Apparatus and method to mitigate thermally induced aberrations in immersion optical system

Abstract

An apparatus includes an imaging assembly, a flow cell, and a temperature regulation assembly. The imaging assembly includes an objective lens assembly and an immersion fluid assembly to provide immersion fluid under a bottom surface of a front element of the objective lens assembly. The immersion fluid maintains immersion fluid between the bottom surface of the front element of the objective lens assembly and an upper surface of the flow cell. The temperature regulation assembly includes a first sensor, a first temperature adjusting feature, and a processor. The first sensor senses a temperature of the objective lens assembly. The first temperature adjusting feature adjusts a temperature of the flow cell. The processor activates the first temperature adjusting feature based on at least data from the first sensor.

Description

APPARATUS AND METHOD TO MITIGATE THERMALLY INDUCED ABERRATIONS IN IMMERSION OPTICAL SYSTEMBACKGROUND

[0001] Aspects of the present disclosure relate generally to devices, systems, and methods providing biological or chemical analysis. Various protocols in biological or chemical research involve performing a large number of controlled reactions on local support surfaces or within predefined reaction chambers. The designated reactions may then be observed or detected, and subsequent analysis may help identify or reveal properties of chemicals involved in the reaction. For example, in some multiplex assays, an unknown analyte having an identifiable label (e.g., fluorescent label) may be exposed to thousands of known probes under controlled conditions. Each known probe may be deposited 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 wells may 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.

[0002] While a variety of devices, systems, and methods have been made and used to perform biological or chemical analysis, it is believed that no one prior to the inventor(s) has made or used the devices and techniques described herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0003] FIG. 1 depicts a schematic view of an example of a system that may be used to provide biological or chemical analysis.

[0004] FIG. 2 depicts a schematic view of an example of a set of components that may cooperate to provide a fluid path in the system of FIG. 1 .

[0005] FIG. 3 depicts a schematic view of another example of a system that may be used toprovide biological or chemical analysis.

[0006] FIG. 4 depicts a cross-sectional view of an example of a flow cell that may be used in the system of FIG. 1.

[0007] FIG. 5 depicts a cross-sectional view of another example of a flow cell that may be used in the system of FIG. 1.

[0008] FIG. 6 depicts a top plan view of the flow cell of FIG. 5, with an upper wafer omitted to reveal a lower wafer.

[0009] FIG. 7 depicts a schematic view of another example of a system that may be used to provide biological or chemical analysis.

[0010] FIG. 8 depicts a schematic view of an example of imaging components that may be integrated into the system of FIG. 7.

[0011] FIG. 9 depicts a schematic view of another example of imaging components that may be integrated into the system of FIG. 7.

[0012] FIG. 10 depicts a schematic view of another example of imaging components that may be integrated into the system of FIG. 7.

[0013] FIG. 11 depicts a schematic view of another example of an arrangement of imaging components that may be integrated into the system of FIG. 7.

[0014] FIG. 12 depicts a schematic view of the imaging components of FIG. 11 with fluid lines coupled with an immersion fluid manifold.

[0015] FIG. 13 depicts a cross-sectional side view of an objective lens assembly of the imaging components of FIG. 11.

[0016] FIG. 14 depicts a bottom plan view of the objective lens assembly of FIG. 13.

[0017] FIG. 15 depicts a perspective view of the imaging components of FIG. 11.

[0018] FIG. 16 depicts a cross-sectional view of the imaging components of FIG. 11, taken along line 16-16 of FIG. 15.

[0019] FIG. 17A depicts a schematic view of a flow cell, with an immersion fluid footprint at a first position along the flow cell.

[0020] FIG. 17B depicts a schematic view of the flow cell of FIG. 17A, with the immersion fluid footprint at a second position along the flow cell.

[0021] FIG. 18A depicts a cross-sectional side view of the imaging components of FIG. 11 during an example of a first stage of operation.

[0022] FIG. 18B depicts a cross-sectional side view of the imaging components of FIG. 11 during an example of a second stage of operation.

[0023] FIG. 18C depicts a cross-sectional side view of the imaging components of FIG. 11 during an example of a third stage of operation.

[0024] FIG. 18D depicts a cross-sectional side view of the imaging components of FIG. 11 during an example of a fourth stage of operation.

[0025] FIG. 19 depicts a schematic view of an example of an arrangement of imaging components with thermal regulation features that may be integrated into the system of FIG. 7.

[0026] FIG. 20 depicts a flow chart representing an example of a method of use of the arrangement of FIG. 19.

[0027] FIG. 21 depicts a schematic view of another example of an arrangement of imaging components with thermal regulation features that may be integrated into the system of FIG. 7.

[0028] FIG. 22 depicts a schematic view of an example of a thermal regulation feature that may be integrated into the arrangement of FIG. 21.

[0029] FIG. 23 depicts a schematic view of another example of a thermal regulation feature that may be integrated into the arrangement of FIG. 21.

[0030] FIG. 24 depicts a schematic view of another example of a thermal regulation feature that may be integrated into the arrangement of FIG. 21.

[0031] FIG. 25 depicts a schematic view of another example of a thermal regulation feature that may be integrated into the arrangement of FIG. 21.

[0032] FIG. 26 depicts a flow chart representing an example of a method of use of the arrangement of FIG. 21.

[0033] FIG. 27 depicts a flow chart representing another example of a method of use of the arrangement of FIG. 21.

[0034] FIG. 28 depicts a cross-sectional view of an example of an objective lens assembly that may be incorporated into any of the arrangements of imaging components described herein.

[0035] FIG. 29 depicts a cross-sectional view of another example of an objective lens assembly that may be incorporated into any of the arrangements of imaging components described herein.

[0036] FIG. 30 depicts a cross-sectional view of another example of an objective lens assembly that may be incorporated into any of the arrangements of imaging components described herein.

[0037] FIG. 31 depicts a cross-sectional view of another example of an objective lens assembly that may be incorporated into any of the arrangements of imaging components described herein.DETAILED DESCRIPTION

[0038] The following detailed description of certain examples will be better understood when read in conjunction with the appended drawings. To the extent that the figures illustrate diagrams of the functional blocks of various examples, the functional blocks are not necessarily indicative of the division between hardware components. Thus, for example, one or more of the functional blocks (e.g., processors or memories) may be implemented in a single piece of hardware (e.g., a general purpose signal processor or random access memory, hard disk, or the like). Similarly, the programs may be stand-alone programs, may be incorporated as subroutines in an operating system, may be functions in an installed software package, and the like. It should be understood that the various examples are not limited to the arrangements and instrumentality shown in the drawings. It should be appreciated 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 to achieve the benefits and advantages disclosed herein.

[0039] I. Overview of System for Biological or Chemical Analysis

[0040] Examples described herein may be used in various biological or chemical processes and systems for academic analysis, commercial analysis, or other analysis. More specifically, examples described herein may be used in various processes and systems where it is desired to detect an event, property, quality, or characteristic that is indicative of a designated reaction. Bioassay systems such as those described herein may be configured to perform a plurality of designated reactions that may be detected individually or collectively. For example, bioassay systems may be used to sequence a dense array of nucleic acid features through iterative cycles of enzymatic manipulation and image acquisition. In some examples, nucleic acids can be attached to a surface and amplified. Examples of such amplification are described in U.S. Pat. No. 7,741,463, entitled “Method of Preparing Libraries of Template Polynucleotides,” issued June 22, 2010, the disclosure of which is incorporated by reference herein, in its entirety; and / or U.S. Pat. No. 7,270,981, entitled “Recombinase Polymerase Amplification,” issued September 18, 2007, the disclosure of which is incorporated by reference herein, in its entirety.

[0041] Components that are used in the bioassay systems may include one or more microfluidic channels that deliver reagents or other reaction components to a reaction site. The reaction sites may be randomly distributed across a substantially planar surface; or may be patterned across a substantially planar surface. Each of the reaction sites may be imaged to detect light from the reaction site. The signals indicating photons emitted from the reaction sites and detected by image sensors may provide illumination values. These illumination values may be combined into an imageindicating photons as detected from the reaction sites. These images may be further analyzed to identify compositions, reactions, conditions, etc., at each reaction site.

[0042] II. Examples of Fluidics Devices and Fluid Flow Paths

[0043] A. Example of System with Higher Volume Throughput

[0044] FIG. 1 illustrates a schematic diagram of an example of a system (100) that may be used to perform an analysis on one or more samples of interest. In some implementations, the sample may include one or more clusters of nucleotides (e.g., DNA) that are linearized to form a single stranded DNA (sstDNA). In the implementation shown, system (100) is configured to receive a flow cell cartridge assembly (102) including a flow cell assembly (103) and a sample cartridge (104). System (100) includes a flow cell receptacle (122) that receives flow cell cartridge assembly (102), a vacuum chuck (124) that supports flow cell assembly (103), and a flow cell interface (126) that is used to establish a fluidic coupling between system (100) and flow cell assembly (103). Flow cell interface (126) may include one or more manifolds. System (100) further includes a sipper manifold assembly (106), a sample loading manifold assembly (108), and a pump manifold assembly (110). System (100) also includes a drive assembly (112), a controller (114), an imaging system (116), and a waste reservoir (118). Controller (114) is electrically and / or communicatively coupled to drive assembly (112) and to imaging system (116); and is configured to cause drive assembly (112) and / or the imaging system (116) to perform various functions as disclosed herein.

[0045] In the present example, flow cell assembly (103) includes a flow cell (128) having a channel (130) and defining a plurality of first openings (132), which are fluidically coupled to the channel (130) and arranged on a first side (134) of the channel (130). Flow cell (128) further includes a plurality of second openings (136) fluidically coupled to the channel (130) and arranged on a second side (138) of the channel (130). Fluid may thus flow through flow cell (128) via channel. While the flow cell (128) is shown including one channel (130), flow cell (128) may include two or more channels (130). Flow cell assembly (103) also includes a flow cell manifold assembly (140) coupled toflow cell (128) and having a first manifold fluidic line (142) and a second manifold fluidic line (144). Flow cell manifold assembly (140) may be in the form of a laminate including a plurality of layers as discussed in more detail below.

[0046] In the implementation shown, first manifold fluidic line (142) has a first fluidic line opening (146) and is fluidically coupled to each of the first openings (132) of flow cell (128); and second manifold fluidic line (144) has a second fluidic line opening (148) and is fluidically coupled to each of the second openings (136). As shown, flow cell assembly (103) includes gaskets (150) coupled to flow cell manifold assembly (140) and fluidically coupled to fluidic line openings (146, 148). In some implementations where flow cell (128) includes a plurality of channels (130), flow cell manifold assembly (140) may include additional fluidic lines (152) that couple first fluidic line openings (146) to a single manifold port (154). In such implementations, a single gasket (150) may be coupled to flow cell manifold assembly (140) that surrounds the manifold port (154) and is in fluidic communication with a plurality of channels (130). In operation, flow cell interface (126) engages with corresponding gaskets (150) to establish a fluidic coupling between system (100) and flow cell (128). The engagement between flow cell interface (126) and gaskets (150) reduces or eliminates fluid leakage between flow cell interface (126) and flow cell (128).

[0047] In the implementation shown, first manifold fluidic line (142) has a portion (156) that is substantially parallel to a longitudinal axis (158) of channel (130); and second manifold fluidic line (144) has a portion (160) that is substantially parallel to longitudinal axis (158) of channel (130). Additionally, first manifold fluidic line (142) is shown being at least partially adjacent a first end (162) of flow cell (128) and spaced from a second end (164) of flow cell (128); and second manifold fluidic line (144) is shown being at least partially adjacent second end (164) of flow cell (128) and spaced from first end (162). Other arrangements of manifold fluidic lines (142, 144) may prove suitable, however.

[0048] In the implementation shown, system (100) includes a sample cartridge receptacle (166) that receives sample cartridge (104) that carries one or more samples of interest (e.g., an analyte). System (100) also includes a sample cartridge interface (168) thatestablishes a fluidic connection with sample cartridge (104). Sample loading manifold assembly (108) includes one or more sample valves (170). Pump manifold assembly (110) includes one or more pumps (172), one or more pump valves (174), and a cache (176). Valves (170, 174) and pumps (172) may take any suitable form. Cache (176) may include a serpentine cache and may temporarily store one or more reaction components during, for example, bypass manipulations of the system (100). While cache (176) is shown being included in pump manifold assembly (110), cache (176) may alternatively be located elsewhere (e.g., in sipper manifold assembly (106) or in another manifold downstream of a bypass fluidic line (178), etc.).

[0049] Sample loading manifold assembly (108) and pump manifold assembly (110) flow one or more samples of interest from sample cartridge (104) through a fluidic line (180) toward flow cell cartridge assembly (102). In some implementations, sample loading manifold assembly (108) may individually load or address each channel (130) of flow cell (128) with a respective sample of interest. The process of loading channel (130) with a sample of interest may occur automatically using system (100). As shown in FIG. 1, sample cartridge (104) and sample loading manifold assembly (108) are positioned downstream of flow cell cartridge assembly (102). In the implementation shown, sample loading manifold assembly (108) is coupled between flow cell cartridge assembly (102) and pump manifold assembly (110). To draw a sample of interest from sample cartridge (104) and toward pump manifold assembly (110), sample valves (170), pump valves (174), and / or pumps (172) may be selectively actuated to urge the sample of interest toward pump manifold assembly (110). Sample cartridge (104) may include a plurality of sample reservoirs that are selectively fluidically accessible via the corresponding sample valves (170). To individually flow the sample of interest toward channel (130) of flow cell (128) and away from pump manifold assembly (110), sample valves (170), pump valves (174), and / or pumps (172) may be selectively actuated to urge the sample of interest toward flow cell cartridge assembly (102) and into respective channels (130) of flow cell (128).

[0050] Drive assembly (112) interfaces with sipper manifold assembly (106) and pump manifold assembly (110) to flow one or more reagents that interact with the samplewithin flow cell (128). In some scenarios, a reversible terminator is attached to the reagent to allow a single nucleotide to be incorporated onto a growing DNA strand. In some such implementations, one or more of the nucleotides has a unique fluorescent label that emits a color when excited. The color (or absence thereof) is used to detect the corresponding nucleotide. In the implementation shown, imaging system (116) excites one or more of the identifiable labels (e.g., a fluorescent label) and thereafter obtains image data for the identifiable labels. The labels may be excited by incident light and / or a laser and the image data may include one or more colors emitted by the respective labels in response to the excitation. The image data (e.g., detection data) may be analyzed by system (100). Examples of features and functionalities that may be incorporated into imaging system (116) will be described in greater detail below.

[0051] After the image data is obtained, drive assembly (112) interfaces with sipper manifold assembly (106) and pump manifold assembly (110) to flow another reaction component (e.g., a reagent) through flow cell (128) that is thereafter received by waste reservoir (118) via a primary waste fluidic line (182) and / or otherwise exhausted by system (100). Some reaction components may perform a flushing operation that chemically cleaves the fluorescent label and the reversible terminator from the sstDNA. The sstDNA may then be ready for another cycle.

[0052] The primary waste fluidic line (182) is coupled between pump manifold assembly (110) and waste reservoir (118). In some implementations, pumps (172) and / or pump valves (174) of pump manifold assembly (110) selectively flow the reaction components from flow cell cartridge assembly (102), through fluidic line (180) and sample loading manifold assembly (108) to primary waste fluidic line (182). Flow cell cartridge assembly (102) is coupled to a central valve (184) via flow cell interface (126). Central valve (184) is coupled with flow cell interface (126) via a fluidic line (185). An auxiliary waste fluidic line (186) is coupled to central valve (184) and to waste reservoir (118). In some implementations, auxiliary waste fluidic line (186) receives excess fluid of a sample of interest from flow cell cartridge assembly (102), via central valve (184), and flows the excess fluid of the sample of interest to waste reservoir (118) when back loading the sample of interest into flow cell (128), as described herein.

[0053] Sipper manifold assembly (106) includes a shared line valve (188) and a bypass valve (190). Shared line valve (188) may be referred to as a reagent selector valve. Central valve (184) and the valves (188, 190) of sipper manifold assembly (106) may be selectively actuated to control the flow of fluid through fluidic lines (192, 194, 196). Sipper manifold assembly (106) may be coupled to a corresponding number of reagent reservoirs (198) via reagent sippers (200). Reagent reservoirs (198) may contain fluid (e.g., reagent and / or another reaction component). In some implementations, sipper manifold assembly (106) includes a plurality of ports. Each port of sipper manifold assembly (106) may receive one of the reagent sippers (200). Reagent sippers (200) may be referred to as fluidic lines. Some forms of reagent sippers (200) may include an array of sipper tubes extending downwardly along the z-dimension from ports in the body of sipper manifold assembly (106). Reagent reservoirs (198) may be provided in a cartridge, and the tubes of reagent sippers (200) may be configured to be inserted into corresponding reagent reservoirs (198) in the reagent cartridge so that liquid reagent may be drawn from each reagent reservoir (198) into the sipper manifold assembly (106).

[0054] Shared line valve (188) of sipper manifold assembly (106) is coupled to central valve (184) via shared reagent fluidic line (196). Different reagents may flow through shared reagent fluidic line (196) at different times. In some versions, when performing a flushing operation before changing between one reagent and another, pump manifold assembly (110) may draw wash buffer through shared reagent fluidic line (196), central valve (184), and flow cell cartridge assembly (102).

[0055] Bypass valve (190) of sipper manifold assembly (106) is coupled to central valve (184) via dedicated reagent fluidic lines (194, 196). Each of the dedicated reagent fluidic lines (194, 196) may be associated with a single reagent. The fluids that may flow through dedicated reagent fluidic lines (194, 196) may be used during sequencing operations and may include a cleave reagent, an incorporation reagent, a scan reagent, a cleave wash, and / or a wash buffer.

[0056] Bypass valve (190) is also coupled to cache (176) of pump manifold assembly (110) via bypass fluidic line (178). One or more reagent priming operations, hydrationoperations, mixing operations, and / or transfer operations may be performed using bypass fluidic line (178). The priming operations, the hydration operations, the mixing operations, and / or the transfer operations may be performed independent of flow cell cartridge assembly (102). Thus, the operations using bypass fluidic line (178) may occur during, for example, incubation of one or more samples of interest within flow cell cartridge assembly (102). That is, shared line valve (188) may be utilized independently of bypass valve (190) such that bypass valve (190) may utilize bypass fluidic line (178) and / or cache (176) to perform one or more operations while shared line valve (188) and / or central valve (184) simultaneously, substantially simultaneously, or offset synchronously perform other operations.

[0057] Drive assembly (112) includes a pump drive assembly (202) and a valve drive assembly (204). Pump drive assembly (202) may be adapted to interface with one or more pumps (172) to pump fluid through flow cell (128) and / or to load one or more samples of interest into flow cell (128). Valve drive assembly (204) may be adapted to interface with one or more of the valves (170, 174, 184, 188, 190) to control the position of the corresponding valves (170, 174, 184, 188, 190).

