Systems and methods for optical scanning and imaging via a fluid medium for nucleic acid sequencing.

Abstract

To provide systems and methods for imaging biochemical reaction.SOLUTION: Embodiments of the disclosure include methods and systems for nucleic acid sequencing that may include an objective coupled to an actuator, wherein the actuator is configured to move the objective over a surface of a substrate. In some embodiments, a droplet may be disposed on the surface of the substrate, and the droplet may be moved along with the objective. The distal end of the objective may include a material that provides a higher friction against the droplet than a material of the surface of the substrate. In some embodiments, the distal end of the objective may be immersed in a fluid as it is moved over the surface of the substrate. The substrate may include vertical walls within a region to retain the fluid.SELECTED DRAWING: Figure 1B

Description

Technical Field

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[0001] (Cross - reference to related applications) This application claims priority to U.S. Provisional Patent Application No. 63 / 045,566, filed Jun. 29, 2020, for “Systems and Methods for Optical Scanning and Imaging Through a Fluid Medium for Nucleic Acid Sequencing”, the entire disclosure of which is hereby incorporated herein by reference.

[0002] (Related fields) The present invention generally relates to imaging systems, and more particularly to imaging systems for use in general nucleic acid sequencing and biochemical experiments.

Background Art

[0005] This disclosure presents a system and method for imaging biochemical reactions. The system and method can be used, for example, for sequencing template nucleic acid molecules placed on a substrate. The substrate may have an array of spots containing biochemical molecules. This disclosure relates to an optical imaging system that can be configured to image a substrate by rapidly scanning it with an objective lens. The disclosed optical imaging system offers improved resolution compared to conventional systems, thereby increasing the spot density on the substrate and resulting in significant cost reductions, as described herein.

[0006] In some embodiments, the optical imaging system may comprise an actuator, a mounting element configured to receive a substrate, and an objective lens having a proximal and distal end, wherein the substrate comprises one or more nucleic acid sequences, the outer surface of the substrate is configured to receive droplets, the outer surface of the substrate comprises a first material, the proximal end of the objective lens is connected to the actuator, the distal end of the objective lens is configured to be located near the outer surface of the substrate, the distal end of the objective lens comprises a second material, the second material is configured to provide a higher frictional force to the droplet than the first material, the actuator is configured to move the objective lens from a first location on the outer surface of the substrate to a second location on the outer surface of the substrate, and the movement of the objective lens is configured to move the droplet together with the distal end of the objective lens such that the objective lens and the outer surface of the substrate maintain contact with the droplet at the second location.

[0007] In some embodiments, a method for optically imaging a substrate for nucleic acid sequencing may include: placing a droplet on the outer surface of a substrate, wherein the substrate has one or more nucleic acid sequences, and the outer surface of the substrate comprises a first material; positioning an objective lens at a first location on the outer surface of the substrate such that the distal end of the objective lens is in contact with the droplet, wherein the distal end of the objective lens comprises a second material, the second material being configured to provide a higher frictional force to the droplet than the first material; and moving the objective lens to a second location on the outer surface of the substrate, wherein the movement of the objective lens is configured to move the droplet together with the distal end of the objective lens such that the objective lens and the outer surface of the substrate maintain contact with the droplet at the second location.

[0008] In some embodiments, the optical imaging system may comprise an actuator, a mounting element configured to receive a substrate, and an objective lens having a proximal and distal end, wherein the substrate comprises a vertical wall defining a region of the substrate, the vertical wall configured to hold fluid within the region, the substrate having one or more nucleic acid sequences, the proximal end of the objective lens being connected to the actuator, the distal end of the objective lens being positioned near the outer surface of the substrate, the distal end of the objective lens being configured to be immersed in the fluid, and the actuator being configured to move the objective lens from a first location on the outer surface of the substrate to a second location on the outer surface of the substrate, while keeping the distal end of the objective lens immersed in the fluid.

[0009] In some embodiments, a method for optically imaging a substrate for nucleic acid sequencing may include: placing a fluid within a region of the substrate, the region being bounded by vertical walls, and the substrate having one or more nucleic acid sequences; positioning the objective lens at a first location on the outer surface of the substrate such that the distal end of the objective lens is in contact with a droplet; and moving the objective lens to a second location on the outer surface of the substrate while keeping the distal end of the objective lens immersed in the fluid.

[0010] This summary is provided to give a simplified overview of the different embodiments of the present disclosure, which are described in more detail below. This summary is not intended to be used to limit the scope of the claimed subject matter. Other features, details, utility, and advantages of the claimed subject matter will become apparent from the detailed description below. [Brief explanation of the drawing]

