening.

[0279] Example 110 The apparatus of any of embodiments 104-109, the air curtain manifold has an annular shape with a circumferentially extending surface with openings positioned along the circumferentially extending surface, the air curtain manifold for expelling pressurized air outward relative to the objective lens via the openings positioned along the circumferentially extending surface.

[0280] Example 111 The apparatus of embodiment 110, the circumferentially extending surface is angled along a vertical plane.

[0281] Example 112 The apparatus of any of embodiments 110-111, the air curtain manifold further comprises an annular channel in fluid communication with the openings positioned along the circumferentially extending surface.

[0282] Example 113 The apparatus of any of embodiments 104-112, the apparatus further comprising a frame to which the imaging assembly is fixed, the air curtain manifold being fixed to the frame independently of the imaging assembly.

[0283] Example 114 The apparatus of any of embodiments 104-113, the air curtain manifold for expelling pressurized air outward relative to the objective lens in the form of a frustoconical air curtain.

[0284] Example 115 The apparatus of embodiment 114, the frustoconical air curtain is centered along a longitudinal axis along which the objective lens is centered.

[0285] Example 116 The apparatus of any of embodiments 104-115, the air curtain manifold is positioned to expel pressurized air downward toward the flow cell.

[0286] Example 117 The apparatus of any of embodiments 92-116, the imaging assembly further comprising an objective lens housing supporting the objective lens, the objective lens housing having a distal face that encircles an outer periphery of the objective lens.

[0287] Example 118 The distal face comprises a hydrophilic material, according to the apparatus of embodiment 117.

[0288] Example 119 The bottom surface of the objective lens comprises a hydrophilic material, according to the apparatus of any one of embodiments 117-118.

[0289] Example 120 The distal face further comprises at least one annular recess spaced outwardly from the outer perimeter of the objective lens, according to the apparatus of any one of embodiments 117-119.

[0290] Example 121 The air delivery assembly comprises an air blade manifold for expelling pressurized air toward a space between the bottom surface of the objective lens and an upper surface of the flow cell, according to the apparatus of any one of embodiments 92-120.

[0291] Example 122 The air blade manifold comprises a plurality of openings for expelling pressurized air toward the space between the bottom surface of the objective lens and an upper surface of the flow cell, according to the apparatus of embodiment 121.

[0292] Example 123 The plurality of openings are positioned along an arc, according to the apparatus of embodiment 122.

[0293] Example 124 The air blade manifold further comprises a plurality of channels in fluid communication with the plurality of openings, according to the apparatus of any one of embodiments 122-123.

[0294] Example 125 Each channel in fluid communication with the plurality of openings is oriented along a respective radius extending toward a center point, such that the plurality of openings all extend along the radii of their orientations toward the center point, according to the apparatus of embodiment 124.

[0295] Example 126 The center point is positioned to correspond to a central region of the space between the bottom surface of the objective lens and an upper surface of the flow cell, according to the apparatus of embodiment 125.

[0296] Example 127 The apparatus of any one of embodiments 121-126, the air blade manifold to expel pressurized air in the form of a substantially flat air blade toward the space between the bottom surface of the objective lens and an upper surface of the flow cell.

[0297] Example 128 The apparatus of any one of embodiments 121-127, further comprising a frame to which the imaging assembly is fixed, the air blade manifold being fixed to the frame independently of the imaging assembly.

[0298] Example 129 The apparatus of any one of embodiments 121-128, further comprising an air curtain manifold to expel pressurized air outward relative to the objective lens.

[0299] Example 130 The apparatus of embodiment 129, further comprising a source of pressurized air to provide pressurized air to the air blade manifold, the source of pressurized air also to provide pressurized air to the air curtain manifold.

[0300] Example 131 The apparatus of embodiment 130, further comprising a valve to direct pressurized air to a selected one of the air blade manifold or the air curtain manifold.

[0301] Example 132 The apparatus of any one of embodiments 92-131, further comprising a sensor to monitor for presence of immersion fluid below the bottom surface of the objective lens.

[0302] Example 133 The apparatus of embodiment 132, the sensor comprising an optical sensor.

[0303] Example 133 The apparatus of embodiment 133, the optical sensor positioned and oriented to view the immersion fluid below the bottom surface of the objective lens along a horizontal dimension.

[0304] Example 134 The apparatus of embodiment 133, the optical sensor positioned and oriented to view light transmitted through the immersion fluid and reflected back through the objective lens.

[0305] Example 135 The apparatus of any one of embodiments 132-134, the sensor comprising a set of electrodes.

[0306] Example 136 The apparatus of any one of embodiments 132-135, the sensor comprising a force sensor.

[0307] Example 137 A method comprising: positioning an imaging assembly relative to a flow cell, the imaging assembly comprising: an objective lens having a bottom surface; and an immersion fluid assembly comprising: a first port; and a sidewall defining an immersion fluid retention region below the objective lens, the immersion fluid retention region having a first horizontal dimension and a second horizontal dimension orthogonal to the first horizontal dimension, the first horizontal dimension being greater than the second horizontal dimension; depositing, via the first port, an immersion fluid to a space between the bottom surface of the objective lens and an upper surface of the flow cell; and providing relative movement between the flow cell and the imaging assembly along a horizontal path in a direction parallel to the first horizontal dimension, the immersion fluid remaining in the space between the bottom surface of the objective lens and the upper surface of the flow cell during the relative movement between the flow cell and the imaging assembly.