[0058] FIG. 2 shows an example of a fluidic arrangement (220) that may be incorporated into a variation of system (100). Fluidic arrangement (220) of this example includes a pump manifold assembly (222), which may operate similar to pump manifold assembly (110) described above; a sample loading manifold assembly (228), which may operate similar to sample loading manifold assembly (108) described above; a flow cell interface (240), which may operate similar to flow cell interface (126) described above; a sipper manifold assembly (250), which may operate similar to sipper manifold assembly (106) described above; and a waste reservoir (270), which may operate similar to waste reservoir (118) described above. Pump manifold assembly (222) is coupled with a port assembly (258) of sipper manifold assembly (250) via a fluidic line (224), which may be similar to fluidic line (178); and with sample loading manifold assembly (228) via a fluidic line (226). Sample loading manifold assembly (228) is coupled with flow cell interface (240) via fluidic line (230), which may be similar to fluidic line (180); and with port assembly (258) via fluidic lines (232, 234). Flow cell interface (240) iscoupled with sipper manifold assembly (250) via fluidic line (242), which may be similar to fluidic line (185). Sipper manifold assembly (250) includes a manifold body (252) and a common output port (256), which provides fluid communication via fluidic line (185). A valve assembly (254) controls fluid flow through common output port (256) and may operate similar to central valve (184). Port assembly (258) of sipper manifold assembly (250) is coupled with waste reservoir (270) via fluidic line (272), which may be similar to fluidic line (186).

[0059] A plurality of reagent sippers (260) extend from manifold body (252) and are fluidically coupled with valve assembly (254) via respective fluid channels (262) in manifold body (252). Reagent sippers (260) may operate similar to reagent sippers (200). Valve assembly (254) is operable to selectively couple fluid channels (262) with flow cell interface (240) via common output port (256) and fluidic line (230), to thereby selectively provide various reagents to flow cell interface (240). In other words, when each reagent sipper (260) is disposed in a different respective reagent (e.g., in a respective reagent reservoir (198)), a flow cell (e.g., like flow cell (128)) that is coupled with flow cell interface (240) may selectively receive those different reagents based on control of valve assembly (254).

[0060] Port assembly (258) may provide a fluidic interface between pump manifold assembly (222) and sipper manifold assembly (250), thereby allowing sipper manifold assembly (250) to receive pressurized fluid from pump manifold assembly (222). Port assembly (258) may also provide a fluidic interface between sample loading manifold assembly (228) and sipper manifold assembly (250), thereby allowing sipper manifold assembly (250) to receive sample fluid from sample loading manifold assembly (228). In addition, port assembly (258) may provide a fluidic interface between waste reservoir (270) and sipper manifold assembly (250), thereby allowing sipper manifold assembly (250) to communicate waste fluid to waste reservoir (270). Communication of fluids via port assembly (258) may be regulated, at least in part, by valve assembly (254).

[0061] Referring back to FIG. 1, 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) toperform various functions including the disclosed implementations. User interface (206), communication interface (133), and memory (212) are electrically and / or communicatively coupled to the one or more processors (210). User interface (206) may be adapted to receive input from a user and to provide information to the user associated with the operation of system (100) and / or an analysis taking place. User interface (206) may include a touch screen, a display, a keyboard, a speaker(s), a mouse, a track ball, and / or a voice recognition system.

[0062] Communication interface (208) is adapted to enable communication between system (100) and a remote system(s) (e.g., computers) via a network(s) (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 may be associated with analysis results, imaging data, etc. generated or otherwise obtained by system (100). Some of the communications provided to system (100) may be associated with a fluidics analysis operation, patient records, and / or a protocol(s) to be executed by system (100).

[0063] The one or more processors (210) and / or system (100) may include one or more of a processor-based system(s) or a microprocessor-based system(s). In some implementations, the one or more processors (210) and / or system (100) includes 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 executing various functions including the ones described herein.

[0064] Memory (212) may include one or more of a semiconductor memory, a magnetically readable memory, an optical memory, a hard disk drive (HDD), an optical storage drive, a solid-state storage device, a solid-state drive (SSD), a flash memory, a readonly memory (ROM), erasable programmable read-only memory (EPROM),electrically erasable programmable read-only memory (EEPROM), a random-access memory (RAM), a non-volatile RAM (NVRAM) memory, a compact disc (CD), a compact disc 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 storage disk in which information is stored for any duration (e.g., permanently, temporarily, for extended periods of time, for buffering, for caching).

[0065] B. Example of System with Lower Volume Throughput

[0066] FIG. 3 illustrates a schematic diagram of another example of a system (300) that may be used to perform an analysis on one or more samples of interest. Except as otherwise described below, system (300) of this example may be configured and operable like system (100) described above with reference to FIG. 1. In some instances, system (100) is used to provide a higher volume throughput; while system (300) is used to provide a lower volume throughput. Alternatively, systems (100, 300) may provide any other suitable amount or degree of throughput. System (300) of the present example receives a reagent cartridge (302) and includes, in part, a gas source (304), a drive assembly (306), a controller (308), an imaging system (310), and a waste reservoir (312). Reagent cartridge (302) may be referred to as a consumable, a reagent reservoir, or a reagent assembly. Controller (308) is electrically and / or communicatively coupled to drive assembly (306) and to imaging system (310) and causes drive assembly (306) and / or imaging system (310) to perform various functions as disclosed herein.

[0067] Reagent cartridge (302) in the implementation shown includes a well assembly (314) having a body (316). Body (316) has a first wall (318) defining a well (320) having a port (322). First wall (318) has a distal end (324) that defines an opening (326) having an opening perimeter (328). A second wall (330) surrounds first wall (318) and has a distal end (332). Distal end (332) may be referred to as an edge or an outer edge. A cover (334) is coupled to distal end (324) of first wall (318) and covers opening (326) along opening perimeter (328) at a connected portion (336); and is uncoupled from distal end (324) of first wall (318) at an unconnected portion (338). Connection portion (336) may be referred to as connection sections or connected segments andunconnected portion (338) may be referred to as unconnected sections or unconnected segments. First wall (318) has a height and second wall (330) has a height that is greater than the height of first wall (318). First well (318) and second well (330) may alternatively be the same or similar heights. An impermeable barrier (340) is coupled to distal end (332) of second wall (330) and covers well (320). Impermeable barrier (340) may be foil, plastic, etc. and may prevent or inhibit moisture from infiltrating wells (320) of reagent cartridge (302).

[0068] Unconnected portion (338) of cover (334) forms a vent (342) that allows air flow out of well (320). Dried reagent (348) is contained within well (320), and vent (342) is sized to substantially retain dried reagent (348) within the well (320). Body (316) may include a plurality of wells (320) while one well (320) is shown in FIG. 3. Liquid (346) may flow into well (320) via port (322) in practice to rehydrate dried reagent (348). Vent (342) may vent gas from well (320) as liquid (346) flows into well (320); and cover (334) prevents or inhibits reagent (348) and / or liquid (346) from escaping from well (320). Put another way, vents (342) retain reagent (348) and / or liquid (346) within wells (320); and prevent or inhibit reagent (348) and / or liquid (346) from migrating out of wells (320). Vent (342) and cover (334) prevent or inhibit cross-contamination between reagents when reagent cartridge (302) includes more than one well (320). Liquid (346) and dried reagent (348) may be flowed into and out of well (320) to mix liquid (346) from liquid reservoir (362) and dried reagent (348). System (300) and / or reagent cartridge (302) may include a mixing chamber that is used to mix liquid (346) and dried reagent (348) in some implementations. Impermeable barrier (340) may be pierced prior to liquid (346) flowing into well (320).

[0069] Gas source (304) may be used to pressurize liquid reservoir (362) to flow liquid (346) into well (320); and / or a pump (350) may draw liquid (346) from liquid reservoir (362) and flow liquid (346) into well (320) to rehydrate reagent (348). Gas source (304) may be provided by system (300) and / or may be carried by reagent cartridge (302). Gas source (304) may alternatively be omitted. Pump (350) may be implemented by a syringe pump, a peristaltic pump, a diaphragm pump, etc. While pump (350) may be positioned downstream of flow cell (368) as shown, pump (350) may be positionedupstream of flow cell (368) or omitted entirely.

[0070] Reagent cartridge (302) and / or system (300) includes valves (352) that may be selectively actuatable to control the flow of fluid through fluidic lines (356). Such valves (352) may 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 piezo valve, etc. A regulator (354) may be positioned between gas source (304) and valve (352); and regulate the pressure of the gas provided to valve (352). Regulator (354) may include a valve that controls the flow of the gas from gas source (304).

[0071] Body (316) of well assembly (314) has an edge (364); and impermeable barrier (340) may be hermetically connected to body (316) along edge (364). Impermeable barrier (340) may include foil, plastic, and / or any other suitable material(s). System (300) may pierce impermeable barrier (340), impermeable barrier (340) may be pierced by an individual prior to use, or impermeable barrier (340) may be pierced by some other structure or methodology. System (300) includes an actuator assembly (360) in the implementation shown that interfaces with impermeable barrier (340) to pierce impermeable barrier (340). System (300) may include a protrusion such as a post having a blunt or sharp end that is movable by actuator assembly (360) to pierce impermeable barrier (340). Impermeable barrier (340) may alternatively be pierced by an operator prior to reagent cartridge (302) being positioned in system (300). System (300) also includes a liquid reservoir (362) containing liquid (346). Liquid (346) may comprise a rehydrating liquid, a wash buffer, and / or any other suitable kind(s) of liquid.

[0072] System (300) further includes a flow cell receptacle (366) that receives a flow cell (368). Flow cell (368) may be configured and operable like flow cell (128). In some variations, flow cell (368) is carried by and / or integrated into reagent cartridge (302). Flow cell (368) may carry the sample of interest. Gas source (304) and / or pump (350) may flow liquid (346) to rehydrate dry reagents (348) and to flow one or more liquid reagents through reagent cartridge (302) that interact with the sample. Imaging system (310) may be configured and operable like imaging system (116), such that imaging system (310) may be used to obtain image data from flow cell (368). After the image data is obtained, drive assembly (306) may interface with reagent cartridge (302) toflow another reaction component (e.g., a reagent) through flow cell (368) that is thereafter received by the waste reservoir (312) and / or otherwise exhausted by reagent cartridge (302). In the present example, drive assembly (306) includes a pump drive assembly (370), a valve drive assembly (372), and actuator assembly (360). Pump drive assembly (370) interfaces with pump (350) to pump fluid through reagent cartridge (302) and / or flow cell (368); and valve drive assembly (372) interfaces with valve (352) to control the position of valve (352).

[0073] Controller (308) of this example includes a user interface (374), a communication interface (376), a processor (378), and a memory (380). User interface (374) may be configured and operable like user interface (206) of system (100). Communication interface (376) may be configured and operable like communication interface (208) of system (100). Processor (378) may be configured and operable like processor (210) of system (100). Memory (380) may be configured and operable like memory (212) of system (100).

[0074] Further examples and details of how various features of each system (100, 300) may be configured and operable will be described below. By way of further example only, the various features of system (100, 300) may be configured and operable in accordance with at least some of the teachings of International Pub. 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. Pat. 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. Pat. App. 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.

[0075] III. Examples of Flow Cell Structures

[0076] As noted above, a system (100, 300) may execute reactions in a flow cell (128, 368) and / or perform analysis on one or more samples of interest in a flow cell (128, 368).The following describes examples of forms that such flow cells (128, 368) may take, it being understood that flow cells (128, 368) may take various other forms and have various other features in addition to or in lieu of the features described below.

[0077] A. Example of Single-Surface Patterned Flow Cell

[0078] FIG. 4 shows an example of a flow cell (400) that includes a patterned substrate (402), which includes depressions (404) separated by interstitial regions (406), and surface chemistry (410, 412) positioned in the depressions (404). Depressions (404) may be in the form of microwells or nanowells. Depressions (404) may be configured to contain nucleic acid strands or other oligonucleotides and thereby provide a reaction site for SBS and / or for other kinds of processes. In some versions, each depression (404) has a cylindraceous configuration, with a generally circular cross-sectional profile. In some other versions, each depression (404) has a polygonal (e.g., hexagonal, octagonal, square, rectangular, elliptical, etc.) cross-sectional profile. Alternatively, depressions (404) may have any other suitable configuration. It should also be understood that depressions (404) may be arranged in any suitable pattern, including but not limited to a grid pattern.

[0079] Surface chemistry (410, 412) of the present example includes functionalized coating layer (410) and primers (412). While not shown, it is to be understood that the depressions (404) may also have surface preparation or treatment chemistry (e.g., silane or a silane derivative) positioned between the substrate (402) and the functionalized coating layer (410). This same surface preparation or treatment chemistry may also be positioned on the interstitial regions (406). In the present example, a hydrogel (440) is applied before lid (420) is bonded to substrate (402). Hydrogel (440) covers surface chemistry (410, 412) in depressions (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, hydrogel (440) may comprise PAZAM, crosslinked polyacrylamide, agarose gel, etc.

[0080] Flow cell (400) of this example further includes a lid (420) bonded to bonding region(s) (422) of patterned substrate (402). In the example shown in FIG. 4,lid (420) includes a top portion (424) that is connected to several sidewalls (426), and these components (424, 426) define a portion of each of the six flow channels (430A, 430B, 430C, 430D, 430E, 430F). The respective sidewalls (426) isolate 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 depressions (404).

[0081] Lid (420) may be bonded to bonding region (422) of substrate (402) using any suitable technique, such as laser bonding, diffusion bonding, anodic bonding, eutectic bonding, plasma activation bonding, glass frit bonding, or other methods known in the art. In some versions, a spacer layer (428) may be used to bond lid (420) to bonding region (422). Spacer layer (428) may comprise any material that will seal at least some of interstitial regions (404) (e.g., bonding region (422)) of substrate (402) and lid (420) together. While not shown, lid (420) or the patterned substrate (402) may include inlet and outlet ports that are to fluidically engage other ports (not shown), such as those of sample cartridge interface (168), for directing fluid(s) into the respective flow channels (430A, 430B, 430C, 430D, 430E, 43 OF) (e.g., from a reagent cartridge or other fluid storage system) and out of the flow channel (e.g., to waste reservoir (118) or another waste removal system). Flow channels (430A, 430B, 430C, 430D, 430E, 430F) may serve to, for example, selectively introduce reaction components or reactants to hydrogel (440) and the underlying surface chemistry (410, 412) in order initiate designated reactions in / at depressions (404).

[0082] While flow cell (400) includes a pattern of depressions (404) to provide an array of reaction sites, other variations may provide reaction sites on or at various other kinds of structural features, including but not limited to continuously planer surfaces and / or protruding surfaces, etc. By way of further example only, flow cell (400) may be constructed and operable in accordance with at least some of the teachings of U.S. Pat. No. 10,919,033, entitled “Flow Cells with Hydrogel Coating,” issued February 16, 2021, the disclosure of which is incorporated by reference herein, in its entirety.

[0083] B. Example of Dual-Surface Patterned Flow Cell

[0084] While FIG. 4 shows an example of a flow cell (400) that has a single surface patterned with reaction sites (i.e., depressions (404) formed in substrate (402)), it may be desirable in some instances to provide a variation of flow cell (400) that provide two surfaces patterned with reaction sites. An example of a dual-surface patterned flow cell (450) is shown in FIGS. 5-6. 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 depressions (462, 464), such that depressions (462) of wafer (452) align with depressions (464) of wafer (454) when flow cell (450) is assembled. Depressions (462) are separated from each other by interstitial regions (466); and depressions (464) are separated from each other by interstitial regions (468). Spacer layer (456) does not contact interstitial regions (466, 468) in this example.

[0085] Depressions (462, 464) of flow cell (450) may be configured and operable like depressions (404) of flow cell (400) described above. Each depression (462, 464) of the present example includes a grafted coating (470), which may be similar to functionalized coating layer ( 10); and primers (472), which may be similar to primers (412) described above. Each depression (462, 464) may further include hydrogel, like hydrogel (440), and / or any other suitable feature(s). As shown in FIGS. 5-6, depressions (462, 464) are provided within a plurality of flow channels (480A, 480B, 480C, 480D). Flow channels (480A, 480B, 480C, 480D) are separated from each other by walls (458) and ends (459) formed by spacer layer (456). In the example shown in FIG. 6, flow cell (450) provides four flow channels (480A, 480B, 480C, 480D), with each flow channel (480A, 480B, 480C, 480D) containing several rows and columns of depressions (462, 464). When flow cell (450) is used in system (100, 300), flow channels (480A, 480B, 480C, 480D) may serve to, for example, selectively introduce reaction components or reactants surface chemistry (470, 472) in order initiate designated reactions in / at depressions (462, 464). In some instances, since each wafer (452, 454) has its own set of depressions (462, 464) providing corresponding reaction sites, flow cell (450) may provide twice as many reactions as a similarly sized flow cell(400) during a given time period.

[0086] The broken lines in FIG. 6 indicate how flow cell (450) may 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). In the present example, however, a single flow cell (450) has more than two flow channels (480A, 480B, 480C, 480D). While flow cell (450) includes a pattern of depressions (462, 464) to provide an array of reaction sites, other variations may provide reaction sites on or at various other kinds of structural features, including but not limited to continuously planer surfaces and / or protruding surfaces, etc. By way of further example only, flow cell (450) may be constructed and operable in accordance with at least some of the teachings of U.S. Pat. No. 10,955,332, entitled “Flow Cell Package and Method for Making the Same,” issued March 23, 2021, the disclosure of which is incorporated by reference herein, in its entirety.

[0087] IV. Examples of Imaging System Features

[0088] As noted above, system (100, 300) includes an imaging system (116, 310) that excites one or more identifiable labels (e.g., a fluorescent label) in samples in reaction sites provided by depressions (404, 462, 464) of a flow cell (128, 368, 400, 450); and thereafter obtains image data for the identifiable labels. This image data is used to identify nucleotides as part of a nucleic acid sequencing process. Alternatively, the image data may be used for various other purposes. The following description provides details on how some versions of imaging system (116, 310) may be configured and operable.

[0089] FIG. 7 illustrates a schematic diagram of another example of a system (500) that may be used to perform an analysis on one or more samples of interest. Except as otherwise described below, system (500) of this example may be configured and operable like systems (100, 300) described above. System (500) is configured to perform a large number of parallel reactions within a flow cell (510). Flow cell (510) may be configured and operable like flow cells (400, 450) described above or may have any other suitable configuration. Flow cell (510) may thus include one or more flow channels that receivea solution from system (500) and direct the solution toward reaction sites of flow cell (510).