[0011] [Figure 1A] This is a schematic diagram of an optical imaging system. [Figure 1B] This is a schematic diagram of an optical imaging system. [Figure 2]This figure shows the substrate shown in Figure 1, divided into an array of sub-regions. [Figure 3A] This figure shows an exemplary embodiment of an optical imaging system in which a droplet is placed and held between the objective lens and the substrate. [Figure 3B] This figure shows an exemplary embodiment of an optical imaging system in which a droplet is placed and held between the objective lens and the substrate. [Figure 3C] This figure shows a cross-section of an exemplary embodiment of an objective lens having a concave distal end. [Figure 4] This is an illustrative schematic diagram of an optical imaging system comprising a droplet monitoring subsystem and a droplet delivery subsystem. [Figure 5A] This figure shows an alternative embodiment of an optical imaging system in which the distal end of an objective lens is immersed in a fluid placed on the outer surface of a substrate. [Figure 5B] This figure shows an alternative embodiment of an optical imaging system in which the distal end of an objective lens is immersed in a fluid placed on the outer surface of a substrate. [Figure 5C] This figure shows an exemplary embodiment of an objective lens having a proximal portion and a distal portion, the distal portion having a distal end that is immersed in a fluid. [Figure 5D] This figure shows an exemplary embodiment having a curved vertical wall. [Figure 6] This figure illustrates an exemplary method for optically imaging a substrate in order to determine the nucleic acid sequence on the substrate. [Figure 7] This figure shows another exemplary method for optically imaging a substrate in order to sequence nucleic acid sequences on the substrate.

[0012] In accordance with common practice, the described features and elements are not depicted to scale, but rather to emphasize the features and elements relevant to this disclosure. [Modes for carrying out the invention]

[0013] This disclosure describes an optical imaging system that may be used to image biochemical reactions. For example, the disclosed optical imaging system may be used to sequence template nucleic acid molecules (e.g., DNA molecules, RNA molecules). In some embodiments, the template nucleic acid molecule may be bound to or placed on the surface of a substrate (e.g., the inner surface of a flow cell) that can be imaged by the optical imaging system. For example, a DNA template may be immobilized at a position (spot) greater than 10e7 on an array on the planar inner surface of a substrate (e.g., a flow cell). In this example, the nucleic acid sequencing method may include performing more than 400 sequencing cycles. In each cycle, one nucleotide (e.g., adenine, guanine, thymine, and cytosine) may flow across the substrate and be incorporated (into the growth chain) at each site where complementary nucleotide bases are present. In one approach, each of the four different nucleotides may be labeled with a different colored fluorescent dye or conjugated with a dye-labeled antibody. In each sequencing cycle, a light source (e.g., a laser) may illuminate the spot (e.g., in series), causing the dyes to emit light corresponding to their respective colors. The color emitted from one of the four dyes at each spot may be detected by a camera (e.g., a time-delay integrated charge-coupled device (TDI-CCD) camera or similar camera), and an imaging system can then record the detection of the nucleotide corresponding to the detected color for each spot. Those familiar with the art will know of variations in sequencing methods, including variations in template types (e.g., see Huang et al., 2017, Gigascience 6:1-9; Mardis et al., 2013, Annu Rev Anal Chem 6:287-303), labeling systems (e.g., see WO2018129214), and labeling strategies (e.g., see US 9,523,125). In conventional systems, emitted light travels from a spot on the substrate, through a glass coverslip (e.g., the top of the substrate), through an air gap, into the microscope's objective lens, and finally to a camera that captures one or more images.In some embodiments, the objective lens comprises a housing configured to collect and focus rays from a substrate, and one or more lenses, which focus the rays to produce a magnified image that can be captured by a camera. The system may be configured to move the objective lens in a pattern over an array of spots on a substrate so that the entire array is imaged during each cycle. While this disclosure focuses on sequencing nucleic acid molecules, the disclosed optical imaging system is intended to be used to image any suitable biochemical experiment.

[0014] A significant portion, or at least a large portion, of the cost associated with nucleic acid sequencing per gigabase is the amount of reagents consumed in the sequencing process. Therefore, increasing the sample density on the substrate significantly reduces the cost of nucleic acid sequencing. In optical detection-based systems, the optical numerical aperture (NA) of the detection system partially determines the system's optical resolution, thereby determining the maximum sample density. High-NA optics tend to be more expensive, larger, and more difficult to align and maintain than low-NA optics.

[0015] In certain optical systems (e.g., confocal optical systems), the numerical aperture (NA) can be limited by the lowest refractive index in the image chain. The reason the NA is limited is the critical angle at each interface between materials. The critical angle is defined as the maximum ray angle at which total internal reflection does not occur at an interface. When the refractive indices of each interface in the image chain are close, the critical angle is large. As the difference in refractive indices increases, the critical angle decreases, and the amount of light transmitted through the interface decreases. Since the objective lens of the optical system has a relatively high refractive index, and segments of the image chain with relatively low refractive indices can form relatively small critical angles, the amount of light transmitted to the objective lens decreases. Therefore, one way to increase the NA of such a system without relying on more expensive, larger, and more complex equipment for high-NA systems is to increase the refractive index of one or more segments of the image chain that tend to have low refractive indices. Methods and systems for doing so are disclosed herein.