[0308] Example 138 The method of embodiment 137, further comprising activating an air curtain comprising pressurized air oriented toward the upper surface of the flow cell and away from the objective lens.

[0309] Example 139 The method of embodiment 138, the act of activating the air curtain being performed prior to the act of depositing immersion fluid.

[0310] Example 140 The method of any one of embodiments 138-139, the act of activating the air curtain being performed concurrently with the providing of relative movement between the flow cell and the imaging assembly.

[0311] Example 141 The method of any one of embodiments 138-140, the air curtain having a frustoconical shape.

[0312] Example 142 The method of any of embodiments 138-141, the air curtain is provided via an air curtain manifold positioned about the imaging assembly.

[0313] Example 143 The method of any of embodiments 137-142, further comprising activating an air blade comprising pressurized air oriented toward the space between the bottom surface of the objective lens and an upper surface of the flow cell.

[0314] Example 144 The method of embodiment 143, the act of activating the air blade is performed after providing relative movement between the flow cell and the imaging assembly.

[0315] Example 145 The method of any of embodiments 143-144, further comprising aspirating the immersion fluid from the space between the bottom surface of the objective lens and the upper surface of the flow cell.

[0316] Example 146 The method of embodiment 145, the immersion fluid is aspirated from the space between the bottom surface of the objective lens and the upper surface of the flow cell via a second port of the immersion fluid assembly.

[0317] Example 147 The method of any of embodiments 145-146, the act of activating the air blade is performed after aspirating the immersion fluid from the space between the bottom surface of the objective lens and the upper surface of the flow cell.

[0318] Example 148 The method of any of embodiments 145-147, the air blade is provided via an air blade manifold positioned adjacent to the imaging assembly.

[0319] Example 149 The method of embodiment 148, the air blade manifold has a plurality of openings positioned along an arc, the air blade comprising pressurized air expelled via the plurality of openings such that an air flow of the air blade is focused toward a central region positioned in the space between the bottom surface of the objective lens and the upper surface of the flow cell.

[0320] Example 150 The method of any one of embodiments 137-149, further comprising monitoring, via a sensor, a presence of immersion fluid in the space between the bottom surface of the objective lens and the upper surface of the flow cell.

[0321] Example 151 The method of embodiment 150, the sensor comprising an optical sensor, the act of monitoring comprising obtaining optical data along a horizontal path.

[0322] Example 152 The method of any one of embodiments 150-151, the sensor comprising an optical sensor, the act of monitoring comprising obtaining optical data from light transmitted through the objective lens and toward the optical sensor.

[0323] Example 153 The method of any one of embodiments 150-152, the sensor comprising an electrode, the act of monitoring comprising monitoring whether an electrical circuit between the electrodes is completed.

[0324] Example 154 The method of any one of embodiments 150-153, the sensor comprising a force sensor, the act of monitoring comprising monitoring a force associated with the immersion fluid.

[0325] Example 155 The method of any one of embodiments 137-154, further comprising: removing the flow cell from a space below the imaging assembly; and positioning another flow cell in the space below the imaging assembly.

[0326] Example 156 The method of any one of embodiments 137-155, further comprising performing nucleotide sequencing in the flow cell.

[0327] Example 157 A method comprising: positioning an imaging assembly relative to a flow cell, the imaging assembly comprising: an objective lens having a bottom surface; and an immersion fluid assembly comprising: a first port; and a sidewall defining an immersion fluid retention region below the objective lens; depositing, via the first port, immersion fluid to a space between the bottom surface of the objective lens and an upper surface of the flow cell; providing relative movement between the flow cell and the imaging assembly along a horizontal path in a direction parallel to the first horizontal dimension, the immersion fluid remaining in the space between the bottom surface of the objective lens and the upper surface of the flow cell during the relative movement between the flow cell and the imaging assembly; and delivering pressurized air to one or both of the objective lens or the upper surface of the flow cell.

[0328] Example 158 The method of embodiment 157, the immersion fluid retention region has a first horizontal dimension and a second horizontal dimension orthogonal to the first horizontal dimension, the first horizontal dimension being greater than the second horizontal dimension.

[0329] Example 159 The method of embodiment 158, the horizontal path is in a direction parallel to the first horizontal dimension.

[0330] Example 160 The method of any one of embodiments 157-159, the act of delivering pressurized air to one or both of the objective lens or the upper surface of the flow cell comprises activating an air curtain comprising pressurized air oriented toward the upper surface of the flow cell and away from the objective lens.

[0331] Example 161 The method of embodiment 160, the act of activating the air curtain is performed prior to the act of depositing immersion fluid.

[0332] Example 162 The method of any one of embodiments 160-161, the act of activating the air curtain is performed concurrently with the providing of relative movement between the flow cell and the imaging assembly.

[0333] Example 163 The method of any one of embodiments 160-162, the air curtain has a frustoconical shape.

[0334] Example 164 The method of any of embodiments 160-163, the air curtain is provided via an air curtain manifold positioned about the imaging assembly.

[0335] Example 165 The method of any of embodiments 157-164, the act of delivering pressurized air into one or both of the objective lens or the upper surface of the flow cell comprises activating an air blade comprising pressurized air oriented toward the space between the bottom surface of the objective lens and the upper surface of the flow cell.

[0336] Example 166 The method of embodiment 165, the act of activating the air blade is performed after providing relative movement between the flow cell and the imaging assembly.

[0337] Example 167 The method of any of embodiments 165-166, further comprising aspirating the immersion fluid from the space between the bottom surface of the objective lens and the upper surface of the flow cell.