[0090] System (500) includes a system controller (520) that may communicate with the various components, assemblies, and sub-systems of the system (500). Controller (520) may be configured and operable like controllers (114, 308) described above. An imaging assembly (522) of system (500) includes a light emitting assembly (550) that emits light that reaches reaction sites on flow cell (510). Light emitting assembly (550) may include an incoherent light emitter (e.g., emit light beams output by one or more excitation diodes), or a coherent light emitter such as emitter of light output by one or more lasers or laser diodes. In some implementations, light emitting assembly (550) may include a plurality of different light sources (not shown), each light source emitting light of a different wavelength range. Some versions of light emitting assembly (550) may also include one or more collimating lenses (not shown), a light structuring optical assembly (not shown), a projection lens (not shown) that is operable to adjust a structured beam shape and path, epifluorescence microscopy components, and / or other components. Although system (500) is illustrated as having a single light emitting assembly (550), multiple light emitting assemblies (550) may be included in some other implementations.

[0091] In the present example, the light from light emitting assembly (550) is directed by dichroic mirror assembly (546) through an objective lens assembly (542) onto a sample of a flow cell (510), which is positioned on a motion stage (570). In the case of fluorescent microscopy of a sample, a fluorescent element associated with the sample of interest fluoresces in response to the excitation light, and the resultant light is collected by objective lens assembly (542) and is directed to an image sensor of camera system (540) to detect the emitted fluorescence. In some implementations, a tube lens assembly may 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 moveable lens element may be translatable along a longitudinal axis of the tube lens assembly to account for focusing on an upper interior surface or lower interior surface of the flow cell (510) and / or spherical aberrationintroduced by movement of the objective lens assembly (542).

[0092] In the present example, a filter switching assembly (544) is interposed between dichroic mirror assembly (546) and camera system (540). Filter switching assembly (544) includes one or more emission filters that may be used to pass through particular ranges of emission wavelengths and block (or reflect) other ranges of emission wavelengths. For example, emission filters may be used to direct different wavelength ranges of emitted light to different image sensors of the camera system (540) of imaging assembly (522). For instance, the emission filters may be implemented as dichroic mirrors that direct emission light of different wavelengths from flow cell (510) to different image sensors of camera system (540). In some variations, a projection lens is interposed between filter switching assembly (544) and camera system (540). Filter switching assembly (544) may be omitted in some versions.

[0093] System (500) further includes a fluid delivery assembly (590) that may direct the flow of reagents (e.g., fluorescently labeled nucleotides, buffers, enzymes, cleavage reagents, etc.) to (and through) flow cell (510) and waste valve (580). Fluid delivery assembly (590) may be configured and operable like the various fluid delivery components described above in the context of FIGS. 1-3. System (500) of the present example also includes a temperature station actuator (530) and heater / cooler (532) that may optionally regulate the temperature of conditions of the fluids within the flow cell (510). In some implementations, the heater / cooler (532) may be fixed to sample stage (570), upon which the flow cell (510) is placed, and / or may be integrated into sample stage (570).

[0094] Flow cell (510) may be removably mounted on sample stage (570), which may provide movement and alignment of flow cell (510) relative to objective lens assembly (542). Sample stage (570) may have one or more actuators to allow sample stage (570) to move in any of three dimensions. For example, actuators may be provided to allow sample stage (570) to move in the x, y, and z directions relative to objective lens assembly (542), tilt relative to objective lens assembly (542), and / or otherwise move relative to objective lens assembly (542). Movement of sample stage (570) may allow one or more sample locations on flow cell (510) to be positioned in optical alignmentwith objective lens assembly (542). Movement of sample stage (570) relative to objective lens assembly (542) may be achieved by moving sample stage (570) itself, by moving objective lens assembly (542), by moving some other component of imaging assembly (522), by moving some other component of system (500), or any combination of the foregoing. For instance, in some implementations, the sample stage (570) may be actuatable in the x and y directions relative to the objective lens assembly (542) while a focus component (562) or z-stage may move the objective lens assembly (542) along the z direction relative to the sample stage (570).

[0095] In some implementations, a focus component (562) may be included to control positioning of one or more elements of objective lens assembly (542) relative to the flow cell (510) in the focus direction (e.g., along the z-axis or z-dimension). Focus component (562) may include one or more actuators physically coupled to the objective lens assembly (542), the optical stage, the sample stage (570), or a combination thereof, to move flow cell (510) on sample stage (570) relative to the objective lens assembly (542) to provide proper focusing for the imaging operation. 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 an in-focus position to the focus component (562) or a component thereof or operable to control the focus component (562), such as controller (520), to move the objective lens assembly (542) to position the corresponding portion of the flow cell (510) in focus of the objective lens assembly (542).

[0096] In some implementations, an actuator of focus component (562) or for sample stage (570) may be physically coupled to objective lens assembly (542), the optical stage, sample stage (570), or a combination thereof, such as, for example, by mechanical, magnetic, fluidic, or other attachment or contact directly or indirectly to or with the stage or a component thereof. The actuator of focus component (562) may be configured to move objective lens assembly (542) in the z-direction while maintaining sample stage (570) in the same plane (e.g., maintaining a level or horizontal attitude, perpendicular to the optical axis). In some implementations, sample stage (570)includes an x direction actuator and a y direction actuator to form an x-y stage. Sample stage (570) may also be configured to include one or more tip or tilt actuators to tip or tilt sample stage (570) and / or a portion thereof, to account for any slope in its surfaces.

[0097] Camera system (540) may include one or more image sensors to monitor and track the imaging (e.g., sequencing) of flow cell (510). Camera system (540) may be implemented, for example, as a CCD or CMOS image sensor camera, but other image sensor technologies (e.g., active pixel sensor) may be used. By way of further example only, camera system (540) may 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 kinds of camera technologies. While camera system (540) and associated optical components are shown as being positioned above flow cell (510) in FIG. 7, one or more image sensors or other camera components may be incorporated into system (500) in numerous other ways as will be apparent to those skilled in the art in view of the teachings herein. For instance, one or more image sensors may be positioned under flow cell (510), such as within the sample stage (570) or below the sample stage (570); or may even be integrated into flow cell (510).

[0098] FIG. 8 shows an example of various components that may be integrated into imaging assembly (522) of system (500). In particular, the arrangement shown in FIG. 8 may represent a variation of light emitting assembly (550). The arrangement shown in FIG. 8 may be particularly useful in scenarios where camera system (540) includes a TDI camera. In the arrangement shown in FIG. 8, a line generation module (LGM) (602) and emission optics module (EOM) (604) are aligned and mechanically coupled to precision mounting plate (610), as well as to each other. EOM (604) includes an objective lens assembly (606) that is aligned, via a mirror (608) with a tube lens (620), which in turn is optically coupled to LGM (602). LGM (602) may include one or more light sources (e.g., coherent light sources such as laser diodes). In some examples, LGM (602) may include a first light source configured to emit light in red wavelengths, and a second light source configured to emit light in green wavelengths. LGM (602) may further include optical components, such as focusing surfaces, lenses, reflective surfaces, or mirrors. The optical components may be positioned within an enclosureof LGM (602) to direct and focus the light emitted from the one or more light sources into an adjacent modular subassembly. One or more of the optical components of LGM (602) may also be configured to shape the light emitted from the one or more light sources into desired patterns. For example, in some implementations, the optical components may shape the light into line patterns (e.g., by using one or more Powell lenses, or other beam shaping lenses, diffractive or scattering components). In some variations, LGM (602) may include one or more laser modules which may be individually removed from LGM (602) and replaced.

[0099] Light beams generated by LGM (602) transmit through an interface baffle between LGM (602) and EOM (604), pass through objective lens assembly (606), and strike an optical target (e.g., 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 light radiation from the target may pass back through objective lens assembly (606) and into tube lens (622). A lens element (622), which may form part of tube lens (620), is configured to articulate along an axis (e.g., a z-axis) to correct for spherical aberration artifacts introduced by objective lens assembly (606) imaging through varied thickness of flow cell (510) components. As illustrated, lens element (622) may be articulated closer to or further away from objective lens assembly (606) to adjust the beam shape and path. Objective lens assembly (606) may emit excitation light toward the optical target (e.g., flow cell (510)) and receive fluorescence emission from the optical target. An actuator may be configured to position objective lens assembly (606) to a region of interest proximate to the optical target. The processor of controller (520) may then execute program instructions for detecting fluorescence emission from the optical target.

[0100] FIG. 9 shows an example of another configuration that may be provided in imaging assembly (522). In particular, FIG. 9 shows an imaging assembly (650) positioned in relation to a flow cell (670). Flow cell (670) may be representative of any of the variations of flow cells (128, 368, 400, 450, 510) described herein. Flow cell (670) has an upper layer (671) and a lower layer (673) that are separated by a fluid filled channel (675). In the configuration shown, upper layer (671) is optically transparent andimaging assembly (650) is focused to an area (676) on inner surface (672) of upper layer (671). In other variations, imaging assembly (650) may be focused on inner surface (674) of lower layer (673). One or both of surfaces (672, 674) may include array features that are to be detected by imaging assembly (650).

[0101] Imaging assembly (650) includes an objective lens assembly (666) that is configured to direct excitation radiation from a light emitting assembly (652) to flow cell (670); and to direct emission from flow cell (670) to a detector (664). In the arrangement shown, excitation radiation from light emitting assembly (652) passes through a lens (658), though a beam splitter (660), and through objective lens assembly (666) on to reach flow cell (670). In the present example, light emitting assembly (652) includes two light emitting diodes (LEDs) (656, 654), which produce radiation at different wavelengths from each other. The emission radiation from flow cell (670) is captured by objective lens assembly (666) and is reflected by beam splitter (660) through conditioning optics (662) and to detector (664) (e.g., a CMOS sensor). Beam splitter (660) functions to direct the emission radiation in a direction that is orthogonal to the path of the excitation radiation. The position of objective lens assembly (666) may be moved in the z dimension to alter focus of imaging assembly (650). The imaging assembly (650) may be moved back and forth in the y direction to capture images of several areas of at least one inner surface (672, 674) of flow cell (670).

[0102] In the present 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 from fluorophore labels in flow cell (670) in response to irradiation at these 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 irradiation at only one single respective wavelength. As another variation, two or more detectors (664) may receive excitation radiation from a common light emitting assembly (652).

[0103] FIG. 10 shows an example of another configuration that may be provided in imaging assembly (522). In particular, FIG. 10 shows an imaging assembly (700) positioned inrelation to a flow cell (774). Flow cell (774) may be representative of any of the variations of flow cells (128, 368, 400, 450, 510) described herein. Flow cell (770) has a translucent cover plate (772), a substrate (774), and a liquid layer (776) that is interposed between cover plate (772) and substrate (774). A biological sample may be located on an inside surface of cover plate (772) (above liquid layer (776)) and / or on an inside surface of substrate (774) (below liquid layer (776)).

[0104] Imaging assembly (700) of this example includes an LGM (710) with two light sources (712, 714), disposed therein. Light sources (712, 714) may include laser diodes, diode pumped solid state lasers, or other light sources as known in the art, which output laser beams at different wavelengths (e.g., red or green light). The light beams output from light sources (712, 714) are directed through a beam shaping lens or lenses (716). In some implementations, one or more light shaping lenses may be used to shape the light beams output from each or both light sources. LGM (710) may use one or more Powell lenses to spread and / or shape the laser beams from single or near-single mode laser light sources. Other beam shaping optics may be used to control uniformity and increase tolerance such as an active beam expander, an attenuator, one relay lenses, cylindrical lenses, actuated mirrors, diffractive elements, and scattering components. Laser beams may intersect at the back focal point of the objective lens to provide better tolerance on surfaces of flow cell (770).

[0105] LGM (710) ofthis example further includes mirrors (718, 720). A light beam generated by light source (712) reflects off mirror (718), as to be directed through an aperture or semi-reflective surface of mirror (720), and into EOM (740) through a single interface port. Similarly, a light beam generated by light source (714) reflects off of mirror (720) as to be directed into EOM (740) through a single interface port. In some examples, an additional set of articulating mirrors may be incorporated adjacent to mirrors (718, 720) to provide additional tuning surfaces. Both light beams may be combined using dichroic mirror (720). Mirrors (718, 720) may each be configured to articulate using manual or automated controls to align the light beams from light sources (712, 714). The light beams also pass through a shutter element (722) in the present example.

[0106] EOM (740) includes an objective lens assembly (756) and a z-stage (758), which movesobjective lens assembly (756) longitudinally closer to or further away from flow cell (770). LGM (710) is configured to generate a uniform line illumination through objective lens assembly (756). Z-stage (758) may then move objective lens assembly (756) as to focus the light beams onto either of the inside surfaces of flow cell (770) (e.g., focused on a biological sample). In some implementations, the objective lens assembly (756) may be configured to focus the light beams at a focal point beyond flow cell (770), such as to increase the line width of the light beams at the surfaces of flow cell (770).

[0107] EOM (740) of the present example also include a semi-reflective mirror (754) to direct light through objective lens assembly (756), while allowing light returned from flow cell (774) to pass through. EOM (740) further includes a tube lens (744) and a corrective lens (748). Corrective lens (748) may be articulated longitudinally by a z- stage (746), either closer to or further away from objective lens assembly (756), to ensure accurate imaging (e.g., to correct spherical aberration caused by moving objective lens assembly (756); and / or from imaging through a thicker substrate, etc.). Light transmitted through corrective lens (748) and tube lens (744) passes through filter element (742) and into camera system (730). Camera system (730) includes one or more optical sensors (732) to detect light emitted from the biological sample in response to the incident light beams.

[0108] In the present example, EOM (740) further includes semi-reflective mirror (752) to reflect a focus tracking light beam emitted from a focus tracking module (FTM) (760) onto flow cell (774), and then to reflect light returned from flow cell (774) back into FTM (760). FTM (760) may include a focus tracking optical sensor to detect characteristics of the returned focus tracking light beam and generate a feedback signal to optimize focus of objective lens assembly (756) on flow cell (774).

[0109] The direction, size, and / or polarization of the laser beams may be adjusted by using lenses, mirrors, and / or polarizers. Optical lenses (e.g., cylindrical, spherical, or aspheric) may be used to actively adjust the illumination focus on dual surfaces of the flow cell (770) target. LGM (710) may also include multiple units, with each unit being designed for particular / different wavelengths and polarization. Stacking multiple unitsmay be used to increase the laser power and wavelength options. Two or more laser wavelengths may be combined with dichroics and polarizers.

[0110] By way of example only, focus tracking module (560) and / or other components of imaging assembly (522) may be constructed and operable in accordance with at least some of the teachings of U.S. Pat. No. 10,416,428, entitled “Systems and Methods for Improved Focus Tracking Using a Light Source Configuration,” issued September 17, 2019, the disclosure of which is incorporated by reference herein, in its entirety; U.S. Pub. No. 2023 / 0228984, entitled “Dynamic Detilt Focus Tracking,” published July 20, 2023, the disclosure of which is incorporated by reference herein, in its entirety; and / or U.S. Pub. No. 2024 / 0114243, entitled “Spot Error Handling for Focus Tracking,” published April 4, 2024, the disclosure of which is incorporated by reference herein, in its entirety. By way of further example only, components of imaging assembly (522) may be configured and operable in accordance with at least some of the teachings of U.S. Pat. No. 10,774,371, entitled “Laser Line Illuminator for High Throughput Sequencing,” issued September 15, 2020, the disclosure of which is incorporated by reference herein, in its entirety; and / U.S. Pat. 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.

[0111] VI. Examples of Immersion Microscopy Features for Sequencing System

[0112] As noted above, some versions of an imaging assembly (522, 650, 700) in a sequencing system (500) may include an objective lens assembly (542, 606, 666, 756) positioned over a flow cell (128, 368, 400, 450, 450A, 510, 670, 770) to capture images with fluorescent emissions from fluorophore labels of nucleotides in flow cell (128, 368, 400, 450, 450A, 510, 670, 770). In some instances, it may be desirable to provide an immersion fluid (e.g., water, oil, etc.) between objective lens assembly (542, 606, 666, 756) and the upper surface of flow cell (128, 368, 400, 450, 450A, 510, 670, 770), to thereby provide an immersion microscopy arrangement. Such an immersion fluid may enhance the resolution and clarity of images captured via objective lens assembly (542, 606, 666, 756), effectively increasing the numerical aperture of objective lens assemblyby increasing the refractive index that would otherwise be provided in an air gap between objective lens assembly (542, 606, 666, 756) positioned over a flow cell (128, 368, 400, 450, 450A, 510, 670, 770). The following provides examples of how immersion fluid features may be integrated into an imaging assembly of a sequencing system, such as any of the imaging assemblies (522, 650, 700) described herein.

[0113] A. Overview

[0114] FIG. 11 shows an example of an arrangement (1000) that may be incorporated into any of the various sequencing systems described herein. Arrangement (1000) of this example includes a frame (1010), a sample stage (1020), a flow cell (1030), and an imaging assembly (1040). Frame (1010) is shown schematically and may take various suitable forms. For instance, frame (1010) may comprise a stationary superstructure within a sequencing instrument or system enclosure, providing a mechanical ground for other features of arrangement (1000).

[0115] Sample stage (1020) may be configured and operable like sample stage (570) described above, such that sample stage (1020) may provide structural support to flow cell (1030); and further provide movement and alignment of flow cell (1030) relative to objective lens assembly (2000). Sample stage (1020) may have one or more actuators to allow sample stage (1020) to move in any of three dimensions. For example, actuators may be provided to allow sample stage (1020) to move in the x, y, and z directions relative to objective lens assembly (2000), tilt relative to objective lens assembly (2000), and / or otherwise move relative to objective lens assembly (2000). Flow cell (1030) may be configured and operable like any of the other flow cells (128, 368, 400, 450, 450A, 510, 670, 770) described herein.

[0116] Imaging assembly (1040) of this example comprises an objective lens assembly (2000), an immersion fluid manifold (2040), and a set of additional imaging components (1044) that are shown schematically in FIG. 11. These additional imaging components (1044) may include any of the various imaging components of any of the various imaging assemblies (522, 650, 700) described herein. Specific features and functionalities of objective lens assembly (2000) and immersion fluid manifold (2040) will be describedin greater detail below. Imaging assembly (1040) of this example is coupled with frame (1010) via a mount (1042) in this example. In some versions, mount (1042) provides a static, fixed relationship between imaging assembly (1040) and frame (1010), such that imaging assembly (1040) does not move relative to frame (1010). In some other versions, at least a portion of imaging assembly (1040) moves relative to frame (1010). For instance, mount (1042) may include one or more motors, servos, or other actuators, etc. that provide(s) controlled movement of imaging assembly (1040) in one or more of the x-dimension, the y-dimension, or the z-dimension relative to frame (1010). In addition, or in the alternative, one or more features within imaging assembly may provide movement of objective lens assembly (2000) and / or one or more focusing lens elements relative to frame (1010), such as to provide controlled focus of an imaging region on or in flow cell (1030), etc.