[0016] In conventional optical systems used for nucleic acid sequencing, the segment of the image chain with the lowest refractive index is often an air gap, which may exist, for example, between the objective lens and the substrate of the optical system. Air gaps generally have a refractive index of approximately 1.00. In this example, the NA of the exemplary optical imaging system may be approximately 0.8. Replacing the air with a substance having a higher refractive index can increase the overall NA of the optical system. For example, replacing the air gap with water may raise the lowest refractive index of the image chain to 1.33. In this example, the NA of a similar optical imaging system using water instead of air gap may be approximately 1.0. As another example, replacing the air gap with standard oil may raise the lowest refractive index of the image chain to 1.51. In this example, the NA of a similar optical imaging system using water instead of air gap may be approximately 1.2. As yet another example, replacing the air gap with high refractive index oil may further increase the NA to 1.4. As yet another example, any suitable aqueous or oily solution may be used to appropriately produce the desired change in the lowest refractive index. Essentially, this disclosure proposes using a fluid with a refractive index higher than air as a medium between the objective lens and the substrate. Increasing the minimum refractive index of the image chain has a direct and measurable effect on the numerical aperture (NA) of the optical imaging system, thereby improving resolution and consequently increasing the density of spots on the substrate. The increased density reduces the amount of reagents required, leading to cost reductions. This can be explained by benchmarking an optical imaging system with an air gap, setting its density to 1.00 and its relative cost to 1.00. With this benchmark in mind, replacing the air gap with water can increase the density to approximately 1.56, and correspondingly reduce the relative cost to approximately 0.64. Replacing the air gap with standard oil can increase the density to approximately 2.25, and correspondingly reduce the relative cost to approximately 0.44. Replacing the air gap with high refractive index oil can increase the density to approximately 3.06, and correspondingly reduce the relative cost to approximately 0.33.

[0017] The use of a fluid medium having a refractive index higher than air is well known in standard microscope applications using immersion objective optical systems, but such applications involve static image processing. In dynamic imaging processes, such as the scanning optical system contemplated herein for imaging a substrate while the objective lens rapidly moves over the substrate, conventional immersion objective optical systems are insufficient. An exemplary optical scanning system can move the objective lens at a speed between 10 mm / second and 60 mm / second. In some cases, a next-generation sequencer capable of moving the objective lens at about 300 mm / second may be equipped. Moving the objective lens of a conventional system at such high speeds within such a fluid medium tends to cause excessive turbulence (and generation of air bubbles), which not only affects the image quality but also may cause loss of the fluid medium. The present disclosure describes two approaches for a solution that replaces the air gap with a fluid medium having a higher refractive index while addressing potential problems that typically arise when the objective lens is moved rapidly. In some embodiments, the approaches disclosed herein are used to move the objective lens at a speed between 10 mm / second and 60 mm / second, or between 10 mm / second and 300 mm / second, while maintaining good image quality using the fluid medium. In some embodiments, very high speeds can be achieved, enabling speeds between 10 mm / second and 3750 mm / second, or between 30 mm / second and 3750 mm / second. This enables high-speed scanning and imaging, for example, at a camera line rate of about 1 M / second.

[0018] Figure 1A is an exemplary schematic diagram of an optical imaging system 100. Referring to Figure 1A, for example, the optical imaging system 100 comprises an objective lens 110. The objective lens 110 may be configured for use in capturing an image of the substrate 120. Referring to Figure 1A, the objective lens 110 may have a proximal end 110-1 and a distal end 110-2. The proximal end 110-1 of the objective lens 110 may be coupled (directly or indirectly) to an actuator, and the distal end 110-2 of the objective lens 110 may be configured to be located near the outer surface 120-1 of the substrate 120. In some embodiments, biochemical molecules may be bonded to the interior of the substrate (e.g., the inner surface of the substrate) or arranged on top of it. For example, the inner surface of the substrate may contain one or more target nucleic acid sequences. In some embodiments, the outer surface of the substrate may be the surface of a separate element that can be removably positioned on top of the rest of the substrate. For example, referring to Figure 1A, the outer surface 120-1 of the substrate 120 may be the surface of a coverslip positioned on top of the rest of the substrate (for example, the bottom of the substrate, referring to Figure 1; this may be a portion other than the top wall) (for example, the substrate is a flow cell whose top is not covered by anything other than a coverslip). In this example, biochemical molecules such as polynucleotides may be placed on the inner surface of the bottom of the substrate, and elements such as the coverslip may be located on top of the bottom of the substrate. In other embodiments, the substrate may be a more integrated structure in which the outer surface is integrated with the substrate. For example, referring to Figure 1A, the outer surface 120-1 may be an integrated, non-removable portion of the substrate 120. In this example, there may be one or more chambers and / or conduits within the substrate 120, and biochemical molecules such as nucleic acid sequences may be placed on the inner surfaces of the chambers and / or conduits.

[0019] In some embodiments, the optical imaging system 100 may include an actuator for moving (i.e., “scanning”) the objective lens over a plurality of locations on the outer surface 120-1 of the substrate 120. For example, referring to FIG. 1A, the objective lens 110 is coupled to an actuator 130. Although the actuator is illustrated as a single block in FIG. 1A, it will be understood that the actuator may be a multi-component subsystem capable of moving the objective lens over the outer surface 120-1 of the substrate 120.