[0338] Example 168 The method of embodiment 167, the immersion fluid is aspirated from the space between the bottom surface of the objective lens and the upper surface of the flow cell via a second port of the immersion fluid assembly.

[0339] Example 169 The method of any of embodiments 167-168, the act of activating the air blade is performed after aspirating the immersion fluid from the space between the bottom surface of the objective lens and the upper surface of the flow cell.

[0340] Example 170 The method of any of embodiments 167-169, the air blade is provided via an air blade manifold positioned adjacent to the imaging assembly.

[0341] Example 171 The method of embodiment 170, the air blade manifold has a plurality of openings positioned along an arc, the air blade comprising pressurized air expelled via the plurality of openings such that an air flow of the air blade is focused toward a central region positioned in the space between the bottom surface of the objective lens and the upper surface of the flow cell.

[0342] Example 172 The method of any one of embodiments 157-171, further comprising monitoring, via the sensor, a presence of immersion fluid in the space between the bottom surface of the objective lens and the upper surface of the flow cell.

[0343] Example 173 The method of embodiment 172, the sensor comprising an optical sensor, the act of monitoring comprising obtaining optical data along a horizontal path.

[0344] Example 174 The method of any one of embodiments 172-173, the sensor comprising an optical sensor, the act of monitoring comprising obtaining optical data from light transmitted through the objective lens and toward the optical sensor.

[0345] Example 175 The method of any one of embodiments 172-174, the sensor comprising an electrode, the act of monitoring comprising monitoring whether an electrical circuit between the electrodes is completed.

[0346] Example 176 The method of any one of embodiments 172-175, the sensor comprising a force sensor, the act of monitoring comprising monitoring a force associated with the immersion fluid.

[0347] Example 177 The method of any one of embodiments 157-176, further comprising: removing the flow cell from a space below the imaging assembly; and positioning another flow cell in the space below the imaging assembly.

[0348] Example 178 The method of any one of embodiments 157-177, further comprising performing nucleotide sequencing in the flow cell.

[0349] VIII. Miscellaneous While the foregoing examples are provided in the context of a system (100) that can be used for a nucleotide sequencing process, the teachings herein can also be readily applied to other contexts, including systems that perform other processes (i.e., other than nucleotide sequencing processes). Thus, the teachings herein are not necessarily limited to systems used to perform nucleotide sequencing processes.

[0350] It should be understood that the subject matter described herein is not limited in its application to the details of construction and the arrangements of components set forth in the description herein or illustrated in the drawings herein. The subject matter described herein is capable of other embodiments and of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. As used herein, an element or step recited in the singular and preceded by the word "a" or "an" should be understood as not excluding plural elements or steps, unless such exclusion is explicitly recited. Furthermore, references to "one example" are not intended to be interpreted as excluding the existence of additional examples that also incorporate the recited features. As used herein, "including," "containing" or "having" and variations thereof mean encompassing without limitation.

[0351] When used in the claims, the term "set" should be understood to mean one or more things grouped together. Similarly, when used in the claims, "based on" should be understood as indicating that a thing is determined at least in part on the thing specified as being "based on." Where a thing is required to be determined exclusively by another thing, then the thing will be referred to as "exclusively based on" the other thing from which it is determined.

[0352] Unless otherwise specified or limited, the terms "mount," "connect," "support," and "couple" and variations thereof are used broadly and encompass both direct and indirect mount, connection, support, and coupling. Further, "connected" and "coupled" are not restricted to physical or mechanical connections or relationships. In addition, it is to be understood that the phraseology and terminology used herein, such as, for example, "above," "below," "front," "back," "side," "top," "bottom," and the like, are only used for descriptive purposes and not meant to be limiting. Further, the terms "external" and "internal" are used herein for descriptive purposes and not meant to indicate or imply relative importance or significance.

[0353] It should be understood that the foregoing description is intended to be illustrative and not limiting. For example, the above examples (and / or aspects thereof) can be used in combination with each other. Furthermore, numerous modifications as become apparent to those skilled in the art once informed by the above description will be made in order to adapt a particular situation or material to the teachings of the subject matter presented herein. While the dimensions, types of materials and coatings described herein are intended to define parameters of the disclosed subject matter, they are by no means limiting but rather illustrative. Many additional examples will be apparent to those of ordinary skill in the art in view of the foregoing description. Accordingly, the scope of the presently disclosed subject matter should be determined by reference to the appended claims and the full scope of equivalents to which those claims are entitled. In the appended claims, the terms "comprise" and "comprising" are used as open-ended terms that are synonymous with the corresponding terms "include" and "including," respectively. Furthermore, in the following claims, the terms "first," "second," and "third," etc. are used merely as labels, and are not intended to impose numerical requirements on their objects. Furthermore, the limitations of the following claims are not written in means-plus-function format and are not intended to be interpreted based on 35 U.S.C. § 112(f) paragraph six, unless and until such claim limitations expressly use the phrase "means for" followed by a statement of function devoid of further structure.

[0354] The following claims set forth certain aspects of examples of the disclosed subject matter and are considered a part of the disclosure. These aspects can be combined with each other.

Claims

1. An apparatus, the apparatus comprising: an imaging assembly, the imaging assembly comprising: an objective lens having a bottom surface, and an immersion fluid assembly, the immersion fluid assembly comprising: a first port for introducing immersion fluid beneath the bottom surface of the objective lens, a second port for removing immersion fluid from beneath the bottom surface of the objective lens, and a sidewall defining an immersion fluid retention region beneath the objective lens, the immersion fluid retention region having a first horizontal dimension and a second horizontal dimension orthogonal to the first horizontal dimension, the first horizontal dimension being greater than the second horizontal dimension; and an actuation assembly for driving relative movement between a flow cell and the imaging assembly along a horizontal path in a direction parallel to the first horizontal dimension.