[0117] As shown in FIG. 12, an immersion fluid source (2080) is fluidically coupled with immersion fluid manifold (2040) via a fluid conduit (2082). Immersion fluid source (2080) may include any suitable kind of immersion fluid, including but not limited to water (e.g., HPLC double distilled water, etc.), oil, etc. A suction source (2090) is also fluidically coupled with immersion fluid manifold (2040) via a fluid conduit (2092). By way of example only, each fluid conduit (2082, 2092) may include a flexible tube and / or any other suitable component s). In some versions, suction source (2090) is also fluidically coupled with immersion fluid source (2080) via a third conduit (not shown) such that fluid communicated from immersion fluid source (2080) to immersion fluid manifold (2040) may be recirculated back to immersion fluid source (2080) via suction source (2090) and the third conduit. In some other versions, suction source (2090) is fluidically coupled with a tank or other fluid container, such that fluid drawn from immersion fluid manifold (2040) by suction source (2090) is deposited in the tank or other fluid container for disposal or other handling. Either or both of immersion fluid source (2080) or suction source (2090) may include a pump that is operable to convey immersion fluid to immersion fluid manifold (2040) or draw immersion fluid from immersion fluid manifold (2040) in accordance with the teachings herein.

[0118] B. Example of Objective Lens Assembly

[0119] FIGS. 13-14 show features of objective lens assembly (2000) in greater detail. As shown, objective lens assembly (2000) of this example includes an objective lens element (2002) that is supported by a housing (2006). Housing (2006) includes a tapered distal portion (2008) that terminates in an annular distal face (2010). Annular distal face (2010) surrounds distal face (2004) of objective lens element (2002). In some versions, distal face (2004) of objective lens element (2002) is substantially flush with annular distal face (2010). In some other versions, objective lens element (2002) is recessed relative to annular distal face (2010). Alternatively, objective lens element (2002) may be recessed relative to annular distal face (2010) at any other suitable depth.

[0120] In the present example, an annular recess (2012) is formed in annular distal face (2010). Annular recess (2012) of has a cross-sectional profde resembling a pyramidal frustum in this example. Alternatively, annular recess (2012) may have any other suitable kind of cross-sectional profile, including but not limited to rounded / concave, triangular, rectangular, etc. While only one annular recess (2012) is provided in this example, other versions may include two or more annular recesses (2012) (e.g., in a concentric arrangement along annular distal face (2010)). Annular recess (2012) of the present example is configured to assist in retention of a volume of immersion fluid (IF) between objective lens assembly (2000) and an upper surface (1032) of flow cell (1030), even during movement of flow cell (1030) relative to objective lens assembly (2000), as described in greater detail below.

[0121] While objective lens element (2002) of the present example has a flat distal face (2004), other versions of objective lens element (2002) may have a concave distal face. In some scenarios, a concave version of distal face (2004) may assist in retention of a volume of immersion fluid (IF) between objective lens assembly (2000) and an upper surface (1032) of flow cell (1030), even during movement of flow cell (1030) relative to objective lens assembly (2000), as described in greater detail below. Some versions of objective lens assembly (2000) may include a combination of a concave distal face and one or more annular recesses (2012) to assist in retention of a volume of immersion fluid (IF) between objective lens assembly (2000) and an upper surface (1032) of flow cell (1030). In addition, or in the alternative, distal face (2004) of objective lenselement (2002) and / or distal face (2010) of housing (2006) may include a hydrophilic material (e.g., polyetherimide, acrylic, Inconel, etc.) and / or other treatment to assist in retention of a volume of immersion fluid (IF) between objective lens assembly (2000) and an upper surface (1032) of flow cell (1030).

[0122] C. Example of Immersion Fluid Manifold

[0123] FIGS. 15-16 show features of immersion fluid manifold (2040) in greater detail. As shown, immersion fluid manifold (2040) of this example includes an annular body(2042) with an inner surface (2043) that is angled to taper inwardly toward the bottom of body (2022). The angle of inner surface (2043) is configured to complement the angle of tapered distal portion (2008) of housing (2006) of objective lens assembly (2000). Inner surface (2043) thus fits together in apposition with tapered distal portion (2008) when immersion fluid manifold (2040) is secured at the bottom of objective lens assembly (2000) as best seen in FIG. 16. In some versions, the fit between inner surface(2043) and tapered distal portion (2008) is substantially fluid tight, thereby preventing undesired migration of immersion fluid, into a space that may otherwise be defined between inner surface (2043) and tapered distal portion (2008), during operation as described in greater detail below.

[0124] A central opening (2045) of immersion fluid manifold (2040) is sized to accommodate annular distal face (2010) of housing (2006) of objective lens assembly (2000), such that immersion fluid manifold (2040) does not obstruct the field of view through objective lens element (2002). Similarly, immersion fluid manifold (2040) does not cover any region of annular recess (2012), such that immersion fluid manifold (2040) does not impede the fluid retention capabilities of annular recess (2012) as described herein.

[0125] Immersion fluid manifold (2040) of the present example further includes a recess(2044) under and around central opening (2045). Recess (2044) is surrounded by a rim (2046) at the bottom of body (2042). As best seen in FIG. 15, recess (2044) has an elliptical shape In some scenarios, the elliptical shape and orientation of recess (2044) may promote retention of a volume of immersion fluid (IF) in the space betweenobjective lens element (2002) and upper surface (1032) of flow cell (1030), including while there is relative motion between flow cell (1030) and objective lens assembly (2000) during an image capture process.

[0126] As best seen in FIG. 16, immersion fluid manifold (2040) of the present example further includes a first port (2050), a first channel (2052), a second port (2056), and a second channel (2058). First port (2050) is positioned on a laterally facing region of body (2042) and is configured to couple with immersion fluid source (2080) via fluid conduit (2082), as described above with reference to FIG. 12. Immersion fluid manifold (2040) thus receives immersion fluid via fluid conduit (2082) and first port (2050). In some versions, first port (2050) includes an outwardly extending rigid fitting and / or other structural feature to facilitate coupling with fluid conduit (2082). First channel (2052) extends from first port (2050) to recess (2044), providing a path for communication of immersion fluid from immersion fluid source (2080) to reach recess (2044). Thus, a volume of immersion fluid from immersion fluid source (2080) may be communicated to the space between objective lens element (2002) and upper surface (1032) of flow cell (1030) via fluid conduit (2082), first port (2050), and first channel (2052).

[0127] Second port (2056) is also positioned on a laterally facing region of body (2042) and is configured to couple with suction source (2090) via fluid conduit (2092), as described above with reference to FIG. 12. Immersion fluid is thus drawn from immersion fluid manifold (2040) via fluid conduit (2092) and second port (2056). In some versions, second port (2056) includes an outwardly extending rigid fitting and / or other structural feature to facilitate coupling with fluid conduit (2092). Second channel (2058) extends from second port (2056) to recess (2044), providing a path for communication of immersion fluid from recess (2044). Thus, suction source (2090) may be activated to draw / remove a volume of immersion fluid from the space between objective lens element (2002) and upper surface (1032) of flow cell (1030) via second channel (2058), second port (2056), and fluid conduit (2092). This drawing / removal of immersion fluid may be provided during a de-priming step of operation as described in greater detail below. In addition, or in the alternative, such drawing of immersion fluid by suctionsource (2090) may be provided simultaneously with communication of immersion fluid from immersion fluid source (2080), such as during circulation of immersion fluid within the space between objective lens element (2002) and upper surface (1032) of flow cell (1030).

[0128] D. Example of Use of Imaging Assembly with Immersion MicroscopyFeatures

[0129] FIGS. 17A-17B schematically depict an example of an imaging process where imaging assembly (1040) is used to capture images with fluorescent emissions from fluorophore labels of nucleotides in flow cell (1030). As shown, flow cell (1030) includes eight channels (1034). By way of example only, each channel (1034) of flow cell (1030) may be configured and operable like each channel (130) of flow cell (128) described above, like each channel (430) of flow cell (400) described above, or like each channel (480) of flow cell (450) described above, or otherwise. At the beginning of an imaging process, imaging assembly (1040) is positioned at a first end of a first channel (1034a), as shown in FIG. 17A. The ellipse (1036) in FIG. 17A represents the footprint of rim (2046) of immersion fluid manifold (2040) over upper surface (1032) of flow cell (1030).

[0130] Through the imaging process, flow cell (1030) moves relative to imaging assembly (1040) along the x-y plane, allowing imaging assembly (1040) to capture numerous images along the length and width of first channel (1034a). As noted above, this relative movement is provided by one or more actuators of sample stage (1020); while imaging assembly (1040) remains stationary along the x-y plane. As also noted above, some other variations may provide driven movement of imaging assembly (1040) along the x-y plane; while flow cell (1030) remains stationary along the x-y plane. In either scenario, after imaging assembly (1040) has captured sufficient images along first channel (1034a), the relative position between imaging assembly (1040) and flow cell (1030) continues to change to allow imaging assembly (1040) to capture images along second channel (1034b), along third channel (1034c), along fourth channel (1034d), along fifth channel (1034e), along sixth channel (1034f), along seventh channel (1034g), and along eighth channel (1034h). At the end of this imaging process, imagingassembly (1040) is positioned at a second end of a eighth channel (1034h), as shown in FIG. 17B.

[0131] FIGS. 18A-18D also schematically depict an example of an imaging process where imaging assembly (1040) is used to capture images with fluorescent emissions from fluorophore labels of nucleotides in flow cell (1030). As shown, in FIG. 18A, the process may begin with positioning of flow cell (1030) under objective lens assembly (2000). With flow cell (1030) suitably positioned under objective lens assembly (2000), and as shown in FIG. 18B, immersion fluid manifold (2040) is primed. While immersion fluid source (2080) is not shown in FIG. 18B, this priming step includes communication of immersion fluid from immersion fluid source (2080) to recess (2044) via conduit (2082) first port (2050) and first channel (2052). This priming step results in a volume of immersion fluid (IF) in the space between objective lens element (2002) and upper surface (1032) of flow cell (1030).

[0132] In some versions, immersion fluid is continuously circulated to this space through continuous deposition of immersion fluid from immersion fluid source (2080) and continuous drawing-off of immersion fluid by suction source (2090). In some such versions, the immersion fluid is provided at a certain temperature, or within a certain temperature range, to regulate the temperature of objective lens element (2002) and / or flow cell (1030). In addition, or in the alternative, one or more thermal elements (e.g., within objective lens assembly (2000), within immersion fluid manifold (2040), and / or elsewhere) may be used to regulate the temperature of objective lens element (2002), the volume of immersion fluid (IF), and / or flow cell (1030). Further examples of features and techniques that may be used to provide thermal regulation are described in greater detail below.

[0133] Once the priming has provided a sufficient volume of immersion fluid (IF) in the space between objective lens assembly (2000) and upper surface (1032) of flow cell (1030), and as shown in FIG. 18C, the process may proceed to a scan of flow cell (1030). During this scanning process, relative movement may be provided between flow cell (1030) and imaging assembly (1040), such that imaging assembly (1040) captures images along the length and width of each channel (1034) of flow cell (1030) asdescribed above with reference to FIGS. 17A-17B. As shown in FIG. 18C, the volume of immersion fluid (IF) may remain in the space between objective lens element (2002) and upper surface (1032) during the relative movement between flow cell (1030) and imaging assembly (1040), though the volume of immersion fluid (IF) may encounter some degree of lateral deformation due to shearing forces. The volume of immersion fluid (IF) may be sufficiently maintained between objective lens element (2002) and upper surface (1032) even when the relative movement between flow cell (1030) and imaging assembly (1040) is substantially fast (e g., between approximately 20 mm / s and approximately 100 mm / s, or faster than approximately 100 mm / s).

[0134] After flow cell (1030) has been sufficiently scanned by imaging assembly (1040), with a sufficient number of images captured, the process may proceed to a de-priming step as shown in FIG. 18D. In some versions, the relative movement between flow cell (1030) and imaging assembly (1040) has ceased when the de-priming step is initiated. To provide the de-priming, suction source (2090) is activated to draw the volume of immersion fluid (IF) out of the space between objective lens assembly (2000) and upper surface (1032). After the de-priming by suction source (2090) and immersion fluid manifold (2040), flow cell (1030) may be removed. In some cases, there are no additional flow cells (1030) to scan, such that the process may then end. In some other cases, there is at least one more flow cell (1030) to scan. In such cases, the process may return to the beginning of the process described above, positioning the next flow cell as shown in FIG. 18A, etc., cycling through the above-described steps until all flow cells (1030) have been scanned.

[0135] VII. Examples of Features to Reduce, Prevent, or Mitigate Thermally Induced Aberrations in Immersion Microscopy System

[0136] In some scenarios, flow cell (128, 368, 400, 450, 450A, 510, 670, 770, 1030) may be heated to achieve a target temperature that is selected to promote a desired performance during the sequencing and imaging processes. During the sequencing and imaging process, it may be desirable to maintain that target temperature. In addition, objective lens assembly (542, 606, 666, 756, 2000) may start at an ambient temperature and then heat up during operation, in response to light passing through objective lens assembly(542, 606, 666, 756, 2000). The initial temperature of objective lens assembly (542, 606, 666, 756, 2000), operating temperature of objective lens assembly (542, 606, 666, 756, 2000), and rate of change in temperature of objective lens assembly (542, 606, 666, 756, 2000) may tend to differ from the initial temperature of flow cell (128, 368, 400, 450, 450A, 510, 670, 770, 1030), operating temperature of flow cell (128, 368, 400, 450, 450A, 510, 670, 770, 1030), and rate of change in temperature of flow cell (128, 368, 400, 450, 450A, 510, 670, 770, 1030), respectively.

[0137] In objective lens assemblies (542, 606, 666, 756) of imaging assemblies (522, 650, 700) that lack immersion fluid features such as immersion fluid manifold (2040), temperature differentials between objective lens assembly (542, 606, 666, 756) and flow cell (128, 368, 400, 450, 450A, 510, 670, 770) may be acceptable, such that the temperature differentials do not adversely affect performance of objective lens assembly (542, 606, 666, 756). To the extent that the temperature changes in objective lens assemblies (542, 606, 666, 756) provide any thermally induced aberrations or other optical effects, such effects may be negligible, accounted for through optics or image processing, or otherwise acceptable. However, in arrangements like arrangement (1000) where immersion fluid is interposed between flow cell (1030) and objective lens assembly (2000), the immersion fluid may serve as a thermal conductor, such that heat may be transferred from flow cell (1030) to objective lens assembly (2000) via the immersion fluid (and vice-versa). Such heat transfer may induce thermal gradients across elements of objective lens assembly (2000), and these thermal gradients may produce optical aberrations or other optical effects that would not otherwise occur in the absence of immersion fluid. These optical aberrations or other optical effects that are thermally induced via the immersion fluid may in turn adversely impact the quality of images obtained through objective lens assembly (2000). For instance, in a system with circularly symmetric heating, defocus or spherical or other optical aberrations may occur. Within a system with asymmetric or large aspect ratio elliptical heating, astigmatism may occur.

[0138] While temperature changes in flow cell (1030) may generate thermal gradients within objective lens assembly (2000) due to thermal conduction through immersion fluid,which may in turn induce new aberrations or changes in aberrations within objective lens assembly (2000), temperature changes in objective lens assembly (2000) may also affect the temperature of flow cell (1030) due to thermal conduction through immersion fluid. For instance, when a laser or other form of light is projected through objective lens assembly (2000) for a certain period of time, the front objective element within objective lens assembly (2000) may tend to heat up, and this increase in temperature may be transferred to flow cell (1030) via the immersion fluid that is interposed between the front objective element within objective lens assembly (2000). In addition, or in the alternative, if a heating or cooling feature is activated to regulate the temperature of objective lens assembly (2000), such heating or cooling effects may also reach flow cell (1030) via the immersion fluid. In some cases, changes in the temperature of flow cell (1030) that are provided through immersion fluid may adversely affect sequencing or other processes occurring within flow cell (1030).

[0139] In view of the foregoing, it may be desirable to provide features that reduce, prevent, or mitigate thermally induced optical aberrations in an immersion microscopy system such as that provided by arrangement (1000), without adversely affecting sequencing or other processes occurring within flow cell (1030). The following describes examples of various features and techniques that may be used to reduce, prevent, or mitigate thermally induced aberrations in an immersion microscopy system without adversely affecting sequencing or other processes occurring within flow cell (1030). The following teachings may be readily incorporated into any of the systems (100, 300, 500) described herein and / or in any other systems where an immersion fluid may tend to serve as a thermal conductor that transfers heat resulting in thermally induced optical aberrations.

[0140] A. Example of Arrangement with Flow Cell Thermal Regulation Feature

[0141] As noted above, flow cell (1030) may be heated to achieve a target temperature that is selected to promote a desired performance during the sequencing and imaging processes. Flow cell (1030) may also be cooled to achieve a target temperature that is selected to promote a desired performance during the sequencing and imaging processes. In scenarios where thermal conduction via immersion fluid may result inthe temperature of flow cell (1030) affecting the temperature of objective lens assembly (2000) and / or the temperature of objective lens assembly (2000) affecting the temperature of flow cell (1030), the temperature of objective lens assembly (2000) and the temperature of flow cell (1030) may both be monitored, and the data representing these temperatures may be used to drive a feature that dynamically heats or cools flow cell (1030) to maintain or otherwise achieve a desired temperature. Actively regulating the temperature of flow cell (1030) based on real-time sensed temperature data may prevent or at least reduce the occurrence of optical thermally induced aberrations in objective lens assembly (2000) that might otherwise occur from thermal conduction via immersion fluid.

[0142] FIG. 19 shows an arrangement (3000) where a processor (3010) is electrically coupled with a set of temperature sensors (3020, 3030) and a thermal regulator (3040). Processor (3010) is thus configured to receive signals from temperature sensors (3020, 3030) and drive thermal regulator (3040) in accordance with one or more control algorithms. Examples of such control algorithms are described in greater detail below. Processor (3010) may 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 executing various functions including the ones described herein. In some versions, processor (3010) is further configured to provide other functionalities. For instance, processor (3010) may constitute the same component as processors (210, 378) described above; or may be configured to provide at least some of the functionalities describe above with respect to processors (210, 378).

[0143] Temperature sensor (3020) is thermally coupled with objective lens assembly (2000) such that temperature sensor (3020) is configured to sense the temperature of objective lens assembly (2000). Temperature sensor (3020) may take any suitable form (e.g., thermocouple, thermistor, etc.) as will be apparent to those skilled in the art in view of the teachings herein. While temperature sensor (3020) is shown as being positionedalong a side of objective lens assembly (2000), this is merely a schematic, illustrative representation. Temperature sensor (3020) may be positioned anywhere on and / or within objective lens assembly (2000). For instance, in some versions of arrangement (3000), temperature sensor (3020) is positioned on housing (2006) near the front objective lens element (2002).[00144J Some versions of temperature sensor (3020) may include two or more temperature sensors, such that the term “temperature sensor” should be read to include a plurality of sensors. Such a plurality of temperature sensors may be configured to sense temperatures at different respective regions of objective lens assembly (2000). It should also be understood that, to the extent the description herein refers to temperature sensor (3020) being configured to sense the temperature of objective lens assembly (2000) in general, some versions of temperature sensor (3020) may only in fact sense the temperature of one or more particular regions of objective lens assembly (2000) (e.g., a housing of objective lens assembly (2000), a certain lens element of objective lens assembly (2000), etc.). In some cases, objective lens assembly (2000) may tend to operate at a temperature within a range from approximately 20° C to approximately 35° C.