[0020] Figure 1B is another exemplary schematic diagram of the optical imaging system 101. In some embodiments, the optical imaging system may include an objective lens configured for use in capturing one or more images of a substrate (e.g., a flow cell). One embodiment of a flow cell is schematically shown in Figure 1B. As shown, the flow cell comprises a first substrate 120, a second substrate 140, and a flow space 150. In one approach, nucleic acid template molecules (e.g., DNB) are immobilized at positions on the inner surface of the substrate (e.g., surface 120-2 or 140-1), and reagents and washing buffers flow through the space 150. Thus, the space 150 is generally an aqueous environment, which may be necessary to preserve the nucleic acid template placed in the space 150. The positions, or spots, may be organized as a regular ordered arrangement on surface 120-2 or 140-1 and adapted to contain the nucleic acid template molecules. For example, the above-mentioned locations may be regions on the substrate surface derivatized for binding nucleic acid molecules (e.g., DNB, template clusters generated by bridge amplification, or other templates), wells, or other structures. In the process of sequencing or other analysis, a detectable optical signal, such as a fluorescence or emission signal, is emitted by a dye associated with the template molecule. For example, in synthetic sequencing methods, the dye may be bound to nucleotides incorporated into the growing chain at each location, or to affinity reagents bound to the incorporated nucleotides. The emitted signal (hereinafter referred to as the "fluorescence" signal) travels from the template immobilized on surface 140-1 or 120-2 through the substrate (e.g., glass coverslip) 120 and the gap 160 to the distal end 110-2 of the objective lens. In this approach, substrate 120 is transparent to the optical signal, but substrate 140 may be opaque. As described elsewhere in this specification, the gap 160 may contain water or oil. In one approach, the nucleic acid template is immobilized on surface 120-2 of substrate 120.In one approach, the nucleic acid template is positioned on a patterned array on surface 120-2. In these approaches (for example, when the nucleic acid template is positioned on surface 120-2), the fluorescence signal does not need to travel through the aqueous environment of space 150. This can be advantageous, in particular, if gap 160 contains oil or another material with a relatively high refractive index. This is because image quality and resolution are partially limited by the medium with the lowest refractive index through which the light ray passes, as will be explained below. Therefore, by eliminating aqueous media from the path of the light ray (by positioning the nucleic acid template on surface 120-2), image quality and resolution can be improved by eliminating relatively low refractive index aqueous media in space 150. For example, when the nucleic acid template is positioned on surface 120-2, the light ray from the nucleic acid template only needs to pass through the substrate 120, oil or other high refractive index material, and the objective lens before being imaged. That is, the light ray does not need to pass through any aqueous media that may need to be present in space 150. In some embodiments, the nucleic acid template may be positioned on surface 140-1. To avoid any ambiguity, the description of a specific flow cell is not intended to limit the invention.

[0021] Figure 2 shows the substrate 120 shown in Figure 1 divided into an array of subregions. In some embodiments, the substrate may be divided into multiple subregions. For example, referring to Figure 2, the substrate 120 may have multiple subregions such as a first subregion 210 and a second subregion 220. In one approach, the substrate has a patterned array of derivatized regions ("spots") to which DNA is templated (e.g., DNB is immobilized). In some embodiments, physical barriers may exist between the subregions. In other embodiments, the subregions may not be separated by physical barriers; that is, the division between regions may be a virtual division not bounded by structural elements. Each subregion may correspond to a spot, and each spot may contain a particular subset of molecules (e.g., a particular set of DNA template strands). In some embodiments, the actuator may be configured to move the objective lens between different subregions of the substrate. For example, referring to Figure 2, the actuator may be configured to move the objective lens from a first location on the outer surface 120-1 of the substrate 120 (e.g., corresponding to sub-region 210) to a second location on the outer surface 120-1 of the substrate 120 (e.g., corresponding to sub-region 220). Although Figure 2 illustrates the substrate 120 divided into discrete sub-regions (e.g., a first sub-region 210 and a second sub-region 220), the disclosure intends that the substrate does not have to be divided into discrete sub-regions at all, and the actuator may simply move continuously from one location to another on the outer surface 120-1 of the substrate 120 while imaging the substrate 120.

[0022] Figures 3A and 3B show exemplary embodiments of an optical imaging system 100 in which a droplet 310 is placed and held between an objective lens 110 and a substrate 120. In some embodiments, the optical imaging system 100 can replace the air gap conventionally present in optical scanning systems with a droplet of fluid having a refractive index greater than that of air. For example, referring to Figure 3A, the optical system 100 is positioned so as to contact both the distal end 110-2 of the objective lens 110 and the outer surface 120-1 of the substrate 120. Thereafter, the fluid in the droplet acts as a medium through which light rays can pass between the objective lens 110 and the substrate 120 without an air gap. In some embodiments, the droplet may be a droplet of water, an oil droplet, a droplet of an aqueous solution, or a droplet of an oily solution.

[0023] In some embodiments, the droplets 310 may be positioned by a droplet delivery subsystem. The droplet delivery subsystem may comprise, for example, a conduit (e.g., a pipe or other suitable channel) connected to a fluid reservoir. The droplet delivery subsystem may comprise any suitable microfluidic droplet generator configured to generate droplets from the fluid in the fluid reservoir. The droplet generator may be configured to generate droplets of a suitable predetermined size. The droplets may be guided through the conduit and positioned at a suitable location on the outer surface 120-1 of the substrate 120 using any suitable means such as electrowetting on a dielectric (EWOD) or a pressure difference.