2. The apparatus of claim 1, further comprising a flow cell having an upper surface beneath the fluid retention region, the immersion fluid assembly for maintaining immersion fluid between the bottom surface of the objective lens and the upper surface of the flow cell.

3. The apparatus of claim 2, the upper surface comprising a hydrophobic material.

4. The apparatus of any one of claims 2-3, the flow cell further comprising a plurality of channels, each channel of the plurality of channels comprising a plurality of reaction sites.

5. The apparatus of claim 4, the plurality of reaction sites comprising a plurality of nucleotides.

6. The apparatus of any one of claims 4-5, at least some channels of the plurality of channels oriented along the first horizontal dimension.

7. The apparatus of claim 6, each channel of the plurality of channels spaced apart from another channel of the plurality of channels along the second horizontal dimension.

8. The apparatus of any one of claims 4-7, the actuation assembly for driving relative movement between the flow cell and the imaging assembly to position the imaging assembly successively over each channel of the plurality of channels.

9. The apparatus of any one of claims 1-8, the bottom surface of the objective lens being planar.

10. The apparatus of any one of claims 1-9, the bottom surface of the objective lens comprising a concave surface.

11. The apparatus of any one of claims 1-10, further comprising an air curtain manifold for expelling pressurized air outward relative to the objective lens.

12. The apparatus of claim 11, the air curtain manifold positioned to encircle the objective lens.

13. The apparatus of claim 12, the objective lens centered along a longitudinal axis, the air curtain manifold centered along the longitudinal axis.

14. The apparatus of any one of claims 11-13, the air curtain manifold defining a central opening.

15. The apparatus of claim 14, a portion of the imaging assembly positioned within the central opening of the air curtain manifold.

16. The apparatus of claim 15, the central opening sized to define a gap between the air curtain manifold and the portion of the imaging assembly positioned within the central opening.

17. The apparatus of any one of claims 11 to 16, the air curtain manifold having an annular shape with a circumferentially extending surface with openings positioned along the circumferentially extending surface, the air curtain manifold for expelling pressurized air outward relative to the objective lens via the openings positioned along the circumferentially extending surface.

18. The apparatus of claim 17, the circumferentially extending surface angled along a vertical plane.

19. The apparatus of any one of claims 17 to 18, the air curtain manifold further comprising an annular channel in fluid communication with the openings positioned along the circumferentially extending surface.

20. The apparatus of any one of claims 11 to 19, further comprising a frame to which the imaging assembly is fixed, the air curtain manifold being fixed to the frame independently of the imaging assembly.

21. The apparatus of any one of claims 11 to 20, the air curtain manifold for expelling pressurized air outward relative to the objective lens in the form of a frustoconical air curtain.

22. The apparatus of claim 21, the frustoconical air curtain centered along a longitudinal axis along which the objective lens is centered.

23. The apparatus of any one of claims 11 to 22, the air curtain manifold positioned to expel pressurized air downward toward the flow cell.

24. The apparatus of any one of claims 1 to 23, the imaging assembly further comprising an objective lens housing supporting the objective lens, the objective lens housing having a distal face encompassing an outer periphery of the objective lens.

25. The apparatus of claim 24, the distal face comprising a hydrophilic material.

26. The apparatus of any one of claims 24 to 25, the bottom surface of the objective lens comprising a hydrophilic material.

27. The apparatus of any one of claims 24 to 26, the distal face further comprising at least one annular recess spaced outward from the outer periphery of the objective lens.

28. The apparatus of any one of claims 1 to 27, further comprising an air blade manifold for expelling pressurized air toward a space between the bottom surface of the objective lens and an upper surface of the flow cell.

29. The apparatus of claim 28, the air blade manifold comprising a plurality of openings for expelling pressurized air toward the space between the bottom surface of the objective lens and an upper surface of the flow cell.

30. The apparatus of claim 29, the plurality of openings positioned along an arc.

31. The apparatus of any one of claims 29 to 30, the air blade manifold further comprising a plurality of channels in fluid communication with the plurality of openings.

32. The apparatus of claim 31, each channel in fluid communication with the plurality of openings being oriented along a respective radius extending toward a center point, such that the plurality of openings all extend along their oriented radiuses toward the center point.

33. The apparatus of claim 32, the center point being positioned to correspond with a central region of the space between the bottom surface of the objective lens and an upper surface of the flow cell.

34. The apparatus of any one of claims 28 to 33, the air blade manifold to expel pressurized air in the form of a substantially flat air blade toward the space between the bottom surface of the objective lens and an upper surface of the flow cell.

35. The apparatus of any one of claims 28 to 34, further comprising a frame to which the imaging assembly is fixed, the air blade manifold being fixed to the frame independently of the imaging assembly.

36. The apparatus of any one of claims 28 to 35, further comprising an air curtain manifold to expel pressurized air outward relative to the objective lens.

37. The apparatus of claim 36, further comprising a pressurized air source to provide pressurized air to the air blade manifold, the pressurized air source further to provide pressurized air to the air curtain manifold.

38. The apparatus of claim 37, further comprising a valve to direct pressurized air to a selected one of the air blade manifold or the air curtain manifold.