[0145] Temperature sensor (3030) is thermally coupled with flow cell (1030) such that temperature sensor (3030) is configured to sense the temperature of flow cell (1030). Temperature sensor (3030) may take any suitable form (e.g., thermocouple, thermistor, etc.) as will be apparent to those skilled in the art in view of the teachings herein. While temperature sensor (3030) is shown as being positioned along an upper surface of flow cell (1030), this is merely a schematic, illustrative representation. Temperature sensor (3030) may be positioned anywhere on and / or within flow cell (1030). Moreover, as with temperature sensor (3020) described above, some versions of temperature sensor (3030) may include two or more temperature sensors, such that the term “temperature sensor” should be read to include a plurality of sensors. Such a plurality of temperature sensors may be configured to sense temperatures at different respective regions of flow cell (1030). It should also be understood that, to the extent the description herein refers to temperature sensor (3030) being configured to sense the temperature of flow cell(1030) in general, some versions of temperature sensor (3030) may only in fact sense the temperature of one or more particular regions of flow cell (1030) (e.g., an upper surface of flow cell (1030), etc.).

[0146] Thermal regulator (3040) is thermally coupled with flow cell (1030) such that thermal regulator (3040) is operable to actively heat or cool flow cell (1030) in response to a drive signal from processor (3010). In some cases, thermal regulator (3040) may be preset to provide flow cell (1030) at or near a certain operating temperature (e.g., approximately 35° C). As with heater / cooler (532) described above, at least a portion of thermal regulator (3040) may be integrated into or onto sample stage (1020), which provides structural support to flow cell (1030). In addition, or in the alternative, at least a portion of thermal regulator (3040) may be integrated into or onto flow cell (1030). Thermal regulator (3040) may include any of various kinds of features to provide active heating or cooling of at least a portion of flow cell (1030) in response to a drive signal from processor (3010). By way of example only, thermal regulator (3040) may include one or more resistive heating elements (e.g., a resistive element encapsulated in a ceramic and metal component, a cartridge heater, etc.), a thermoelectric cooler, one or more conduits to contain or convey heated or cooled fluid, one or more conduits to convey heated or cooled air, and / or any other suitable features. Some versions of thermal regulator (3040) may only provide heating without providing cooling; while other versions may only provide cooling without providing heating. The portion of thermal regulator (3040) that is in direct contact with flow cell (1030) may include a thermally conductive material such as ceramic, etc.

[0147] While thermal regulator (3040) is shown as being positioned under flow cell (1030), this is merely a schematic, illustrative representation. Thermal regulator (3040) may be positioned anywhere on and / or within flow cell (1030). Moreover, while thermal regulator (3040) is schematically depicted as a single element, some versions of thermal regulator (3040) may include two or more elements, such that the term “thermal regulator” should be read to include a plurality of thermal regulating elements. Such a plurality of thermal regulating elements may be configured to heat and / or cool different respective regions of flow cell (1030).

[0148] As noted above, processor (3010) may receive and process signals from temperature sensor (3020), which may indicate the temperature of one or more regions of objective lens assembly (3000). Processor (3010) may also receive and process signals from temperature sensor (3030), which may indicate the temperature of one or more regions of flow cell (1030). Based on these signals from temperature sensors (3020, 3030), processor (3010) may execute one or more control algorithms, and thereby drive thermal regulator (3040) in accordance with such one or more control algorithms.

[0149] FIG. 20 shows an example of a process (3050) that may be executed by processor (3010). As shown, processor (3010) senses (block 3052) the temperature of flow cell (1030) based on a signal from temperature sensor (3030). Processor (3010) further senses (block 3054) the temperature of objective lens assembly (2000) based on a signal from temperature sensor (3020). While FIG. 20 shows these sensing steps (blocks 3052, 3054) as being performed in a sequence, these sensing steps (blocks 3052, 3054) may be performed simultaneously or in a sequence that is reversed from the sequence shown. With temperature data from both sensors (3020, 3030), processor (3010) calculates (block 3056) a difference between the temperature of flow cell (1030) and the temperature of objective lens assembly (2000). Processor (3010) then determines (block 3058) whether this temperature difference exceeds a threshold. By way of example only, such a threshold may include approximately 0° C, approximately 20° C, approximately 35° C, or any other suitable temperature threshold.

[0150] If the temperature difference does not exceed the threshold, process (3050) may reiterate through the steps described above, such that processor (3010) may continue to monitor the temperature data and determine (block 3058) whether / when any temperature differences exceed the threshold. In the event that the temperature difference exceeds the threshold, processor (3010) may adjust (block 3060) the temperature of flow cell (1030) by activating thermal regulator (3040). In some cases, this may include activating thermal regulator (3040) to reduce the temperature of flow cell (1030). In some other cases, this may include activating thermal regulator (3040) to increase the temperature of flow cell (1030). Regardless of whether thermal regulator (3040) is activated to reduce or increase the temperature of flow cell (1030),such activation may be provided in a pulsed fashion or in a continuous fashion.

[0151] Processor (3010) may continue to monitor the temperature data and determine (block 3058) whether / when the temperature difference no longer exceeds the threshold. When this occurs, processor (3010) may deactivate thermal regulator (3040), drive thermal regulator (3040) to maintain the current temperature of flow cell (1030), or provide some other kind of response.

[0152] While process (3050) depicted in FIG. 20 includes reliance on data from temperature sensors (3020, 3030), a variation of process (3050) may be carried out without data from temperature sensors (3020, 3030). This variation of process (3050) may still provide minimization of the difference between the temperature of flow cell (1030) and the temperature of objective lens assembly (2000). For instance, the temperature of flow cell (1030) may be maintained at approximately 65° C during a sequencing process; and then reduced by thermal regulator (3040) to a temperature in a range from approximately 20° C to approximately 35° C during a scanning process to more closely match with a temperature of objective lens assembly (2000) during the scanning process. It should be understood that, in some cases, objective lens assembly (2000) is not used during a sequencing process; and immersion fluid may not be interposed between objective lens assembly (2000) and flow cell (1030) during the sequencing process.

[0153] In some cases, changes to flow cell (1030) temperature that are driven by thermal regulator (3040) may tend to result in changes to the intensity of dyes in flow cell (1030) and / or induce other changes in flow cell (1030). In some such scenarios, the intensity and / or exposure time of excitation light (e.g., laser light) projected through objective lens assembly (2000) toward flow cell (1030) may be adjusted so that the collected light (e.g., photons emitted by fluorophores excited by the excitation light) are constant across the temperature range.

[0154] B. Example of Arrangement with Flow Cell Thermal Regulation Feature and Objective Lens Assembly Thermal Regulation Feature

[0155] While arrangement (3000) of FIG. 19 provides monitoring of temperatures of objectivelens assembly (2000) and flow cell (1030), and actively driven regulation of the temperature of flow cell (1030), it may be desirable to further provide actively driven regulation of the temperature of objective lens assembly (2000). To that end, FIG. 21 shows an example of an arrangement (4000) where a processor (4010) is electrically coupled with a set of temperature sensors (4020, 4030) and a set of thermal regulators (4022, 4040). Processor (4010) is thus configured to receive signals from temperature sensors (4020, 4030) and drive either or both of thermal regulators (4022, 4040) in accordance with one or more control algorithms. Examples of such control algorithms are described in greater detail below.

[0156] Processor (4010) may be configured and operable like processor (3010) described above. Temperature sensor (4020) may be configured and operable like temperature sensor (3020) described above. Temperature sensor (4030) may be configured and operable like temperature sensor (3030) described above. Thermal regulator (4040) may be configured and operable like thermal regulator (3040) described above. Thermal regulator (4022) may also be configured and operable like thermal regulator (3040) described above, though thermal regulator (4022) of this example is thermally coupled with objective lens assembly (2000) such that thermal regulator (4022) is operable to actively heat or cool objective lens assembly (2000) in response to a drive signal from processor (4010).

[0157] Thermal regulator (4022) may include any of various kinds of features to provide active heating or cooling of at least a portion of objective lens assembly (2000) in response to a drive signal from processor (4010). By way of example only, thermal regulator (4022) may comprise one or more thermoelectric coolers, one or more cartridge heaters, resistive heating elements embedded in an insulating layer and / or embedded within housing (2006) of objective lens assembly (2000), etc. Several additional features that may be used to provide thermal regulator (4022) are described in greater detail below, though it is contemplated that thermal regulator (4022) may alternatively take any other suitable form.

[0158] While only one thermal regulator (4022) is shown in FIG. 21, some variations of arrangement (4000) may include two or more thermal regulators (4022). In some suchvariations, thermal regulators (4022) are positioned at different respective positions along the length of objective lens assembly (2000). For instance, one thermal regulator (4022) may be positioned at or near the front objective element of objective lens assembly while one or more other thermal regulators (4022) may be positioned proximally in relation to the front objective element. In some such versions, different thermal regulators (4022) may be activated at different times and / or at different temperatures to more precisely counteract thermal gradients that are induced by immersion fluid and / or heating from light passing through objective lens assembly (2000). It should also be understood that two or more temperature sensors (4020) may be positioned at different respective positions along the length of objective lens assembly (2000), regardless of whether two or more thermal regulators (4022) are positioned at different respective positions along the length of objective lens assembly (2000).

[0159] 1. Example of Objective Lens Assembly Thermal RegulationFeature Providing Airflow

[0160] FIG. 22 depicts an example of a form that thermal regulator (4022) may take. In this example, thermal regulator (4022) includes an airflow heater / cooler (4050) that is configured to regulate the temperature of objective lens assembly (2000) by blowing heated or cooled air toward objective lens assembly (2000). Airflow heater / cooler (4050) may include one or more integral heating elements (e.g., resistive heating elements, one or more conduits to contain or convey heated fluid or air, etc.), one or more integral cooling elements (e.g., a thermoelectric cooler, one or more conduits to contain or convey cooled fluid or air, etc.), and one or more blowing elements (e.g., a fan, etc.). The one or more blowing elements may be positioned and operable to blow heat from the one or more heating elements toward objective lens assembly (2000) to thereby increase the temperature of objective lens assembly (2000); and / or blow coolness from the one or more cooling elements toward objective lens assembly (2000) to thereby reduce the temperature of objective lens assembly (2000). Airflow heater / cooler (4050) may thus regulate the temperature of objective lens assembly (2000) without directly contacting objective lens assembly (2000).

[0161] In some variations, airflow heater / cooler (4050) only includes one or more integral cooling elements without also including one or more integral heating elements. In some other variations, airflow heater / cooler (4050) only includes one or more integral cooling elements without also including one or more integral heating elements. Thus, use of the term “airflow heater / cooler” should not be understood to require airflow heater / cooler (4050) to necessarily include both heating and cooling capabilities.

[0162] While not shown in FIG. 22, airflow heater / cooler (4050) may be electrically coupled with processor (4010) (like thermal regulator (4022)), such that processor (4010) may drive airflow heater / cooler (4050) in accordance with the teachings provided above with reference to thermal regulator (4022); and in accordance with the teachings provided below with reference to processes (5000, 5010, 5020). Alternatively, airflow heater / cooler (4050) may be used in any other suitable fashion.

[0163] 2. Example of Example of Objective Lens Assembly ThermalRegulation Feature Providing Immersion Fluid Flow

[0164] FIG. 23 depicts an example of another form that thermal regulator (4022) may take. In this example, thermal regulator (4022) regulates the temperature of objective lens assembly (2000) by providing a flow of temperature-controlled immersion fluid via immersion fluid manifold (2040). To provide this effect, thermal regulator (4022) of this example includes an immersion fluid source (4060), a suction source (4062), and a fluid heater / cooler (4064) positioned in the fluid path between immersion fluid source (4060) and suction source (4062). Immersion fluid source (4060) may be configured and operable like immersion fluid source (2080) described above. Similarly, suction source (4062) may be configured and operable like suction source (2090) described above.

[0165] Fluid heater / cooler (4064) may include one or more integral heating elements (e.g., resistive heating elements, etc.) and one or more integral cooling elements (e.g., a thermoelectric cooler, a refrigerant-based unit, etc.). Fluid heater / cooler (4064) is thus operable to actively heat or cool fluid communicated from suction source (4062) to immersion fluid source (4060). While not shown, fluid heater / cooler (4064) may alsoinclude an integral temperature sensor that is electrically coupled with processor (4010), such that processor (4010) may drive fluid heater / cooler (4064) based on a combination of temperature data from the temperature sensor of fluid heater / cooler (4064), temperature data from temperature sensor (4020), and temperature data from temperature sensor (4030).[00166J In some variations, fluid heater / cooler (4064) only includes one or more integral cooling elements without also including one or more integral heating elements. In some other variations, fluid heater / cooler (4064) only includes one or more integral cooling elements without also including one or more integral heating elements. Thus, use of the term “fluid heater / cooler” should not be understood to require fluid heater / cooler (4064) to necessarily include both heating and cooling capabilities.

[0167] While fluid heater / cooler (4064) is shown as being interposed in the fluid path between suction source (4062) and immersion fluid source (4060), fluid heater / cooler (4064) may alternatively be positioned in the fluid path between immersion fluid source (4060) and immersion fluid manifold (2040) or elsewhere. As another example of a variation, while immersion fluid source (4060) is shown as being positioned directly in the fluid path between fluid heater / cooler (4064) and immersion fluid manifold (2040), one or more valves, junctions, other conduits, and / or other components may be positioned between fluid heater / cooler (4064) and immersion fluid source (4060).

[0168] In the present example, immersion fluid source (4060), immersion fluid manifold (2040), suction source (4062), and fluid heater / cooler (4064) together form a closed loop for communication of temperature-regulated immersion fluid. The immersion fluid is thus recirculated in the present example. In some other versions, the immersion fluid is not recirculated. For instance, as another variation, fluid heater / cooler (4064) may be placed in the fluid path between immersion fluid source (4060) and immersion fluid manifold (2040), and immersion fluid drawn from immersion fluid manifold (2040) by suction source (4062) may be deposited in a separate reservoir or vessel, etc. It should therefore be understood that the components shown in FIG. 23 may be rearranged, supplemented, or substituted, in any other suitable way to provide active regulation of the temperature of immersion fluid that flows through immersion fluidmanifold (2040).

[0169] Regardless of how the temperature of the immersion fluid is regulated, the immersion fluid in the present example enters immersion fluid manifold (2040) via first port (2050) and first channel (2052), passes through recess (2044), and exits immersion fluid manifold (2040) via second channel (2058) and second port (2058). By directly contacting distal face (2004) of objective lens element (2002) and the underlying region of upper surface (1032) of flow cell (1030), the temperature-controlled immersion fluid may regulate the temperatures of at least those corresponding regions of objective lens assembly (2000) and flow cell (1030). In cases where immersion fluid manifold (2040) comprises a thermally conductive material, the temperature-controlled immersion fluid may also regulate the temperature of immersion fluid manifold (2040), which may in turn affect the temperature of regions of objective lens assembly (2000) that are in contact with immersion fluid manifold (2040).

[0170] While not shown in FIG. 23, fluid heater / cooler (4064) may be electrically coupled with processor (4010) (like thermal regulators (4022, 4040)), such that processor (4010) may drive fluid heater / cooler (4064) in accordance with the teachings provided above with reference to thermal regulators (4022, 4040); and in accordance with the teachings provided below with reference to processes (5000, 5010, 5020). Alternatively, fluid heater / cooler (4064) may be used in any other suitable fashion.

[0171] 3. Example of Objective Lens Assembly Thermal RegulationFeature Providing Coiled Fluid Flow

[0172] FIG. 24 depicts an example of another form that thermal regulator (4022) may take. In this example, thermal regulator (4022) regulates the temperature of objective lens assembly (2000) by providing a flow of temperature-controlled thermal fluid via a coil pipe (4070). To provide this effect, thermal regulator (4022) of this example includes coil pipe (4070), a fluid heater / cooler (4072), and a pump (4074) positioned in the fluid path between fluid heater / cooler (4072) and coil pipe (4070). These components are configured to circulate a thermal fluid that may include water, oil, and / or any other suitable kind of fluid. Coil pipe (4070) comprises a thermally conductive material andis shown as being wrapped around the exterior of objective lens assembly (2000). In some other versions, at least a portion of coil pipe (4070) is integrated within one or more interior regions of objective lens assembly (2000). Pump (4074) is operable to drive movement of fluid through the closed-loop fluid circuit formed by coil pipe (4070), fluid heater / cooler (4072), and pump (4074). Pump (4074) may comprise any suitable kind of pump.

[0173] Fluid heater / cooler (4072) may include one or more integral heating elements (e.g., resistive heating elements, etc.) and one or more integral cooling elements (e.g., a thermoelectric cooler, a refrigerant-based unit, etc ). Fluid heater / cooler (4072) is thus operable to actively heat or cool fluid communicated from pump (4074) to coil pipe (4070). While not shown, fluid heater / cooler (4072) may also include an integral temperature sensor that is electrically coupled with processor (4010), such that processor (4010) may drive fluid heater / cooler (4072) based on a combination of temperature data from the temperature sensor of fluid heater / cooler (4072), temperature data from temperature sensor (4020), and temperature data from temperature sensor (4030).

[0174] In some variations, fluid heater / cooler (4072) only includes one or more integral cooling elements without also including one or more integral heating elements. In some other variations, fluid heater / cooler (4072) only includes one or more integral cooling elements without also including one or more integral heating elements. Thus, use of the term “fluid heater / cooler” should not be understood to require fluid heater / cooler (4072) to necessarily include both heating and cooling capabilities.

[0175] While fluid heater / cooler (4072) is shown as being interposed in the fluid path between pump (4074) and coil pipe (4070), fluid heater / cooler (4072) may alternatively be positioned in the fluid path between coil pipe (4070) and pump (4074) or elsewhere. As another example of a variation, coil pipe (4070) is shown as being positioned directly in the fluid path between fluid heater / cooler (4072) and pump (4074), one or more valves, junctions, other conduits, and / or other components may be positioned between fluid heater / cooler (4072) and immersion pump (4074). It should therefore be understood that the components shown in FIG. 24 may be rearranged, supplemented,or substituted, in any other suitable way to provide active regulation of the temperature of immersion fluid that flows through coil pipe (4070). As yet another variation, pump (4074) may be omitted, and fluid heater / cooler (4072) may heat or cool thermal fluid that remains stationarily contained in coil pipe (4070) without circulating.

[0176] Regardless of how the temperature of the thermal fluid is regulated, the thermal fluid regulates the temperature of coil pipe (4070). By directly contacting objective lens assembly (2000), the temperature-controlled coil pipe (4070) may regulate the temperatures of at least the corresponding region of objective lens assembly (2000). While coil pipe (4070) is shown as extending vertically along an entire height of objective lens assembly (2000), in some other variations coil pipe (4070) may extend along only a portion of the height of objective lens assembly.