[0024] Figures 3A and 3B illustrate a method for moving the objective lens 110 from a first location (Figure 3A) to a second location (Figure 3B) on the substrate 120 when capturing one or more images of the substrate 120. As described above, this movement may be performed at high speed (e.g., 15 mm / sec, 300 mm / sec). Despite this high-speed movement, the optical imaging system 100 can maintain the droplet 310 (either entirely or substantially entirely with minimal fluid loss) below the distal end 110-2 of the objective lens 110, such that the droplet 310 remains between the distal end 110-2 of the objective lens 110 and the outer surface 120-1 of the substrate 120. When moving the objective lens 110, both the distal end 110-2 and the outer surface 120-1 of the substrate may maintain contact with the droplet during and after the movement. The optical imaging system 100 can image the substrate 120 over multiple such movements while allowing the droplet 310 to function as a fluid medium, without an air gap existing between the objective lens 110 and the substrate 120.

[0025] While Figures 3A and 3B illustrate the above concept relating to a single-substrate design in the simplified schematic diagram of Figure 1A (for example, the nucleic acid sequence to be imaged may be located on or inside substrate 120), it should be noted that this disclosure intends for the same concept to apply to the schematic diagram of Figure 1B, in which two substrates exist. For example, referring to Figure 1B, the nucleic acid sequence to be imaged according to Figures 3A and 3B may be located in space 150 (for example, it may be bound to surface 120-2 or surface 140-1).

[0026] In some embodiments, the optical imaging system 100 may be able to retain the droplet 310 in part as described above by engineering the surface chemistry of the distal end 110-2 of the objective lens 110 and / or the outer surface 120-1 of the substrate 120. More specifically, the surface chemistry may be selected to optimize the friction between the fluid medium and the distal end 110-2 of the objective lens 110 and / or the friction between the fluid medium and the outer surface 120-1 of the substrate 120 to enhance droplet retention. For the purposes of this disclosure, friction (frictional force) may be characterized as a force resisting the relative motion of the fluid medium sliding against the distal end 110-2 or the outer surface 120-1. In some embodiments, the outer surface 120-1 of the substrate may include a first material, and the distal end 110-2 of the objective lens may include a second material, the second material may be configured to provide a higher frictional force to the droplet than the first material. In other words, the surface chemistry may be configured such that the droplet 310 receives a relatively high frictional force at the distal end 110-2 of the objective lens and a relatively low frictional force at the outer surface 120-1 of the substrate 120. With such a configuration, when the objective lens 110 moves, the portion of the droplet 310 that is in contact with the distal end 110-2 of the objective lens is attracted to the distal end, thereby moving the droplet together with the objective lens 110. In contrast, the portion of the droplet 310 that is in contact with the outer surface 120-1 of the substrate is not similarly attracted to the outer surface 120-1 of the substrate, and the outer surface 120-1 of the substrate may actually repel the droplet 310 so that the droplet 310 can move freely on the outer surface 120-1 without adhering to it. In other words, the surface chemistry may be configured such that the distal end 110-2 of the objective lens attracts the droplet 310, and the outer surface 120-1 of the substrate repels (is repelled) the droplet 310. In some embodiments, the material may be selected such that when a droplet of a particular fluid medium (e.g., oil or aqueous solution) is placed on the surface, the droplet remains at a desired contact angle. For example, a droplet of a fluid medium placed on surface 120-1 may have a contact angle between 93 and 160 degrees. A larger contact angle is desirable because it results in a greater repulsive force from the droplet surface.As another example, the contact angle may be between 11 and 120 degrees. Similarly, the material of the distal end 110-2 of the objective lens 110 may be selected such that a droplet of a fluid medium placed on a flat surface made of that material has a contact angle between 20 and 80 degrees.

[0027] The degree of friction between various surfaces and fluids can be determined and compared by known means. Surfaces with different material compositions may have different degrees of friction with respect to a particular fluid. Based on the relative friction with respect to a particular fluid medium, a suitable material may be selected to achieve the attractive / repulsive effect described above. In some embodiments, when the droplet 310 is, for example, water or aqueous solution, the first material of the outer surface 120-1 of the substrate may include a hydrophobic material (e.g., glass, silicon dioxide, sapphire, calcium oxide, Teflon, polytetrafluoroethylene, fluorinated ethylene propylene, perfluoroalkoxyalkane), and the second material of the distal end 110-2 of the objective lens may include a hydrophilic material (e.g., polyester, polyurethane, polyether, Sympatex). In some embodiments, if the droplet 310 is, for example, oil or oily, the first material of the outer surface 120-1 of the substrate may include an oleophilic material (e.g., barium sulfate, polyethylene terephthalate (PET), polypropylene, carbon-based sponge, functional polymer sponge, kapok / milkweed seed hair), and the second material of the distal end 110-2 of the objective lens may include an oleophobic material (e.g., Teflon and Teflon derivatives, hexadecane, polytetrafluoroethylene).