39. The apparatus of any one of claims 1 to 38, further comprising a sensor to monitor for the presence of immersion fluid below the bottom surface of the objective lens.

40. The apparatus of claim 39, the sensor comprising an optical sensor.

41. The apparatus of claim 40, the optical sensor being positioned and oriented to view the immersion fluid below the bottom surface of the objective lens along a horizontal dimension.

42. The apparatus of claim 40, the optical sensor being positioned and oriented to view light transmitted through the immersion fluid and reflected back through the objective lens.

43. The apparatus of any one of claims 39 to 42, the sensor comprising a set of electrodes.

44. The apparatus of any one of claims 39 to 43, the sensor comprising a force sensor.

45. An apparatus comprising: an imaging assembly comprising: an objective lens assembly comprising: an objective lens element having a bottom surface, and a housing having a bottom surface adjacent to the bottom surface of the objective lens element, the bottom surface of the housing comprising a fluid retention feature, and an immersion fluid assembly comprising: a first port to introduce immersion fluid below the objective lens assembly, a second port to remove immersion fluid from below the objective lens assembly, and a third port to introduce immersion fluid below the objective lens assembly. a side wall defining an immersion fluid retention region below the objective lens assembly; the fluid retention feature of the housing of the objective lens assembly is laterally interposed between the objective lens element of the objective lens assembly and the side wall of the immersion fluid assembly.

46. The apparatus of claim 45, the fluid retention feature comprising an annular recess surrounding the objective lens element.

47. The apparatus of claim 46, the fluid retention feature positioned on the bottom surface of the housing.

48. The apparatus of any one of claims 45-47, the bottom surface of the housing comprising a hydrophilic material.

49. The apparatus of any one of claims 45-48, the bottom surface of the objective lens element comprising a hydrophilic material.

50. The apparatus of any one of claims 45-49, the immersion fluid retention region having a first horizontal dimension and a second horizontal dimension orthogonal to the first horizontal dimension, the first horizontal dimension being greater than the second horizontal dimension.

51. The apparatus of claim 50, further comprising an actuation assembly for driving relative movement between a flow cell and the imaging assembly along a path.

52. The apparatus of claim 51, the path comprising a horizontal path in a direction parallel to the first horizontal dimension.

53. The apparatus of any one of claims 45-52, further comprising a flow cell having an upper surface below the fluid retention region, the immersion fluid assembly for maintaining immersion fluid between the bottom surface of the objective lens and the upper surface of the flow cell.

54. The apparatus of claim 53, the upper surface comprising a hydrophobic material.

55. The apparatus of any one of claims 53-54, the flow cell further comprising a plurality of channels, each channel of the plurality of channels comprising a plurality of reaction sites.

56. The apparatus of claim 55, the plurality of reaction sites comprising a plurality of nucleotides.

57. The apparatus of any one of claims 55-56, the immersion fluid retention region having a first horizontal dimension and a second horizontal dimension orthogonal to the first horizontal dimension, the first horizontal dimension being greater than the second horizontal dimension, at least some channels of the plurality of channels oriented along the first horizontal dimension.

58. The apparatus of claim 57, each channel of the plurality of channels spaced apart from another channel of the plurality of channels along the second horizontal dimension.

59. The apparatus of any one of claims 55-58, further comprising an actuation assembly for driving relative movement between the flow cell and the imaging assembly to position the imaging assembly successively over each channel of the plurality of channels.

60. The apparatus of any one of claims 45-59, the bottom surface of the objective lens element being planar.

61. The apparatus of any one of claims 45-60, the bottom surface of the objective lens element comprising a concave surface.

62. The apparatus of any one of claims 45-61, further comprising an air curtain manifold for expelling pressurized air outward relative to the objective lens element.

63. The apparatus of claim 62, the air curtain manifold positioned to surround the objective lens assembly.

64. The apparatus of claim 63, the objective lens element centered along a longitudinal axis, the air curtain manifold centered along the longitudinal axis.

65. The apparatus of any one of claims 62-64, the air curtain manifold defining a central opening.

66. The apparatus of claim 65, a portion of the imaging assembly positioned within the central opening of the air curtain manifold.

67. The apparatus of claim 66, the central opening sized to define a gap between the air curtain manifold and the portion of the imaging assembly positioned within the central opening.

68. The apparatus of any one of claims 62-67, the air curtain manifold having an annular shape with a circumferentially extending surface with openings positioned along the circumferentially extending surface, the air curtain manifold for expelling pressurized air outward relative to the objective lens via the openings positioned along the circumferentially extending surface.

69. The apparatus of claim 68, the circumferentially extending surface angled along a vertical plane.

70. The apparatus of any one of claims 68-69, the air curtain manifold further comprising an annular channel in fluid communication with the openings positioned along the circumferentially extending surface.

71. The apparatus of any one of claims 62-70, further comprising a frame, the imaging assembly fixed to the frame, the air curtain manifold fixed to the frame independently of the imaging assembly.

72. The apparatus of any one of claims 62-71, the air curtain manifold for expelling pressurized air outward relative to the objective lens element in the form of a frustoconical air curtain.

73. The apparatus of claim 72, the frustoconical air curtain centered along a longitudinal axis, the objective lens element centered along the longitudinal axis.

74. The apparatus of any one of claims 62-73, further comprising an actuation assembly for driving relative movement along a path between a flow cell and the imaging assembly, the air curtain manifold positioned to expel pressurized air downward toward the flow cell.

75. The apparatus of any one of claims 45-74, further comprising an air blade manifold for expelling pressurized air toward a space between the bottom surface of the objective lens element and an upper surface of the flow cell.