[0177] In some variations, housing (2006) of objective lens assembly (2000) includes one or more milled channels in which coil pipe (4070) is seated. Such milled channels may be positioned in an exterior region of housing (2006) or in an interior region of housing (2006). In some other variations, coil pipe (4070) is integrally formed in housing (2006) of objective lens assembly (2000). For instance, housing (2006) may include one or more integral channels through which thermally regulated fluid may be circulated or otherwise provided. As yet another variation, coil pipe (4070) may be replaced with an outer casing that surrounds housing (2006) of objective lens assembly (2000), such that the thermal fluid may be circulated within (or remain stationary within) a gap defined between an inner wall of the outer casing and the outer wall of the housing of objective lens assembly (2000). Alternatively, any other suitable structures and arrangements may be used to regulate the temperature of objective lens assembly (2000) via a thermal fluid.

[0178] As yet another variation, coil pipe (4070) may be replaced with a wrap that includes a thermoelectric cooler, one or more resistive heating elements, and / or other features that are operable to regulate the temperature of objective lens assembly (2000). Some such versions may operate similar to an electric blanked wrapped around the exterior of objective lens assembly (2000).

[0179] While not shown in FIG. 24, fluid heater / cooler (4072) may be electrically coupled with processor (4010) (like thermal regulator (4022)), such that processor (4010) may drive fluid heater / cooler (4072) in accordance with the teachings provided above with reference to thermal regulator (4022); and in accordance with the teachings provided below with reference to processes (5000, 5010, 5020). Alternatively, fluid heater / cooler (4072) may be used in any other suitable fashion.

[0180] 4. Example of Objective Lens Assembly Thermal RegulationFeature Providing Spacer Element

[0181] FIG. 25 depicts an example of another form that thermal regulator (4022) may take. In this example, thermal regulator (4022) regulates the temperature of objective lens assembly (2000) through a spacer (4080), which is interposed between the distal portion of objective lens assembly (2000) and immersion fluid manifold (2040). In some versions, spacer (4080) comprises a passive element formed of a thermally insulating material, such that spacer (4080) insulates objective lens assembly (2000) from temperature changes that would otherwise be induced through direct contact between objective lens assembly (2000) and immersion fluid manifold (2040). In such versions, while objective lens assembly (2000) may still receive or provide a transfer of heat from or to flow cell (1030) through direct contact between the immersion fluid and distal face (2004) of objective lens element (2002), the presence of spacer (4080) may at least substantially prevent transfers of heat between objective lens assembly (2000) and immersion fluid manifold (2040). By way of example only, spacer (4080) may include graphene, invar, closed cell foam, urethane foam, cellulose, aerogel, fiberglass, and / or any other suitable material(s).

[0182] In some other versions, spacer (4080) includes one or more features that are operable to heat and / or cool spacer (4080). In such versions, a driver (4082) may be coupled with spacer (4080) to drive such features. For instance, some versions of spacer (4080) may include resistive heating elements, and driver (4082) may include a power source that is operable to provide electrical power to such resistive heating elements. In addition, or in the alternative, spacer (4080) may include one or more thermoelectric cooling elements, and driver (4082) may include a power source that is operable toprovide electrical power to such thermoelectric cooling elements. As another example, spacer (4080) may include one or more conduits containing a thermal fluid, and driver (4082) may include a heater and / or cooler that is operable to heat and / or cool such fluid. In some such versions, the thermal fluid is stationarily contained in spacer (4080). In some other versions, the thermal fluid is circulated through spacer (4080). In some such versions, driver (4082) further includes a pump to drive such circulation of the thermal fluid. Alternatively, spacer (4080) and driver (4082) may include any other suitable components to actively heat and / or cool spacer (4080).

[0183] In some variations where spacer (4080) is active, spacer (4080) only includes one or more integral cooling elements without also including one or more integral heating elements. In some other variations where spacer (4080) is active, spacer (4080) only includes one or more integral cooling elements without also including one or more integral heating elements. Thus, an active version of spacer (4080) need not necessarily include both heating and cooling capabilities.

[0184] While not shown in FIG. 25, driver (4082) may be electrically coupled with processor (4010) (like thermal regulator (4022)), such that processor (4010) may drive driver (4082) in accordance with the teachings provided above with reference to thermal regulator (4022); and in accordance with the teachings provided below with reference to processes (5000, 5010, 5020). Alternatively, driver (4082) may be used in any other suitable fashion.

[0185] 5. Example of Method of Thermal Regulation with TargetTemperatures

[0186] In some scenarios, it may be desirable to regulate the temperature of flow cell (1030) such that the temperature of flow cell (1030) remains at a target temperature value (or within a target range); and / or to regulate the temperature of objective lens assembly (2000) such that the temperature of objective lens assembly (2000) remains at a target temperature value (or within a target range). To that end, FIG. 26 shows an example of two processes (5000, 5010) that may be carried out using arrangement (4000) of FIG. 21. Processes (5000, 5010) may be executed by processor (4010). Process (5000)begins with processor (4010) sensing (block 5002) the temperature of flow cell (1030) based on a signal from temperature sensor (4030).

[0187] Processor (4010) then determines (block 5004) whether the sensed temperature of flow cell (1030) is at a target temperature. By way of example only, the target temperature may be approximately 35° C; may be approximately 65° C; may range from approximately 15° C to approximately 70° C; may range from approximately 20° C to approximately 35° C; or may range from approximately 22° C to approximately 32° C. Alternatively, any other suitable target temperature may be used. In some variations, rather than determining (block 5004) whether the sensed temperature of flow cell (1030) is at a target temperature, processor (4010) may instead determine whether the sensed temperature of flow cell (1030) is within a predetermined range. The predetermined range may have any suitable upper and / or lower limit.

[0188] If the sensed temperature of flow cell (1030) is at the target value (or is outside the target range), then process (5000) may reiterate through the steps described above, such that processor (4010) may continue to monitor the sensed temperature of flow cell (1030) and determine (block 5004) whether / when the temperature deviates from the target value (or target range). In the event that the sensed temperature of flow cell (1030) deviates from the target value (or target range), processor (4010) may adjust (block 5006) the temperature of flow cell (1030) by activating thermal regulator (4030). In some cases, this may include activating thermal regulator (4030) to reduce the temperature of flow cell (1030). In some other cases, this may include activating thermal regulator (4030) to increase the temperature of flow cell (1030). Regardless of whether thermal regulator (4030) is activated to reduce or increase the temperature of flow cell (1030), such activation may be provided in a pulsed fashion or in a continuous fashion.

[0189] Processor (4010) may continue to monitor the temperature data and determine (block 5004) whether / when the temperature of flow cell (1030) no longer deviates from the target value (or target range). When this occurs, processor (4010) may deactivate thermal regulator (4030), drive thermal regulator (4030) to maintain the current temperature of flow cell (1030), or provide some other kind of response.

[0190] Process (5010) begins with processor (4010) sensing (block 5012) the temperature of objective lens assembly (2000) based on a signal from temperature sensor (4020). Processor (4010) then determines (block 5014) whether this sensed temperature of objective lens assembly (2000) is at a target temperature. Any suitable target temperature may be used. In some variations, rather than determining (block 5014) whether the sensed temperature of objective lens assembly (2000) is at a target temperature, processor (4010) may instead determine (block 5014) whether the sensed temperature of objective lens assembly (2000) is within a predetermined range. The predetermined range may have any suitable upper and / or lower limit.

[0191] If the sensed temperature of objective lens assembly (2000) is at the target value (or is outside the target range), then process (5010) may reiterate through the steps described above, such that processor (4010) may continue to monitor the sensed temperature of objective lens assembly (2000) and determine (block 5014) whether / when the temperature deviates from the target value (or target range). In the event that the sensed temperature of objective lens assembly (2000) deviates from the target value (or target range), processor (4010) may adjust (block 5016) the temperature of objective lens assembly (2000) by activating thermal regulator (4022). In some cases, this may include activating thermal regulator (4022) to reduce the temperature of objective lens assembly (2000). In some other cases, this may include activating thermal regulator (4022) to increase the temperature of objective lens assembly (2000). Regardless of whether thermal regulator (4022) is activated to reduce or increase the temperature of objective lens assembly (2000), such activation may be provided in a pulsed fashion or in a continuous fashion.

[0192] Processor (4010) may continue to monitor the temperature data and determine (block 5014) whether / when the temperature of objective lens assembly (2000) no longer deviates from the target value (or target range). When this occurs, processor (4010) may deactivate thermal regulator (4022), drive thermal regulator (4022) to maintain the current temperature of objective lens assembly (2000), or provide some other kind of response.

[0193] While FIG. 26 shows processes (5000, 5010) as being separate from each other, itshould be understood that processes (5000, 5010) may be performed simultaneously, may be performed in an alternating fashion, may be performed completely independently of each other, or may be performed in any other suitable relationship relative to each other. In some scenarios, process (5000) is performed while process (5010) is not performed. In some other scenarios, process (5010) is performed while process (5000) is not performed.

[0194] 6. Example of Method of Thermal Regulation with TemperatureEqualization

[0195] In some scenarios, it may be desirable to regulate the temperature of flow cell (1030) and / or the temperature of objective lens assembly (2000) such that the temperature of flow cell (1030) and the temperature of objective lens assembly (2000) are approximately equal to each other. In other words, it may be desirable to prevent or at least minimize any temperature differential between flow cell (1030) and objective lens assembly (2000). To that end, FIG. 27 shows an example of another process (5020) that may be carried out using arrangement (4000) of FIG. 21. Process (5020) may be executed by processor (4010). Process (5020) begins with processor (4010) sensing (block 5022) the temperature of flow cell (1030) based on a signal from temperature sensor (4030); and sensing (block 5024) the temperature of objective lens assembly (2000) based on a signal from temperature sensor (4020). While FIG. 27 shows these two steps (blocks 5022, 5024) being performed simultaneously, these steps (blocks 5022, 5024) may instead be performed in a sequence or in any other suitable fashion.

[0196] Once the temperature of flow cell (1030) and the temperature of objective lens assembly (2000) have been sensed (blocks 5022, 5024), processor (4010) then determines (block 5026) whether there is any difference between the temperature of flow cell (1030) and the temperature of objective lens assembly (2000). In some other variations, processor (4010) determines whether any temperature differential between flow cell (1030) and objective lens assembly (2000) is above a threshold (e.g., similar to the step (block (3058) of process (3050) described above with reference to FIG. 20).

[0197] If there is no difference between the sensed temperature of flow cell (1030) and thesensed temperature of objective lens assembly (2000) (or if there is a difference that falls below a predetermined threshold), then process (5020) may reiterate through the steps described above, such that processor (4010) may continue to sense (blocks 5022, 5024) temperatures of flow cell (1030) and objective lens assembly (2000) and determine (block 5026) whether / when there is any difference between these temperatures (or whether / when any difference between the temperatures exceeds a predetermined threshold).

[0198] In the event that processor (4010) identifies a difference between the sensed temperature of flow cell (1030) and the sensed temperature of objective lens assembly (2000) (or if there is a difference that falls below a predetermined threshold), then processor (4010) may adjust (block 5028) the temperature of flow cell (1030) by activating thermal regulator (4030) and / or adjust (block 5030) the temperature of objective lens assembly (2000) by activating thermal regulator (4022). In some cases, this may include activating thermal regulator (4030) to reduce the temperature of flow cell (1030). In some other cases, this may include activating thermal regulator (4030) to increase the temperature of flow cell (1030). Regardless of whether thermal regulator (4030) is activated to reduce or increase the temperature of flow cell (1030), such activation may be provided in a pulsed fashion or in a continuous fashion.

[0199] In some cases where processor (4010) adjusts (block 5030) the temperature of objective lens assembly (2000) by activating thermal regulator (4022), this may include activating thermal regulator (4022) to reduce the temperature of objective lens assembly (2000). In some other cases, this may include activating thermal regulator (4022) to increase the temperature of objective lens assembly (2000). Regardless of whether thermal regulator (4022) is activated to reduce or increase the temperature of objective lens assembly (2000), such activation may be provided in a pulsed fashion or in a continuous fashion.

[0200] While FIG. 27 shows a combination of adjusting (block 5028) the temperature of flow cell (1030) and adjusting (block 5030) the temperature of objective lens assembly (2000), there may be some scenarios where only the temperature of flow cell (1030) is adjusted (block 5028). There may be other scenarios where only the temperature ofobjective lens assembly (2000) is adjusted (block 5030). In some cases, processor (4010) may determine whether to only adjust (block 5028) the temperature of flow cell (1030), to only adjust (block 5030) the temperature of objective lens assembly (2000), or to adjust (blocks 5028, 5030) the temperatures of both flow cell (1030) and objective lens assembly (2000) based on temperature data from temperature sensors (4020, 4030). Moreover, in cases where processor (4010) determines that the temperature of flow cell (1030) should be adjusted (block 5028) and that the temperature of objective lens assembly (2000) should be adjusted (block 5030), these two adjustment steps may (blocks 5028, 5030) may be performed simultaneously, in a sequence, or in any other suitable fashion.

[0201] In some cases where the temperature adjustment steps (blocks 5028, 5030) are performed simultaneously, processor (4010) may activate thermal regulator (4022) to reduce the temperature of objective lens assembly (2000) while simultaneously activating thermal regulator (4030) to increase the temperature of flow cell (1030). Alternatively, processor (4010) may activate thermal regulator (4022) to increase the temperature of objective lens assembly (2000) while simultaneously activating thermal regulator (4030) to reduce the temperature of flow cell (1030). In either scenario, the simultaneous, opposing thermal driving of flow cell (1030) and objective lens assembly (2000) may facilitate the speed with which the temperature difference between flow cell (1030) and objective lens assembly (2000) is reduced or eliminated.

[0202] When executing temperature adjustments (blocks 5028, 5030), processor (4010) may provide a set point temperature based on any suitable algorithm that factors in temperature data from temperature sensors (4020, 4030). In some versions, the set point temperature is a mean value of the temperature values of flow cell (1030) and objective lens assembly (2000), such that processor (4010) may activate thermal regulator (4022) and thermal regulator (4030) such that the temperatures of flow cell (1030) and objective lens assembly (2000) effectively “meet in the middle.” As one example of how this may be carried out, if the sensed temperature of flow cell (1030) is 23° C and the sensed temperature of objective lens assembly (2000) is 27° C, processor (4010) may activate thermal regulator (4022) to reduce the temperature ofobjective lens assembly (2000) to 25° C while simultaneously activating thermal regulator (4030) to increase the temperature of flow cell (1030) to 25° C. As another example, rather than providing a mean value temperature as the set point, processor (4010) may calculate a weighted average of the temperature values of flow cell (1030) and objective lens assembly (2000), and establish that calculated weighted average as the set point temperature for executing temperature adjustments (blocks 5028, 5030).

[0203] Regardless of whether the temperature of only one or both of flow cell (1030) or objective lens assembly (2000) is / are adjusted (blocks 5028, 5030), processor (4010) may continue to monitor the temperature data and determine (block 5026) whether / when there is a difference between the sensed temperature of flow cell (1030) and the sensed temperature of objective lens assembly (2000) (or if there is a difference that falls below a predetermined threshold). Once the sensed temperatures are equalized (or brought within a predetermined range of each other), processor may deactivate one or both of thermal regulators (4022, 4040), drive one or both of thermal regulators (4022, 4040) to maintain the current temperature of objective lens assembly (2000) or flow cell (1030), or provide some other kind of response.

[0204] C. Examples of Integral Objective Lens Assembly Features to Reduce, prevent, or mitigate Thermally Induced Aberrations

[0205] The examples provided above relate primarily to features that may be used as an addon to a preexisting objective lens assembly (2000), such that the above-described examples may be retrofit with respect to a conventional objective lens assembly (2000). In some cases, it may be desirable to provide a modified version of an objective lens assembly (2000) that directly integrates one or more features to reduce, prevent, or mitigate thermally induced aberrations. Several examples of such features are described in greater detail below. While the features and processes described above with reference to FIGS. 19-27 may be provided as a retrofit with respect to a conventional objective lens assembly (2000), the features and processes described above with reference to FIGS. 19-27 may also be combined with the modified objective lens assemblies described below. It should therefore be understood that the examples described above with reference to FIGS. 19-27 are not mutually exclusive with respectto the examples described below.

[0206] 1. Examples of Passive Integral Objective Lens Assembly Features

[0207] FIG. 28 shows an example of an objective lens assembly (5050) that may be substituted for objective lens assembly (2000) described above. Objective lens assembly (5050) of this example may include one or more passive integral features that reduce, prevent, or mitigate the occurrence of thermally induced aberrations in images captured through objective lens assembly (5050). Objective lens assembly (5050) may be configured and operable like objective lens assembly (2000) except for the differences described below. Objective lens assembly (5050) of this example includes a primary outer housing (5052), several inner housings (5054), and several lens elements (5060). Primary outer housing (5052), which may be considered analogous to housing (2006) shown in FIG. 13 and described above, contains inner housings (5054) and lens elements (5060). Inner housings (5054) support lens elements (5060) within primary outer housing (5052) and thereby maintain appropriate spacing between lens elements (5060), such that gaps (G) are defined between certain lens elements (5060). In some versions, inner housings (5054) may comprise a thermally insulative material, such as graphene, invar, etc. Lens elements (5060) include a front lens element (5060a), which may be considered analogous to lens element (2002) shown in FIG. 13 and described above.

[0208] In some versions, one or more gaps (G) may provide thermal insulation between front lens element (5060a) and other lens elements (5060), thereby reducing the risk of thermally induced aberrations in those other lens elements (5060). By way of example only, one or more gaps (G) may contain a thermally insulating fluid. By way of further example only, one or more gaps (G) may contain a thermally insulating gas (e g., argon, krypton, xenon, etc.). By way of further example only, a vacuum may be provided in one or more gaps (G), with such vacuum providing thermal insulation in such gap (G) or gaps (G).

[0209] In addition to, or as an alternative to, a thermally insulating gas or vacuum being provided in gaps (G), some versions objective lens assembly (5150) may include anisolated chamber surrounding housing (5152). A gap may be defined between the exterior wall of housing (5152) and the inner wall of the chamber. A thermally insulating gas or vacuum may be provided in this gap to further thermally insulate lens elements (5060). In other words, the isolated chamber and insulating gas or vacuum may prevent or reduce thermal transfers from the external environment to lens elements (5060) via housings (5152, 5154).

[0210] In addition, or in the alternative, one or more of lens elements (5060) may be configured to provide substantial resistance to thermally induced aberrations. By way of example only, front lens element (5060a) may comprise an inverse GRIN lens. In some such versions, as the temperature of the inverse GRIN lens increases, the refractive index of the inverse GRIN lens becomes near uniform.