[0028] Figure 3C shows a cross-section of an exemplary embodiment of an objective lens 110 having a concave distal end 110-2. In some embodiments, the distal end 110-2 of the objective lens 110 may be concave to facilitate maintaining contact between the objective lens 110 and the droplet 310 as the objective lens 110 moves. In these embodiments, the curvature of the distal end 110-2 may play a role in increasing the attracting force and assisting in pulling the droplet together with the objective lens 110. The radius of curvature and depth of the distal end 110-2 may be optimized based on the size of the droplet 310 generated by the droplet generator of the droplet delivery subsystem. For example, the distal end 110-2 may have a radius of curvature between 45 mm and 160 mm and a depth between 0.6 mm and 2.1 mm. These ranges have been found to be advantageous, for example, in that they help to retain the droplet as the objective lens moves. In some embodiments, the distal end 110-2 may be concave and may also have the surface chemistry described above (for example, the distal end 110-2 may be made of a material that has a higher frictional and attractive force to the droplet). This enhances the distal end 110-2's ability to move the droplet 310 together with the objective lens 110. In these embodiments, the concave shape of the distal end 110-2 provides a larger surface area in contact with the droplet 310 than if the distal end 110-2 were a flat surface (which can impart frictional and attractive forces to the droplet 310), thus providing a certain level of synergistic effect to achieve this objective.

[0029] Figure 4 is an exemplary schematic diagram of an optical imaging system 100 comprising a droplet monitoring subsystem and a droplet delivery subsystem. In some embodiments, as shown in Figure 4, the optical imaging system 100 may include a droplet monitoring subsystem that (continuously or periodically) monitors an optical scanner (equipped with hardware for imaging the substrate 120, such as an objective lens 110 and an actuator 130) to determine whether the objective lens 110 and the outer surface 120-1 of the substrate 120 are in contact with a droplet 310. The droplet monitoring subsystem may use any suitable means to make this determination. For example, a resistance or capacitance sensor may be used to determine the presence of the droplet 310, or an optical sensor may be used to detect the presence of the droplet 310. Alternatively, the image quality captured through the objective lens 110 may simply be analyzed to confirm that the light rays are properly refracted (indicating that the droplet 310 is still present). If it is determined that both the objective lens 110 and the outer surface 120-1 of the substrate 120 are not in contact with the droplet 310, the droplet delivery subsystem may be triggered to deliver an additional droplet to the optical scanner at an appropriate location on the outer surface 120-1 of the substrate 120 (e.g., under the objective lens 110). In this way, the droplet monitoring subsystem and the droplet delivery subsystem may work in conjunction with the optical scanner to form a feedback control loop that operates to hold the droplet 310 for an extended period and / or to correct a system error that caused the droplet 310 to move away from its position under the objective lens 110.

[0030] Figures 5A-5B illustrate alternative embodiments of the optical imaging system 100, in which the distal end 110-2 of the objective lens is immersed in a fluid 510 placed on the outer surface 120-1 of the substrate 120. In some embodiments, referring to Figure 5A, the substrate includes a vertical wall 525 that borders (encloses) a region of the substrate. The vertical wall 525 may be configured to hold the fluid 510 within that region. The fluid may be maintained at a level sufficient to immerse the distal end 110-2 of the objective lens 110 to a desired depth. The fluid may be any suitable fluid, including the fluid described above with respect to the droplet 310. In some embodiments, the vertical wall 525 may be part of the substrate (e.g., molded into the substrate). In alternative embodiments, the vertical wall 525 may not be part of the substrate, but instead may be part of the housing of the optical imaging system 100 extending over the substrate, thereby achieving the same or similar effect.

[0031] In these embodiments, when the actuator is moved from a first location (Figure 5A) to a second location (Figure 5B) on the substrate 120, the distal end 110-2 may remain immersed in the fluid. Thus, the light ray can travel between the objective lens 110 and the substrate 120 through the fluid without passing through an air gap. In some embodiments, the distal end 110-2 of the objective lens 110 may be flat to reduce turbulence as the objective lens moves from the first location to the second location. As mentioned above, high-throughput imaging applications may require rapid movement of the objective lens, which can amplify turbulence. Also, as mentioned above, turbulence can negatively affect image quality (for example, by forming bubbles or flow aberrations that can produce undesirable image artifacts) and / or cause loss of fluid 510 over time. Reducing this turbulence addresses these problems. Unlike embodiments of the optical imaging system 100 that use droplets, in embodiments in which the objective lens is moved while immersed in a fluid, the concave distal end 110-2 can cause problems. This is because the fluid 510 around the objective lens 110 with the concave distal end 110-2 has a flow profile in which the fluid 510 in contact with the concave distal end 110-2 flows at different velocities along points of different curvatures, resulting in a non-laminar flow. This flow pattern propagates throughout the region as the objective lens 110 moves, thereby generating excessive turbulence. In contrast, a flat distal end 110-2 can reduce such turbulence by providing a more laminar flow.

[0032] Figure 5C shows an exemplary embodiment of an objective lens 110 having a proximal portion 112 and a distal portion 114, the distal portion 114 having a distal end 110-2 immersed in the fluid 510. In such embodiments, the entire distal portion (or alternatively, at least a portion of the distal portion expected to be immersed in the fluid 510) may be non-tapered (e.g., cylindrical). This can further facilitate laminar flow as the objective lens 110 moves and reduce turbulence.

[0033] Figure 5D shows an exemplary embodiment comprising a curved vertical wall 526. In some embodiments, the interior of the vertical wall is perpendicular to the outer surface 120-1 of the substrate 120, as shown by the vertical wall 525 in Figures 5A-5C. In other embodiments, the interior of the vertical wall does not have to be perpendicular to the outer surface 120-1 of the substrate 120, but may be positioned at an angle. In some embodiments, the interior of the vertical wall may be flat, as shown by the vertical wall 525 in Figures 5A-5C. In other embodiments, the interior of the vertical wall may be curved, as shown by the vertical wall 526 in Figure 5D. The interior of the vertical wall may be formed according to any suitable profile.