76. The apparatus of claim 75, the air blade manifold comprising a plurality of openings for expelling pressurized air toward the space between the bottom surface of the objective lens element and an upper surface of the flow cell.

77. The apparatus of claim 76, the plurality of openings positioned along an arc.

78. The apparatus of any one of claims 76-77, the air blade manifold further comprising a plurality of channels in fluid communication with the plurality of openings.

79. The apparatus of claim 78, each channel in fluid communication with the plurality of openings oriented along a respective radius extending toward a center point, such that the plurality of openings all extend along the radius of their orientation toward the center point.

80. The apparatus of claim 79, the center point positioned to correspond to a central region of the space between the bottom surface of the objective lens element and an upper surface of the flow cell.

81. The apparatus of any one of claims 75-80, the air blade manifold for expelling pressurized air toward the space between the bottom surface of the objective lens element and an upper surface of the flow cell in the form of a substantially flat air blade.

82. The apparatus of any one of claims 75-81, further comprising a frame to which the imaging assembly is fixed, the air blade manifold being fixed to the frame independently of the imaging assembly.

83. The apparatus of any one of claims 75-82, further comprising an air curtain manifold for expelling pressurized air outward relative to the objective lens element.

84. The apparatus of claim 83, further comprising a source of pressurized air for providing pressurized air to the air blade manifold, the source of pressurized air also for providing pressurized air to the air curtain manifold.

85. The apparatus of claim 84, further comprising a valve for directing pressurized air to a selected one of the air blade manifold or the air curtain manifold.

86. The apparatus of any one of claims 45-85, further comprising a sensor for monitoring the presence of immersion fluid beneath the bottom surface of the objective lens element.

87. The apparatus of claim 86, the sensor comprising an optical sensor.

88. The apparatus of claim 87, the optical sensor positioned and oriented to view the immersion fluid beneath the bottom surface of the objective lens element along a horizontal dimension.

89. The apparatus of claim 88, the optical sensor positioned and oriented to view light transmitted through the immersion fluid and reflected back through the objective lens element.

90. The apparatus of any one of claims 86-89, the sensor comprising a set of electrodes.

91. The apparatus of any one of claims 86-90, the sensor comprising a force sensor.

92. An apparatus, the apparatus comprising: an imaging assembly, the imaging assembly comprising: an objective lens having a bottom surface; an immersion fluid assembly comprising: a first port for introducing immersion fluid beneath the bottom surface of the objective lens, a second port for removing immersion fluid from beneath the bottom surface of the objective lens, and a sidewall defining an immersion fluid retention region beneath the objective lens; and an air delivery assembly coupled with the objective lens assembly for delivering pressurized air into one or both of the objective lens or an upper surface of a flow cell, thereby purging one or both of debris or immersion fluid relative to one or both of the objective lens or an upper surface of a flow cell.

93. The apparatus of claim 92, the immersion fluid retention region having a first horizontal dimension and a second horizontal dimension orthogonal to the first horizontal dimension, the first horizontal dimension being greater than the second horizontal dimension.

94. The apparatus of claim 93, further comprising an actuation assembly for driving relative movement between a flow cell and the imaging assembly along a horizontal path in a direction parallel to the first horizontal dimension.

95. The apparatus of any one of claims 92-94, further comprising a flow cell having an upper surface beneath the fluid retention region, the immersion fluid assembly for maintaining immersion fluid between the bottom surface of the objective lens and the upper surface of the flow cell.

96. The apparatus of claim 95, the upper surface comprising a hydrophobic material.

97. The apparatus of any one of claims 95-96, the flow cell further comprising a plurality of channels, each channel of the plurality of channels comprising a plurality of reaction sites.

98. The apparatus of claim 97, the plurality of reaction sites comprising a plurality of nucleotides.

99. The apparatus of any one of claims 97-98, at least some channels of the plurality of channels oriented along the first horizontal dimension.

100. The apparatus of claim 99, each channel of the plurality of channels spaced apart from another channel of the plurality of channels along the second horizontal dimension.

101. The apparatus of any one of claims 97-100, the actuation assembly for driving relative movement between the flow cell and the imaging assembly to position the imaging assembly successively over each channel of the plurality of channels.

102. The apparatus of any one of claims 92-101, the bottom surface of the objective lens being planar.

103. The apparatus of any one of claims 92-102, the bottom surface of the objective lens comprising a concave surface.

104. The apparatus of any one of claims 92-103, the air delivery assembly comprising an air curtain manifold for expelling pressurized air outward relative to the objective lens.

105. The apparatus of claim 104, the air curtain manifold positioned to surround the objective lens.

106. The apparatus of claim 105, the objective lens centered along a longitudinal axis, the air curtain manifold centered along the longitudinal axis.

107. The apparatus of any of claims 104-20+, the air curtain manifold defining a central opening.

108. The apparatus of claim 107, a portion of the imaging assembly positioned within the central opening of the air curtain manifold.

109. The apparatus of claim 108, the central opening sized to define a gap between the air curtain manifold and the portion of the imaging assembly positioned within the central opening.

110. The apparatus of any of claims 104-109, the air curtain manifold having an annular shape with a circumferentially extending surface with openings positioned along the circumferentially extending surface, the air curtain manifold for expelling pressurized air outward relative to the objective lens via the openings positioned along the circumferentially extending surface.

111. The apparatus of claim 110, the circumferentially extending surface angled along a vertical plane.

112. The apparatus of any of claims 110-111, the air curtain manifold further comprising an annular channel in fluid communication with the openings positioned along the circumferentially extending surface.