[0211] FIG. 29 shows another example of an objective lens assembly (5150) that may be substituted for objective lens assembly (2000) described above. Objective lens assembly (5150) may be configured and operable like objective lens assembly (5050) except for the differences described below. Like objective lens assembly (5050), objective lens assembly (5150) of this example includes a primary outer housing (5152), several inner housings (5154), and several lens elements (5160). Primary outer housing (5152), which may be considered analogous to housing (2006) shown in FIG. 13 and described above, contains inner housings (5154) and lens elements (5160). Inner housings (5154) support lens elements (5160) within primary outer housing (5152) and thereby maintain appropriate spacing between lens elements (5160), such that gaps (G) are defined between certain lens elements (5160). Lens elements (5160) include a front lens element (5160a), which may be considered analogous to lens element (2002) shown in FIG. 13 and described above.

[0212] Objective lens assembly (5150) may further include any of the passive features described above with respect to objective lens assembly (5050) to reduce, prevent, or mitigate the occurrence of thermally induced aberrations in images captured through objective lens assembly (5150). In addition, objective lens assembly (5150) of this example includes an insulating spacer (5170) interposed between an inner housing (5152) that supports front lens element (5160a) and the adjacent lens element (5160b).Insulating spacer (5170) of this example comprises a thermally insulating material and has an annular shape such that insulating spacer (5170) does not impede the optical path of objective lens assembly (5150). Insulating spacer (5170) may substantially reduce or prevent the transfer of heat from the inner housing (5152) that supports front lens element (5160a) and the adjacent lens element (5160b). In some versions, insulating spacer (5170) also extends between the inner housing (5152) that supports front lens element (5160a) and the inner housing (5152) that supports the adj acent lens element (5160b).

[0213] While only one insulating spacer (5170) is shown in only one position in FIG. 29, variations of objective lens assembly (5150) may provide one or more insulating spacers (5170) in one or more different respective locations within objective lens assembly (5150). For instance, an insulating spacer (5170) may be interposed between front lens element (5160a) and the inner housing (5152) that supports front lens element (5160a). In addition, or in the alternative, an insulating spacer (5170) may be interposed between any inner housings (5152) that would otherwise directly contact each other. In addition, or in the alternative, an insulating spacer (5170) may be interposed between any lens element (5160) and the inner housing (5152) that supports the lens element (5160). Alternatively, insulating spacers (5170) may be positioned at any other suitable locations within objective lens assembly (5150).

[0214] It should also be understood that insulating spacer (5170) may provide some degree of compliance, such that insulating spacer (5170) may accommodate thermally induced expansions and contractions of adjacent components. In some versions, insulating spacer (5170) expands and contracts only radially without also expanding and contracting along the z-direction. This may provide consistent spacing between lens elements (5160) throughout operation.

[0215] In some versions where an insulating feature such as insulating spacer (5170) is interposed between front lens element (5160a) and the rest of lens elements (5160), this thermal insulation may facilitate heating of front lens element (5160a) to a working temperature that is at or near the working temperature of flow cell (1030) (e.g., within approximately 2° C of the working temperature of flow cell (1030)). This heating offront lens element (5160a) may keep the temperature of front lens element (5160a) substantially constant during operation, while the thermal insulation of the rest of lens elements (5160) may keep the temperature of those lens elements (5160) substantially constant during operation. To the extent that the operating temperature of front lens element (5160a) is different from the operating temperature of the rest of lens elements (5160), this difference may remain substantially constant during operation. Objective lens assembly (5150) may thus be optically designed to account for this substantially constant temperature differential.

[0216] 2. Examples of Active Integral Objective Lens Assembly Features

[0217] While the examples described above with respect to FIGS. 28-29 provide passive integral features that reduce, prevent, or mitigate the occurrence of thermally induced aberrations in images captured through an objective lens assembly, it may be desirable to provide an objective lens assembly that includes one or more active integral features that reduce, prevent, or mitigate the occurrence of thermally induced aberrations in images captured through an objective lens assembly. To that end, FIG. 30 shows an example of an objective lens assembly (5100) that includes an active integral element (5110) that is electrically coupled with a processor (5120). Objective lens assembly (5100) may be substituted for objective lens assembly (2000) described above. Objective lens assembly (5100) may be configured and operable like objective lens assembly (2000) except for the inclusion of active integral element (5110).

[0218] Processor (5120) may be configured and operable like processors (3010, 4010) described above. While not shown, processor (5120) may also be electrically coupled with one or more temperature sensors like temperature sensors (3020, 3040, 4020, 4030). In such cases, processor (5120) may drive active integral element (5110) based at least in part on temperature data from one or more of the temperature sensors. In addition, or in the alternative, processor (5120) may drive active integral element (5110) based at least in part on processed image data, where the image data is from images captured via objective lens assembly (5100). For instance, the image data from images captured via objective lens assembly (5100) may indicate the presence of one or more thermally induced aberrations, and processor (5120) may accordingly driveactive integral element (5110) to dynamically eliminate, mitigate, or otherwise account for such thermally induced aberrations. In some other scenarios, the temperature changes in objective lens assembly (5100) may be generally known or assumed, such that processor (5120) may drive active integral element (5110) based on a fixed routine that does not rely on data from any temperature sensors.[00219J Active integral element (5110) may take numerous forms. By way of example only, to address scenarios where temperature fluctuations in objective lens assembly (5100) may tend to induce an astigmatism, active integral element (5110) may include a driven astigmatism compensation assembly. Such an astigmatism compensation assembly may include a pair of flat, optically transmissive plates in the optical path within objective lens assembly (5100). Such optically transmissive plates may be positioned at opposite yet oblique angles relative to the optical axis of objective lens assembly (5100); and may be rotated in opposite directions about respective axes that are orthogonal to the optical axis of objective lens assembly (5100). This arrangement may induce an astigmatism that offsets an astigmatism that is thermally induced in objective lens assembly (5100). Actuators may be coupled with such plates to drive rotation of the plates; and processor (5120) may activate those actuators in response to temperature sensor data, image processing data, and / or other data indicating a change in the astigmatism in objective lens assembly (5100). In other words, the optically transmissive plates may be driven to rotate to dynamically compensate for thermally induced changes to an astigmatism by providing a variable offsetting astigmatism. Such an arrangement may be provided in accordance with at least some of the teachings of U.S. Pat. App. No. 18 / 652,880, entitled “Optical Arrangement for Compensation of Thermally-Induced Astigmatism of Lens,” filed May 2, 2024, the disclosure of which is incorporated by reference herein, in its entirety.

[0220] To address scenarios where temperature fluctuations in objective lens assembly (5100) may tend to induce changes in focus, active integral element (5110) may include a focus adjustment assembly. For instance, the focus adjustment assembly may include an objective refocus and / or zooming tube lens element that is adjustable to provide spherical aberration compensation. Such focus adjustment elements may beautomatically driven by one or more actuators that are in electrical communication with a processor (e.g., like processor (4010), etc ). Such a processor may drive the one or more focus adjustment elements based on data from one or more temperature sensors (4020, 4030), based on image processing data indicating new aberrations of objective lens assembly (5100) or changes in aberrations of objective lens assembly (5100), based on a fixed, predetermined workflow (e.g., not based on sensed data), or based on any other suitable criteria. As another example, the processor may execute an integration through focus algorithms, reiteratively taking images at different z- positions, and automatically selecting the best-focus image plane along the z-axis at different points of operation. This integration through focus routine may be executed on any suitable periodic basis with any suitable frequency at any suitable stages of operation.

[0221] As another example, active integral element (5110) may include one or more transmissive deformable phase plates, one or more deformable lenses, one or more wave plates, one or more molded lenses, and / or one or more deformable mirrors. Alternatively, active integral element (5110) may take any other suitable form. Moreover, while FIG. 30 only shows one active integral element (5110), other variations of objective lens assembly (5100) may include two or more active integral elements (5110).

[0222] While the example depicted in FIG. 30 shows active integral element (5110) as being positioned within objective lens assembly (5100), some variations may provide an active element that is configured and operable like active integral element (5110) but positioned outside objective lens assembly (5100). For instance, such an active element may be positioned in the optical path between objective lens assembly (5110) and camera system (540). Such separate active elements may be activated to optically correct or otherwise mitigate aberrations that are thermally induced within objective lens assembly (5100), despite such active elements being physically separate (i.e., spaced away from) objective lens assembly (5100).

[0223] D. Example of Multi -Housing Objective Lens Assembly

[0224] FIG. 31 shows an example of another objective lens assembly (5200) that may be substituted for objective lens assembly (2000) described above. Objective lens assembly (5200) may be configured and operable like objective lens assembly (2000) except for the differences described below. Objective lens assembly (5200) of this example includes a first subassembly (5210) and a second subassembly (5220). Each subassembly (5210, 5220) may include any suitable number of respective lens elements. Second subassembly (5220) includes a front lens element, which is not shown but may be similar to front lens elements (2002, 5060a, 5160a) described above. In some versions, second subassembly (5220) may also include one or more additional lens elements in addition to including the front lens element. Second subassembly (5220) of the present example also includes a thermal regulator (5222), which may be configured and operable like thermal regulator (4022) described above.

[0225] Subassemblies (5210, 5220) are aligned with each other along a shared longitudinal axis (LA); and are separated from each other along that longitudinal axis (LA) by a gap (G). Objective lens assembly (5200) further includes one or more frame elements (5230) interposed between subassemblies (5210, 5220) to maintain this gap (G) and to maintain alignment between subassemblies (5210, 5220). Frame elements (5230) may take any suitable form, including but not limited to struts, posts, beams, etc. The gap (G) may serve as an insulator, such that heat from second subassembly (5220) will not be transferred to first subassembly (5120). The gap (G) may thus prevent the occurrence of thermally induced aberrations in first subassembly (5120) that might otherwise occur in the absence of the gap (G). Moreover, since the mass of second subassembly (5220) may be less than the mass of a single unitary objective lens assembly that contains all the objective lens elements, thermal regulator (5222) may regulate the temperature of second subassembly (5220) with greater precision that might otherwise be achieved through regulating the temperature of a single unitary objective lens assembly that contains all the objective lens elements.

[0226] The thermal insulation provided by the gap (G) may further facilitate use of thermal regulator (5222) to heat lens elements of second subassembly (5220) to a working temperature that is at or near the working temperature of flow cell (1030) (e.g., withinapproximately 2° C of the working temperature of flow cell (1030)). This heating of lens elements of second subassembly (5220) may keep the temperature of such lens elements substantially constant during operation, while the thermal insulation of the lens elements of first subassembly (5120) by the gap (G) may keep the temperature of those lens elements of first subassembly (5120) substantially constant during operation. To the extent that the operating temperature of lens elements of second subassembly (5220) is different from the operating temperature of lens elements of first subassembly (5120), this difference may remain substantially constant during operation. Objective lens assembly (5220) may thus be optically designed to account for this substantially constant temperature differential.

[0227] While not shown, a shroud may encompass the gap (G) along the exterior regions of subassemblies (5210, 5220). Such a shroud may prevent debris or stray light from entering the gap (G), to thereby mitigate the risk of external features adversely affecting the optical qualities of objective lens assembly (5200) due to the presence of the gap (G). Such a shroud may also be formed of an insulative material and / or otherwise be positioned such that the shroud does not substantially conduct heat from second subassembly (5220) to first subassembly (5120).

[0228] VIII. Examples of Combinations

[0229] The following examples relate to various non-exhaustive ways in which the teachings herein may be combined or applied. The following examples are not intended to restrict the coverage of any claims that may be presented at any time in this application or in subsequent filings of this application. No disclaimer is intended. The following examples are being provided for nothing more than merely illustrative purposes. It is contemplated that the various teachings herein may be arranged and applied in numerous other ways. It is also contemplated that some variations may omit certain features referred to in the below examples. Therefore, none of the aspects or features referred to below should be deemed critical unless otherwise explicitly indicated as such at a later date by the inventors or by a successor in interest to the inventors. If any claims are presented in this application or in subsequent filings related to this application that include additional features beyond those referred to below, thoseadditional features shall not be presumed to have been added for any reason relating to patentability.

[0230] Example 1

[0231] An apparatus comprising: an imaging assembly, the imaging assembly including: an objective lens assembly, the objective lens assembly including a front element having a bottom surface, and an immersion fluid assembly to provide immersion fluid under the bottom surface of the front element of the objective lens assembly; a flow cell having an upper surface, the immersion fluid assembly to maintain immersion fluid between the bottom surface of the front element of the objective lens assembly and the upper surface of the flow cell; and a temperature regulation assembly, the temperature regulation assembly including: a first sensor to sense a temperature of the objective lens assembly, a first temperature adjusting feature to adjust a temperature of the flow cell, and a processor to activate the first temperature adjusting feature based on at least data from the first sensor.

[0232] Example 2

[0233] The apparatus of Example 1, the temperature regulation assembly further including a second sensor to sense a temperature of the flow cell, the processor to activate the first temperature adjusting feature based on at least data from the second sensor.

[0234] Example 3

[0235] The apparatus of Example 2, the processor to determine a difference between the sensed temperature of the objective lens assembly and the sensed temperature of the flow cell.

[0236] Example 4

[0237] The apparatus of Example 3, the processor to compare a difference between the sensed temperature of the objective lens assembly and the sensed temperature of the flow cell to a threshold.

[0238] Example 5

[0239] The apparatus of Example 4, the processor to activate the first temperature adjusting feature in response to the difference between the sensed temperature of the objective lens assembly and the sensed temperature of the flow cell exceeding the threshold.

[0240] Example 6

[0241] The apparatus of any of Examples 4 through 5, the temperature regulation assembly further comprising a second temperature adjusting feature to adjust a temperature of the objective lens assembly, the processor to activate the second temperature adjusting feature in response to the difference between the sensed temperature of the objective lens assembly and the sensed temperature of the flow cell exceeding the threshold.

[0242] Example 7

[0243] The apparatus of Example 3, the processor to activate the first temperature adjusting feature in response to the processor determining a difference between the sensed temperature of the objective lens assembly and the sensed temperature of the flow cell.

[0244] Example 8

[0245] The apparatus of Example 7, the temperature regulation assembly further comprising a second temperature adjusting feature to adjust a temperature of the objective lens assembly, the processor to activate the second temperature adjusting feature in response to the processor determining a difference between the sensed temperature of the objective lens assembly and the sensed temperature of the flow cell.

[0246] Example 9

[0247] The apparatus of any of Examples 2 through 8, the processor to: determine whether the flow cell is at a first target temperature, and activate the first temperature adjusting feature based on at least data indicating that the flow cell is not at the first target temperature.

[0248] Example 10

[0249] The apparatus of Example 9, the temperature regulation assembly further comprising a second temperature adjusting feature to adjust a temperature of the objective lensassembly, the processor to: determine whether the objective lens assembly is at a second target temperature, and activate the second temperature adjusting feature based on at least data indicating that the objective lens assembly is not at the second target temperature.

[0250] Example 11

[0251] The apparatus of Example 10, the second target temperature being equal to the first target temperature.

[0252] Example 12

[0253] The apparatus of any of Examples 1 through 11, the first sensor being positioned to sense a temperature of the front element of the objective lens assembly.

[0254] Example 13

[0255] The apparatus of any of Examples 1 through 12, the temperature regulation assembly further including a second temperature adjusting feature to adjust a temperature of the objective lens assembly.

[0256] Example 14

[0257] The apparatus of Example 13, the processor to activate the first temperature adjusting feature based on at least data from the first sensor.

[0258] Example 15

[0259] The apparatus of any of Examples 13 through 14, the temperature regulation assembly further including a second sensor to sense a temperature of the flow cell, the processor to activate the second temperature adjusting feature based on at least data from the second sensor.

[0260] Example 16

[0261] The apparatus of any of Examples 13 through 15, the second temperature adjusting feature being positioned to adjust a temperature of the front element of the objective lens assembly.

[0262] Example 17

[0263] The apparatus of Example 16, the second temperature adjusting feature being interposed between the objective lens assembly and the immersion fluid assembly.

[0264] Example 18

[0265] The apparatus of any of Examples 13 through 17, the second temperature adjusting feature comprising a resistive heating element.

[0266] Example 19

[0267] The apparatus of any of Examples 13 through 18, the second temperature adjusting feature comprising: a fan or blower, and one or both of a heating element or a cooling element, the fan being operable to blow heated or cooled air toward the objective lens assembly.

[0268] Example 20

[0269] The apparatus of any of Examples 13 through 19, the second temperature adjusting feature comprising an immersion fluid temperature adjusting feature, the second temperature adjusting feature to adjust a temperature of the objective lens assembly by adjusting a temperature of the immersion fluid.

[0270] Example 21

[0271] The apparatus of any of Examples 13 through 20, the objective lens assembly comprising a housing, the second temperature adjusting feature comprising one or more heating elements positioned adjacent to the housing.

[0272] Example 22

[0273] The apparatus of Example 21, the second temperature adjusting feature comprising a coil pipe configured to contain a heated or cooled fluid.

[0274] Example 23

[0275] The apparatus of any of Examples 21 through 22, the second temperature adjustingfeature comprising one or more of a resistive heating element, a cartridge heater, or a thermoelectric cooler.

[0276] Example 24

[0277] The apparatus of any of Examples 13 through 23, further comprising a spacer interposed between the objective lens assembly and the immersion fluid assembly.

[0278] Example 25

[0279] The apparatus of Example 24, the spacer comprising a thermally insulating material.

[0280] Example 26

[0281] The apparatus of any of Examples 24 through 25, the second temperature adjusting feature being positioned in the spacer.

[0282] Example 27

[0283] The apparatus of any of Examples 1 through 26, the objective lens assembly further including one or more thermally insulating elements.

[0284] Example 28

[0285] The apparatus of Example 27, at least one of the thermally insulating elements being positioned adjacent to the front element.

[0286] Example 29

[0287] The apparatus of any of Examples 27 through 28, the objective lens assembly further comprising a plurality of lens elements, the plurality of lens elements including the front element, at least some of the lens elements of the plurality of lens elements being separated by gaps.

[0288] Example 30

[0289] The apparatus of Example 29, the objective lens assembly further comprising a thermally insulating gas in at least one of the gaps.

[0290] Example 31

[0291] The apparatus of any of Examples 29 through 30, the objective lens assembly further comprising a thermally insulating liquid in at least one of the gaps.

[0292] Example 32

[0293] The apparatus of any of Examples 29 through 31, the objective lens assembly providing a vacuum in at least one of the gaps.

[0294] Example 33

[0295] The apparatus of any of Examples 1 through 32, the objective lens assembly further including an active integral element, the active integral element to actively adjust an optical property of the objective lens assembly.

[0296] Example 34

[0297] The apparatus of Example 33, the processor to activate the active integral element based on at least data from the first sensor.

[0298] Example 35

[0299] The apparatus of any of Examples 33 through 34, the active integral element comprising one or more of a pair of opposingly rotatable optically transmissive plates to provide an astigmatism adjustment, a focus adjustment assembly, a transmissive deformable phase plate, a deformable lens, a wave plate, a molded lens, or a deformable mirror.