[0034] In some embodiments, the height of the vertical wall 525 may be optimized to be sufficient to hold the fluid 510 within the region bounded (enclosed) by the vertical wall 525 and to prevent the fluid 510 from splashing or otherwise flowing out of the region as the objective lens 110 moves within the region.

[0035] Embodiments in which the objective lens 110 is immersed in a fluid (e.g., embodiments illustrated in Figures 5A-5D) may also have a feedback control loop similar to that illustrated in Figure 4. In such cases, the droplet monitoring subsystem and the droplet delivery subsystem may be replaced by a fluid monitoring subsystem and a fluid delivery subsystem, respectively. In some embodiments, the fluid monitoring subsystem may continuously or periodically determine that the level of fluid 510 in the region bounded by the vertical wall 525 is sufficient to immerse the objective lens 110. For example, the fluid monitoring subsystem may optically or electrically monitor the fluid level, monitor the weight of the fluid 510 (e.g., using a scale under the substrate that measures the weight of the substrate 120 and the fluid 510), or simply analyze the image quality captured through the objective lens 110 to confirm that the light rays are properly refracted (indicating that the objective lens 110 is still immersed in the fluid 510). As another example, the fluid monitoring subsystem may use an optical sensor to detect the presence of bubbles in the fluid, and since bubbles may indicate fluid evaporation, it may determine whether a certain amount of fluid has evaporated or flowed out of the area. The fluid monitoring subsystem can correlate the amount of bubbles with the amount of evaporated fluid. In some embodiments, when the fluid monitoring subsystem determines that a threshold amount of fluid has evaporated or flowed out (for example, based on the detection of a threshold number of bubbles, or based on the electrical or optical detection that the fluid level line (liquid level) has dropped by a threshold amount), the fluid delivery subsystem may be triggered to automatically replenish the lost fluid. For example, the fluid delivery subsystem may include a conduit connected to a fluid reservoir, and an appropriate amount of replacement fluid may be delivered to the area by any suitable means (e.g., EWOD, pressure difference created by a pump).

[0036] While Figures 5A to 5D illustrate the above concepts in relation to a single-substrate design in the simplified schematic diagram of Figure 1A (for example, the imaged nucleic acid sequence may be located on or inside substrate 120), it should be noted that this disclosure intends for the same concepts to apply to the schematic diagram of Figure 1B in which two substrates are present. For example, referring to Figure 1B, the nucleic acid sequence imaged according to Figures 5A to 5D may be located in space 150 (for example, bound to surface 120-2 or surface 140-1).

[0037] Figure 6 shows an exemplary method 600 for optically imaging a substrate for sequencing nucleic acid sequences. The method may include, in step 610, placing a droplet on the outer surface of the substrate. The substrate contains one or more nucleic acid sequences, and the outer surface of the substrate contains a first material. In step 620, the method may include positioning an objective lens at a first location on the outer surface of the substrate such that the distal end of the objective lens is in contact with the droplet. The distal end of the objective lens contains a second material, which is configured to provide a higher frictional force to the droplet than the first material. In step 630, the method may include moving the objective lens to a second location on the outer surface of the substrate. The movement of the objective lens is configured to move the droplet together with the distal end of the objective lens such that the objective lens and the outer surface of the substrate maintain contact with the droplet at the second location.

[0038] In certain embodiments, one or more steps of the method in Figure 6 may be repeated as needed. While this disclosure describes and illustrates certain steps of the method in Figure 6 as occurring in a specific order, this disclosure intends that any suitable steps of the method in Figure 6 may occur in any suitable order. Furthermore, while this disclosure describes and illustrates exemplary methods for optically imaging a substrate to sequence nucleic acid sequences on the substrate, including certain steps of the method in Figure 6, this disclosure intends that any suitable method for optically imaging a substrate to sequence nucleic acid sequences on the substrate may include all, some, or none of the steps of the method in Figure 6, as needed. Furthermore, while this disclosure describes and illustrates certain components, devices, or systems for performing certain steps of the method in Figure 6, this disclosure intends that any suitable combination of any suitable components, devices, or systems may perform any suitable steps of the method in Figure 6.

[0039] Figure 7 shows another exemplary method 700 for optically imaging a substrate to sequence nucleic acid sequences on the substrate. The method may include, in step 710, placing a fluid within a region of the substrate. The region is bounded by a vertical wall, and the substrate contains one or more nucleic acid sequences (for example, at locations within the boundary corresponding to the region on the substrate). In step 720, the method may include positioning an objective lens at a first location on the outer surface of the substrate such that the distal end of the objective lens is in contact with the fluid. In step 730, the method may include moving the objective lens to a second location on the outer surface of the substrate while the distal end of the objective lens remains immersed in the fluid.