113. The apparatus of any of claims 104-112, the apparatus further comprising a frame, the imaging assembly secured to the frame, the air curtain manifold secured to the frame independently of the imaging assembly.

114. The apparatus of any of claims 104-113, the air curtain manifold for expelling pressurized air outward relative to the objective lens in the form of a frustoconical air curtain.

115. The apparatus of claim 114, the frustoconical air curtain centered along a longitudinal axis, the objective lens centered along the longitudinal axis.

116. The apparatus of any of claims 104-115, the air curtain manifold positioned to expel pressurized air downward toward the flow cell.

117. The apparatus of any of claims 92-116, the imaging assembly further comprising an objective lens housing supporting the objective lens, the objective lens housing having a distal face encompassing an outer periphery of the objective lens.

118. The apparatus of claim 117, the distal face comprising a hydrophilic material.

119. The apparatus of any of claims 117-118, the bottom surface of the objective lens comprising a hydrophilic material.

120. The apparatus of any of claims 117-119, the distal face further comprising at least one annular recess spaced outward from the outer periphery of the objective lens.

121. The apparatus of any one of claims 92-120, the air delivery assembly comprising an air vane manifold for expelling pressurized air toward a space between the bottom surface of the objective lens and an upper surface of the flow cell.

122. The apparatus of claim 121, the air vane manifold comprising a plurality of openings for expelling pressurized air toward the space between the bottom surface of the objective lens and an upper surface of the flow cell.

123. The apparatus of claim 122, the plurality of openings being positioned along an arc.

124. The apparatus of any one of claims 122-123, the air vane manifold further comprising a plurality of channels in fluid communication with the plurality of openings.

125. The apparatus of claim 124, each channel in fluid communication with the plurality of openings being oriented along a respective radius extending toward a center point, such that the plurality of openings all extend along the radii of their orientations toward the center point.

126. The apparatus of claim 125, the center point being positioned to correspond to a central region of the space between the bottom surface of the objective lens and an upper surface of the flow cell.

127. The apparatus of any one of claims 121-126, the air vane manifold for expelling pressurized air toward the space between the bottom surface of the objective lens and an upper surface of the flow cell in the form of a substantially flat air vane.

128. The apparatus of any one of claims 121-127, further comprising a frame to which the imaging assembly is fixed, the air vane manifold being fixed to the frame independently of the imaging assembly.

129. The apparatus of any one of claims 121-128, further comprising an air curtain manifold for expelling pressurized air outward relative to the objective lens.

130. The apparatus of claim 129, further comprising a source of pressurized air for providing pressurized air to the air vane manifold, the source of pressurized air also for providing pressurized air to the air curtain manifold.

131. The apparatus of claim 130, further comprising a valve for directing pressurized air to a selected one of the air vane manifold or the air curtain manifold.

132. The apparatus of any one of claims 92-131, further comprising a sensor for monitoring the presence of immersion fluid beneath the bottom surface of the objective lens.

133. The apparatus of claim 132, the sensor comprising an optical sensor.

134. The apparatus of claim 133, the optical sensor being positioned and oriented to view the immersion fluid beneath the bottom surface of the objective lens along a horizontal dimension.

135. The apparatus of claim 133, the optical sensor positioned and oriented to view light transmitted through the immersion fluid and reflected back through the objective lens.

136. The apparatus of any one of claims 132 to 134, the sensor comprising a set of electrodes.

137. The apparatus of any one of claims 132 to 135, the sensor comprising a force sensor.

138. A method comprising: positioning an imaging assembly relative to a flow cell, the imaging assembly comprising: an objective lens having a bottom surface, and an immersion fluid assembly comprising: a first port, and a sidewall defining an immersion fluid retention region below the objective lens, the immersion fluid retention region having a first horizontal dimension and a second horizontal dimension orthogonal to the first horizontal dimension, the first horizontal dimension being greater than the second horizontal dimension; depositing, via the first port, immersion fluid to a space between the bottom surface of the objective lens and an upper surface of the flow cell; and providing relative movement between the flow cell and the imaging assembly along a horizontal path in a direction parallel to the first horizontal dimension, the immersion fluid remaining in the space between the bottom surface of the objective lens and the upper surface of the flow cell during the relative movement between the flow cell and the imaging assembly.

139. The method of claim 137, further comprising activating an air curtain comprising pressurized air oriented toward the upper surface of the flow cell and away from the objective lens.

140. The method of claim 138, the act of activating the air curtain performed prior to the act of depositing immersion fluid.

141. The method of any one of claims 138 to 139, the act of activating the air curtain performed while providing relative movement between the flow cell and the imaging assembly.

142. The method of any one of claims 138 to 140, the air curtain having a frustoconical shape.

143. The method of any one of claims 138 to 141, the air curtain provided via an air curtain manifold positioned about the imaging assembly.

144. The method of any one of claims 137 to 142, further comprising activating an air blade comprising pressurized air oriented toward the space between the bottom surface of the objective lens and the upper surface of the flow cell.

145. The method of claim 143, the act of activating the air blade performed after providing relative movement between the flow cell and the imaging assembly.

146. The method of any one of claims 143 to 144, further comprising aspirating the immersion fluid from the space between the bottom surface of the objective lens and the upper surface of the flow cell.

147. The method of claim 145, the immersion fluid being aspirated from the space between the bottom surface of the objective lens and the upper surface of the flow cell via a second port of the immersion fluid assembly.