[0300] Example 36

[0301] The apparatus of any of Examples 1 through 35, the objective lens assembly including a first subassembly and a second subassembly, the front element being located in the second subassembly, the first subassembly including at least one additional lens element, the first subassembly and the second subassembly being separated from each other by a gap.

[0302] Example 37

[0303] The apparatus of Example 36, the objective lens assembly further comprising one ormore frame elements, the one or more frame elements maintaining separation between the first subassembly and the second subassembly, the one or more frame elements further maintaining alignment between the first subassembly and the second subassembly.

[0304] Example 38

[0305] The apparatus of any of Examples 36 through 37, the temperature regulation assembly further comprising a second temperature adjusting feature to adjust a temperature of the objective lens assembly.

[0306] Example 39

[0307] The apparatus of Example 38, the second temperature adjusting feature being integrated with the second subassembly such that the second temperature adjusting feature is to adjust a temperature of the front element.

[0308] Example 40

[0309] The apparatus of Example 39, the gap being sized to prevent thermal transfer of temperature changes of the second subassembly induced by the second temperature adjusting feature to the first subassembly.

[0310] Example 41

[0311] A method comprising: capturing an image of a region of a flow cell with an imaging assembly, the flow cell having an upper surface, the imaging assembly including: an objective lens assembly, the objective lens assembly including a front element having a bottom surface, and an immersion fluid assembly providing immersion fluid between the bottom surface of the front element of the objective lens assembly and the upper surface of the flow cell while the image is captured; receiving data indicating a temperature of the objective lens assembly; and adjusting a temperature of the flow cell, based at least in part on the temperature of the objective lens assembly.

[0312] Example 42

[0313] The method of Example 41, further comprising receiving data indicating a temperatureof the flow cell.

[0314] Example 43

[0315] The method of Example 42, further comprising adjusting the temperature of the flow cell based on at least the temperature of the flow cell.

[0316] Example 44

[0317] The method of any of Examples 42 through 43, further comprising determining a difference between the temperature of the objective lens assembly and the temperature of the flow cell.

[0318] Example 45

[0319] The method of Example 44, further comprising comparing the difference between the temperature of the objective lens assembly and the temperature of the flow cell to a threshold.

[0320] Example 46

[0321] The method of Example 45, the act of adjusting a temperature of the flow cell being performed in response to the difference between the temperature of the objective lens assembly and the temperature of the flow cell exceeding the threshold.

[0322] Example 47

[0323] The method of any of Examples 45 through 46, further comprising adjusting a temperature of the objective lens assembly in response to the difference between the temperature of the objective lens assembly and the temperature of the flow cell exceeding the threshold.

[0324] Example 48

[0325] The method of Example 44, the act of adjusting a temperature of the flow cell being performed in response to the difference between the temperature of the objective lens assembly and the temperature of the flow cell.

[0326] Example 49

[0327] The method of Example 48, further comprising adjusting a temperature of the objective lens assembly in response to the difference between the temperature of the objective lens assembly and the temperature of the flow cell.

[0328] Example 50

[0329] The method of Example 49, the temperature of one or both of the flow cell or the objective lens assembly being adjusted until the temperature of the flow cell and the temperature of the objective lens assembly are substantially equal.

[0330] Example 51

[0331] The method of any of Examples 42 through 50, further comprising: determining whether the flow cell is at a first target temperature; and if the flow cell is not at the first target temperature, adjusting the temperature of the flow cell toward the first target temperature.

[0332] Example 52

[0333] The method of Example 51, further comprising: determining whether the objective lens assembly is at a second target temperature; and if the objective lens assembly is not at the second target temperature, adjusting the temperature of the objective lens assembly toward the second target temperature.

[0334] Example 53

[0335] The method of Example 52, the second target temperature being equal to the first target temperature.

[0336] Example 54

[0337] The method of any of Examples 41 through 53, the received data indicating a temperature of the front element of the objective lens assembly.

[0338] Example 55

[0339] The method of any of Examples 41 through 54, further comprising adjusting a temperature of the objective lens assembly, based at least in part on the temperature of the objective lens assembly.

[0340] Example 56

[0341] The method of Example 55, the act of adjusting a temperature of the objective lens assembly including adjusting a temperature of the front element of the objective lens assembly.

[0342] Example 57

[0343] The method of any of Examples 55 through 56, the act of adjusting a temperature of the objective lens assembly including adjusting a temperature of the immersion fluid.

[0344] Example 58

[0345] The method of any of Examples 41 through 57, further comprising activating an integral optical element of the objective lens assembly to thereby actively adjust an optical property of the objective lens assembly.

[0346] Example 59

[0347] The method of Example 58, the integral optical element being activated based at least in part on the temperature of the objective lens assembly.

[0348] Example 60

[0349] The method of any of Examples 58 through 59, the integral optical element being activated to compensate for a thermally induced aberration in the objective lens assembly.

[0350] Example 61

[0351] An apparatus comprising: an imaging assembly, the imaging assembly including: an objective lens assembly, the objective lens assembly including a front element having a bottom surface, an adjustable optical element within an image path of the front element of the objective lens assembly, the adjustable optical element being adjustableto thereby adjust one or more characteristics of an image within the image path of the front element, and an immersion fluid assembly to provide immersion fluid under the bottom surface of the objective lens; a flow cell having an upper surface, the immersion fluid assembly to maintain immersion fluid between the bottom surface of the front element of the objective lens assembly and the upper surface of the flow cell; a first sensor to sense a temperature of the objective lens assembly; and a processor to activate the adjustable optical element based on at least data from the first sensor.

[0352] IX. Miscellaneous

[0353] While the foregoing examples are provided in the context of a system (100) that may be used in nucleotide sequencing processes, the teachings herein may also be readily applied in other contexts, including in systems that perform other processes (i.e., other than nucleotide sequencing procedures). The teachings herein are thus not necessarily limited to systems that are used to perform nucleotide sequencing processes.

[0354] It is to be understood that the subject matter described herein is not limited in its application to the details of construction and the arrangement of components set forth in the description herein or illustrated in the drawings hereof. The subject matter described herein is capable of other implementations and of being practiced or of being 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 proceeded with the word “a” or “an” should be understood as not excluding plural of said elements or steps, unless such exclusion is explicitly stated. Furthermore, references to “one example” are not intended to be interpreted as excluding the existence of additional examples that also incorporate the recited features. The use of “including,” “comprising,” or “having” and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

[0355] When used in the claims, the term “set” should be understood as one or more things which are grouped together. Similarly, when used in the claims “based on” should be understood as indicating that one thing is determined at least in part by what it isspecified as being “based on.” Where one thing is required to be exclusively determined by another thing, then that thing will be referred to as being “exclusively based on” that which it is determined by.

[0356] Unless specified or limited otherwise, the terms “mounted,” “connected,” “supported,” and “coupled” and variations thereof are used broadly and encompass both direct and indirect mountings, connections, supports, and couplings. Further, “connected” and “coupled” are not restricted to physical or mechanical connections or couplings. Also, it is to be understood that phraseology and terminology used herein with reference to device or element orientation (such as, for example, terms like “above,” “below,” “front,” “rear,” “distal,” “proximal,” and the like) are only used to simplify description of one or more examples described herein, and do not alone indicate or imply that the device or element referred to must have a particular orientation. In addition, terms such as “outer” and “inner” are used herein for purposes of description and are not intended to indicate or imply relative importance or significance.

[0357] It is to be understood that the above description is intended to be illustrative, and not restrictive. For example, the above-described examples (and / or aspects thereof) may be used in combination with each other. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the presently described subject matter without departing from its scope. While the dimensions, types of materials and coatings described herein are intended to define the parameters of the disclosed subject matter, they are by no means limiting and instead illustrations. Many further examples will be apparent to those of skill in the art upon reviewing the above description. The scope of the disclosed subject matter should, therefore, be determined with reference to the appended claims, along with the full scope of equivalents to which such claims are entitled. In the appended claims, the terms “including” and “in which” are used as the plain-English equivalents of the respective terms “comprising” and “wherein.” Moreover, 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. Further, 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, unless and until such claim limitations expressly use the phrase “means for” followed by a statement of function void of further structure.

[0358] The following claims recite aspects of certain examples of the disclosed subject matter and are considered to be part of the above disclosure. These aspects may be combined with one another.

Claims

What is claimed is:

1. An apparatus comprising: an imaging assembly, the imaging assembly including: an objective lens assembly, the objective lens assembly including a front element having a bottom surface, and an immersion fluid assembly to provide immersion fluid under the bottom surface of the front element of the objective lens assembly; a flow cell having an upper surface, the immersion fluid assembly to maintain immersion fluid between the bottom surface of the front element of the objective lens assembly and the upper surface of the flow cell; and a temperature regulation assembly, the temperature regulation assembly including: a first sensor to sense a temperature of the objective lens assembly, a first temperature adjusting feature to adjust a temperature of the flow cell, and a processor to activate the first temperature adjusting feature based on at least data from the first sensor.

2. The apparatus of claim 1, the temperature regulation assembly further including a second sensor to sense a temperature of the flow cell, the processor to activate the first temperature adjusting feature based on at least data from the second sensor.

3. The apparatus of claim 2, the processor to determine a difference between the sensed temperature of the objective lens assembly and the sensed temperature of the flow cell.

4. The apparatus of claim 3, the processor to compare a difference between the sensed temperature of the objective lens assembly and the sensed temperature of the flow cell to a threshold.

5. The apparatus of claim 4, the processor to activate the first temperature adjusting feature in response to the difference between the sensed temperature of the objective lens assembly and the sensed temperature of the flow cell exceeding the threshold.

6. The apparatus of any of claims 4 through 5, the temperature regulation assembly further comprising a second temperature adjusting feature to adjust a temperature of the objective lens assembly, the processor to activate the second temperature adjusting feature in response to the difference between the sensed temperature of the objective lens assembly and the sensed temperature of the flow cell exceeding the threshold.

7. The apparatus of claim 3, the processor to activate the first temperature adjusting feature in response to the processor determining a difference between the sensed temperature of the objective lens assembly and the sensed temperature of the flow cell.

8. The apparatus of claim 7, the temperature regulation assembly further comprising a second temperature adjusting feature to adjust a temperature of the objective lens assembly, the processor to activate the second temperature adjusting feature in response to the processor determining a difference between the sensed temperature of the objective lens assembly and the sensed temperature of the flow cell.

9. The apparatus of any of claims 2 through 8, the processor to: determine whether the flow cell is at a first target temperature, and activate the first temperature adjusting feature based on at least data indicating that the flow cell is not at the first target temperature.

10. The apparatus of claim 9, the temperature regulation assembly further comprising a second temperature adjusting feature to adjust a temperature of the objective lens assembly, the processor to: determine whether the objective lens assembly is at a second target temperature, andactivate the second temperature adjusting feature based on at least data indicating that the objective lens assembly is not at the second target temperature.

11. The apparatus of claim 10, the second target temperature being equal to the first target temperature.

12. The apparatus of any of claims 1 through 11, the first sensor being positioned to sense a temperature of the front element of the objective lens assembly.

13. The apparatus of any of claims 1 through 12, the temperature regulation assembly further including a second temperature adjusting feature to adjust a temperature of the objective lens assembly.

14. The apparatus of claim 13, the processor to activate the first temperature adjusting feature based on at least data from the first sensor.

15. The apparatus of any of claims 13 through 14, the temperature regulation assembly further including a second sensor to sense a temperature of the flow cell, the processor to activate the second temperature adjusting feature based on at least data from the second sensor.

16. The apparatus of any of claims 13 through 15, the second temperature adjusting feature being positioned to adjust a temperature of the front element of the objective lens assembly.

17. The apparatus of claim 16, the second temperature adjusting feature being interposed between the objective lens assembly and the immersion fluid assembly.

18. The apparatus of any of claims 13 through 17, the second temperature adjusting feature comprising a resistive heating element.

19. The apparatus of any of claims 13 through 18, the second temperature adjusting feature comprising: a fan or blower, and one or both of a heating element or a cooling element, the fan being operable to blow heated or cooled air toward the objective lens assembly.

20. The apparatus of any of claims 13 through 19, the second temperature adjusting feature comprising an immersion fluid temperature adjusting feature, the second temperature adjusting feature to adjust a temperature of the objective lens assembly by adjusting a temperature of the immersion fluid.

21. The apparatus of any of claims 13 through 20, the objective lens assembly comprising a housing, the second temperature adjusting feature comprising one or more heating elements positioned adjacent to the housing.

22. The apparatus of claim 21, the second temperature adjusting feature comprising a coil pipe configured to contain a heated or cooled fluid.

23. The apparatus of any of claims 21 through 22, the second temperature adjusting feature comprising one or more of a resistive heating element, a cartridge heater, or a thermoelectric cooler.

24. The apparatus of any of claims 13 through 23, further comprising a spacer interposed between the objective lens assembly and the immersion fluid assembly.

25. The apparatus of claim 24, the spacer comprising a thermally insulating material.

26. The apparatus of any of claims 24 through 25, the second temperature adjusting feature being positioned in the spacer.

27. The apparatus of any of claims 1 through 26, the objective lens assembly further including one or more thermally insulating elements.

28. The apparatus of claim 27, at least one of the thermally insulating elements being positioned adjacent to the front element.

29. The apparatus of any of claims 27 through 28, the objective lens assembly further comprising a plurality of lens elements, the plurality of lens elements including the front element, at least some of the lens elements of the plurality of lens elements being separated by gaps.

30. The apparatus of claim 29, the objective lens assembly further comprising a thermally insulating gas in at least one of the gaps.

31. The apparatus of any of claims 29 through 30, the objective lens assembly further comprising a thermally insulating liquid in at least one of the gaps.

32. The apparatus of any of claims 29 through 31, the objective lens assembly providing a vacuum in at least one of the gaps.

33. The apparatus of any of claims 1 through 32, the objective lens assembly further including an active integral element, the active integral element to actively adjust an optical property of the objective lens assembly.

34. The apparatus of claim 33, the processor to activate the active integral element based on at least data from the first sensor.

35. The apparatus of any of claims 33 through 34, the active integral element comprising one or more of a pair of opposingly rotatable optically transmissive plates to providean astigmatism adjustment, a focus adjustment assembly, a transmissive deformable phase plate, a deformable lens, a wave plate, a molded lens, or a deformable mirror.

36. The apparatus of any of claims 1 through 35, the objective lens assembly including a first subassembly and a second subassembly, the front element being located in the second subassembly, the first subassembly including at least one additional lens element, the first subassembly and the second subassembly being separated from each other by a gap.

37. The apparatus of claim 36, the objective lens assembly further comprising one or more frame elements, the one or more frame elements maintaining separation between the first subassembly and the second subassembly, the one or more frame elements further maintaining alignment between the first subassembly and the second subassembly.

38. The apparatus of any of claims 36 through 37, the temperature regulation assembly further comprising a second temperature adjusting feature to adjust a temperature of the objective lens assembly.

39. The apparatus of claim 38, the second temperature adjusting feature being integrated with the second subassembly such that the second temperature adjusting feature is to adjust a temperature of the front element.

40. The apparatus of claim 39, the gap being sized to prevent thermal transfer of temperature changes of the second subassembly induced by the second temperature adjusting feature to the first subassembly.

41. A method comprising: capturing an image of a region of a flow cell with an imaging assembly, the flow cell having an upper surface, the imaging assembly including: an objective lens assembly, the objective lens assembly including a front element having a bottom surface, andan immersion fluid assembly providing immersion fluid between the bottom surface of the front element of the objective lens assembly and the upper surface of the flow cell while the image is captured; receiving data indicating a temperature of the objective lens assembly; and adjusting a temperature of the flow cell, based at least in part on the temperature of the objective lens assembly.

42. The method of claim 41, further comprising receiving data indicating a temperature of the flow cell.

43. The method of claim 42, further comprising adjusting the temperature of the flow cell based on at least the temperature of the flow cell.

44. The method of any of claims 42 through 43, further comprising determining a difference between the temperature of the objective lens assembly and the temperature of the flow cell.

45. The method of claim 44, further comprising comparing the difference between the temperature of the objective lens assembly and the temperature of the flow cell to a threshold.

46. The method of claim 45, the act of adjusting a temperature of the flow cell being performed in response to the difference between the temperature of the objective lens assembly and the temperature of the flow cell exceeding the threshold.

47. The method of any of claims 45 through 46, further comprising adjusting a temperature of the objective lens assembly in response to the difference between the temperature of the objective lens assembly and the temperature of the flow cell exceeding the threshold.

48. The method of claim 44, the act of adjusting a temperature of the flow cell being performed in response to the difference between the temperature of the objective lens assembly and the temperature of the flow cell.

49. The method of claim 48, further comprising adjusting a temperature of the objective lens assembly in response to the difference between the temperature of the objective lens assembly and the temperature of the flow cell.

50. The method of claim 49, the temperature of one or both of the flow cell or the objective lens assembly being adjusted until the temperature of the flow cell and the temperature of the objective lens assembly are substantially equal.

51. The method of any of claims 42 through 50, further comprising: determining whether the flow cell is at a first target temperature; and if the flow cell is not at the first target temperature, adjusting the temperature of the flow cell toward the first target temperature.

52. The method of claim 51, further comprising: determining whether the objective lens assembly is at a second target temperature; and if the objective lens assembly is not at the second target temperature, adjusting the temperature of the objective lens assembly toward the second target temperature.

53. The method of claim 52, the second target temperature being equal to the first target temperature.

54. The method of any of claims 41 through 53, the received data indicating a temperature of the front element of the objective lens assembly.

55. The method of any of claims 41 through 54, further comprising adjusting a temperature of the objective lens assembly, based at least in part on the temperature of the objective lens assembly.

56. The method of claim 55, the act of adjusting a temperature of the objective lens assembly including adjusting a temperature of the front element of the objective lens assembly.

57. The method of any of claims 55 through 56, the act of adjusting a temperature of the objective lens assembly including adjusting a temperature of the immersion fluid.

58. The method of any of claims 41 through 57, further comprising activating an integral optical element of the objective lens assembly to thereby actively adjust an optical property of the objective lens assembly.

59. The method of claim 58, the integral optical element being activated based at least in part on the temperature of the objective lens assembly.

60. The method of any of claims 58 through 59, the integral optical element being activated to compensate for a thermally induced aberration in the objective lens assembly.

61. An apparatus comprising: an imaging assembly, the imaging assembly including: an objective lens assembly, the objective lens assembly including a front element having a bottom surface, an adjustable optical element within an image path of the front element of the objective lens assembly, the adjustable optical element being adjustable to thereby adjust one or more characteristics of an image within the image path of the front element, and an immersion fluid assembly to provide immersion fluid under the bottom surface of the objective lens; a flow cell having an upper surface, the immersion fluid assembly to maintain immersion fluid between the bottom surface of the front element of the objective lens assembly and the upper surface of the flow cell; a first sensor to sense a temperature of the objective lens assembly; anda processor to activate the adjustable optical element based on at least data from the first sensor.

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