[0040] In certain embodiments, one or more steps of the method in Figure 7 may be repeated as needed. While this disclosure describes and illustrates the specific steps of the method in Figure 7 as occurring in a specific order, this disclosure intends that any suitable steps of the method in Figure 7 may occur in any suitable order. Furthermore, while this disclosure describes and illustrates an exemplary method for optically imaging a substrate to sequence nucleic acid sequences on the substrate, including the specific steps of the method in Figure 7, this disclosure intends that any suitable method for optically imaging a substrate to sequence nucleic acid sequences on the substrate may include all, some, or none of the steps of the method in Figure 7, as needed. Furthermore, while this disclosure describes and illustrates specific components, devices, or systems for performing specific steps of the method in Figure 6, this disclosure intends that any suitable combination of any suitable components, devices, or systems may perform any suitable steps of the method in Figure 7.

[0041] The examples and embodiments described herein are for illustrative purposes only, and various modifications or changes taking them into account are suggested to those skilled in the art, are understood to be within the spirit and scope of this application and within the scope of the appended claims. All publications, patents, and patent applications referenced herein are incorporated herein by reference in their entirety for all purposes.

[0042] It will be understood that the above description is illustrative and not restrictive. Many embodiments will be apparent to those skilled in the art upon consideration of the above description. Therefore, the scope of the invention should not be determined by reference to the above description, but rather by reference to the appended claims, together with their equivalent entire scope.

[0043] While the foregoing disclosures illustrate exemplary aspects of the present disclosure, it should be noted that various changes and modifications can be made without departing from the scope of the present disclosure as defined by the appended claims. The functions, steps, and / or operations claimed in the methods according to the aspects of the present disclosure described herein do not need to be performed in any particular order. Furthermore, elements of the present disclosure may be described or claimed in the singular form, but the plural form is also intended unless explicitly limited to the singular form.

Claims

1. (a) Using an optical imaging system equipped with an objective lens, image multiple sub-regions on the substrate, each corresponding to a nucleic acid immobilization spot. (b) During imaging, the liquid fills the space between the distal end of the objective lens and the substrate, (c) During imaging, the optical imaging system operates such that the liquid fills the space between the distal end of the objective lens and the substrate, and the substrate moves relative to the objective lens at a scanning speed of 10 mm / sec to 3750 mm / sec while the objective lens moves through the liquid. It includes, During imaging, the distal end of the objective lens is immersed in the liquid. The distal end of the objective lens is flat, A nucleic acid sequencing method wherein the distal end of the objective lens has a non-tapered shape to reduce the generation of turbulence that occurs when the objective lens moves through the liquid during scanning.

2. The nucleic acid sequence determination method according to claim 1, wherein the substrate is a flow cell with a cover or a substrate without a cover.

3. The nucleic acid sequencing method according to claim 2, wherein the sub-region is located on at least one inner surface of the covered flow cell or on the outer surface of the uncovered substrate.

4. The sub-region is such that the substrate is at least 10 ７ The nucleic acid sequencing method according to claim 2, wherein the subregions are arranged at a density that includes 100 subregions.

5. The nucleic acid sequencing method according to claim 4, wherein the optical imaging system comprises an optical system having an numerical aperture (NA) of at least 1.

0.

6. The nucleic acid sequencing method according to claim 4, wherein the optical imaging system comprises an optical system having an numerical aperture (NA) in the range of 1.0 to 1.

56.

7. The nucleic acid sequence determination method according to claim 1, wherein during imaging, the optical imaging system operates such that the substrate moves relative to the objective lens at a scanning speed of 10 mm / second to 300 mm / second.

8. The nucleic acid sequencing method according to claim 1, wherein during imaging, the optical imaging system operates such that the substrate moves relative to the objective lens at a scanning speed of at least 15 mm / second.

9. The nucleic acid sequence determination method according to claim 1, wherein the liquid comprises oil or water.

10. The nucleic acid sequencing method according to claim 1, further comprising monitoring the air between the distal end of the objective lens and the substrate.

11. During imaging, the distal end of the objective lens is immersed in the liquid. Monitoring the air between the distal end of the objective lens and the substrate, (a) Using a sensor to optically or electronically monitor the level of the liquid, (b) Using a sensor to monitor the weight of the liquid, (c) Use of an image analysis device, and, (d) Use a sensor to detect bubbles. A nucleic acid sequencing method according to claim 10, comprising one or more of the following.

12. The nucleic acid sequencing method according to claim 10, further comprising delivering the liquid to the substrate using a liquid delivery subsystem configured to deliver the liquid to the substrate when it is determined that air is present between the distal end of the objective lens and the substrate.

13. (a) Using an optical imaging system equipped with an objective lens, image multiple sub-regions on the substrate, each corresponding to a nucleic acid immobilization spot. (b) During imaging, the liquid fills the space between the distal end of the objective lens and the substrate, (c) During imaging, the optical imaging system operates such that the substrate moves relative to the objective lens at a scanning speed of 10 mm / sec to 3750 mm / sec while the liquid fills the space between the distal end of the objective lens and the substrate. It includes, During imaging, the distal end of the objective lens is immersed in the liquid. The distal end of the objective lens is flat, A nucleic acid sequencing method wherein the distal end of the objective lens has a non-tapered shape to reduce the generation of turbulence that occurs when the objective lens moves through the liquid during scanning.

14. The liquid is placed within the first region of the substrate, The first region includes the plurality of sub-regions, The nucleic acid sequencing method according to claim 1 or 13, wherein the objective lens is moved relative to the substrate from a first location within the first region to a second location within the first region, with the distal end of the objective lens immersed in the liquid.

Images