148. The method of any one of claims 145-146, the act of activating the air blade being performed after aspirating the immersion fluid from the space between the bottom surface of the objective lens and the upper surface of the flow cell.

149. The method of any one of claims 145-147, the air blade being provided via an air blade manifold positioned adjacent to the imaging assembly.

150. The method of claim 148, the air blade manifold having a plurality of openings positioned along an arc, the air blade comprising pressurized air expelled via the plurality of openings such that an air flow of the air blade is focused toward a central region positioned in the space between the bottom surface of the objective lens and the upper surface of the flow cell.

151. The method of any one of claims 137-149, the method further comprising monitoring, via a sensor, a presence of an immersion fluid in the space between the bottom surface of the objective lens and the upper surface of the flow cell.

152. The method of claim 150, the sensor comprising an optical sensor, the act of monitoring comprising obtaining optical data along a horizontal path.

153. The method of any one of claims 150-151, the sensor comprising an optical sensor, the act of monitoring comprising obtaining optical data from light transmitted through the objective lens and toward the optical sensor.

154. The method of any one of claims 150-152, the sensor comprising an electrode, the act of monitoring comprising monitoring whether an electrical circuit between the electrodes is completed.

155. The method of any one of claims 150-153, the sensor comprising a force sensor, the act of monitoring comprising monitoring a force associated with the immersion fluid.

156. The method of any one of claims 137-154, the method further comprising: removing the flow cell from a space below the imaging assembly; and positioning another flow cell in the space below the imaging assembly.

157. The method of any one of claims 137-155, the method further comprising performing nucleotide sequencing in the flow cell.

158. A method, the method comprising: positioning an imaging assembly relative to a flow cell, the imaging assembly comprising: an objective lens having a bottom surface, and an immersion fluid assembly comprising: a first port, and a sidewall defining an immersion fluid retention region below the objective lens; depositing an immersion fluid to a space between the bottom surface of the objective lens and an upper surface of the flow cell via the first port; ​ providing relative movement between the flow cell and the imaging assembly along a horizontal path in a direction parallel to the first horizontal dimension, the immersion fluid remaining in the space between the bottom surface of the objective lens and the upper surface of the flow cell during the relative movement between the flow cell and the imaging assembly; and delivering pressurized air into one or both of the objective lens or the upper surface of the flow cell.

159. The method of claim 157, the immersion fluid retention region having a first horizontal dimension and a second horizontal dimension orthogonal to the first horizontal dimension, the first horizontal dimension being greater than the second horizontal dimension.

160. The method of claim 158, the horizontal path being in a direction parallel to the first horizontal dimension.

161. The method of any of claims 157 to 159, the act of delivering pressurized air into one or both of the objective lens or the upper surface of the flow cell comprising activating an air curtain comprising pressurized air oriented toward the upper surface of the flow cell and away from the objective lens.

162. The method of claim 160, the act of activating the air curtain being performed prior to the act of depositing immersion fluid.

163. The method of any of claims 160 to 161, the act of activating the air curtain being performed while providing relative movement between the flow cell and the imaging assembly.

164. The method of any of claims 160 to 162, the air curtain having a frustoconical shape.

165. The method of any of claims 160 to 163, the air curtain being provided via an air curtain manifold positioned about the imaging assembly.

166. The method of any of claims 157 to 164, the act of delivering pressurized air into one or both of the objective lens or the upper surface of the flow cell comprising activating an air blade comprising pressurized air oriented toward the space between the bottom surface of the objective lens and the upper surface of the flow cell.

167. The method of claim 165, the act of activating the air blade being performed after providing relative movement between the flow cell and the imaging assembly.

168. The method of any of claims 165 to 166, the method further comprising aspirating the immersion fluid from the space between the bottom surface of the objective lens and the upper surface of the flow cell.

169. The method of claim 167, the immersion fluid being aspirated from the space between the bottom surface of the objective lens and the upper surface of the flow cell via a second port of the immersion fluid assembly.

170. The method of any of claims 167-168, the act of activating the air blade is performed after suctioning the immersion fluid from the space between the bottom surface of the objective lens and the upper surface of the flow cell.

171. The method of any of claims 167-169, the air blade is provided via an air blade manifold positioned adjacent to the imaging assembly.

172. The method of claim 170, the air blade manifold has a plurality of openings positioned along an arc, the air blade includes pressurized air expelled via the plurality of openings such that an air flow of the air blade is focused toward a central region positioned in the space between the bottom surface of the objective lens and the upper surface of the flow cell.

173. The method of any of claims 157-171, the method further comprises monitoring, via a sensor, a presence of an immersion fluid in the space between the bottom surface of the objective lens and the upper surface of the flow cell.

174. The method of claim 172, the sensor includes an optical sensor, the act of monitoring includes obtaining optical data along a horizontal path.

175. The method of any of claims 172-173, the sensor includes an optical sensor, the act of monitoring includes obtaining optical data from light transmitted through the objective lens and toward the optical sensor.

176. The method of any of claims 172-174, the sensor includes an electrode, the act of monitoring includes monitoring whether an electrical circuit between the electrodes is completed.

177. The method of any of claims 172-175, the sensor includes a force sensor, the act of monitoring includes monitoring a force associated with the immersion fluid.

178. The method of any of claims 157-176, the method further comprises: removing the flow cell from a space below the imaging assembly; and positioning another flow cell in the space below the imaging assembly.

179. The method of any of claims 157-177, the method further comprises performing nucleotide sequencing in the flow cell. ​

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