Systems and methods for optical scanning and imaging through fluid media for nucleic acid sequencing - Patents.com

Abstract

Embodiments of the present disclosure include methods and systems for nucleic acid sequencing, comprising an objective lens coupled to an actuator, the actuator configured to move the objective lens 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 lens. A distal end of the objective lens may comprise a material that imparts a higher friction force to the droplet than a material of the surface of the substrate. In some embodiments, the distal end of the objective lens may be immersed in a fluid as it is moved over the surface of the substrate. The substrate may comprise a vertical wall within a region for holding a fluid.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Patent Application No. 63 / 045,566, filed June 29, 2020, for “Systems and Methods for Optical Scanning and Imaging Through a Fluid Medium for Nucleic Acid Sequencing,” the entirety of which is incorporated herein by reference.

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

[0003] Obtaining useful data from images of biochemical experiments requires high spatial resolution, accuracy, and speed. Such images typically need to be acquired at a high enough magnification to clearly resolve individual samples. At the same time, the images need to cover a wide enough field of view to correctly identify the samples. For large-scale studies, imaging and image processing must be performed quickly enough to be commercially viable.

[0004] Step-and-repeat imagers and time-delay integration (TDI) imagers are two broad types of imaging systems that can be used for imaging biochemical experiments. Step-and-repeat systems can acquire about 10 megapixels of image data per second with an alignment accuracy of about 5 μm. TDI systems can acquire about 30 megapixels of image data per second with an alignment accuracy of about 50 nm. While these two types of systems work well for some applications, for others they have structural and functional disadvantages that negatively impact overall throughput. For example, applications involving the study of large-scale biochemical experiments (e.g., massively parallel whole genome sequencing) typically require a higher overall throughput than step-and-repeat and TDI imaging systems can currently provide. Summary of the Invention

[0005] The present disclosure presents a system and method for imaging biochemical reactions. The system and method may be used, for example, for sequencing template nucleic acid molecules disposed on a substrate. The substrate may have an array of spots with biochemical molecules. The present disclosure relates to an optical imaging system that may be configured to image the substrate by rapidly scanning an objective lens over the substrate. The disclosed optical imaging system provides improved resolution over conventional systems, thereby allowing for increased spot density on the substrate, resulting in significant cost savings as described herein.

[0006] In some embodiments, an optical imaging system may include an actuator, a mounting element configured to receive a substrate, and an objective lens having a proximal end and a distal end, the substrate comprising one or more nucleic acid sequences, an outer surface of the substrate configured to receive a droplet, the outer surface of the substrate comprising a first material, the proximal end of the objective lens coupled to the actuator, the distal end of the objective lens configured to be located near the outer surface of the substrate, the distal end of the objective lens comprising a second material, the second material configured to provide a higher frictional force against the droplet than the first material, the actuator 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 moving the objective lens configured to move the droplet with the distal end of the objective lens such that the objective lens and the outer surface of the substrate hold contact with the droplet at the second location.

[0007] In some embodiments, a method of optically imaging a substrate for nucleic acid sequencing may include disposing a droplet on an outer surface of a substrate, the substrate having one or more nucleic acid sequences, the outer surface of the substrate comprising a first material; positioning an objective lens at a first location on the outer surface of the substrate such that a distal end of the objective lens contacts the droplet, the distal end of the objective lens comprising a second material, the second material configured to provide a higher frictional force against the droplet than the first material; and moving the objective lens to a second location on the outer surface of the substrate, wherein moving the objective lens is configured to move the droplet 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, an optical imaging system may include an actuator, a mounting element configured to receive a substrate, and an objective lens having a proximal end and a distal end, the substrate including a vertical wall bounding a region of the substrate, the vertical wall configured to hold a fluid within the region, the substrate having one or more nucleic acid sequences, the proximal end of the objective lens coupled to the actuator, the distal end of the objective lens configured to be located near an outer surface of the substrate, the distal end of the objective lens configured to be immersed in the fluid, and the actuator 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 of optically imaging a substrate for nucleic acid sequencing may include disposing a fluid within a region of the substrate, the region being bounded by vertical walls, the substrate having one or more nucleic acid sequences; positioning an objective lens at a first location on an outer surface of the substrate such that a distal end of the objective lens contacts 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 introduce in a simplified form different embodiments of the disclosure that 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, utilities, and advantages of the claimed subject matter will become apparent from the following Detailed Description. [Brief description of the drawings]

[0011] [Figure 1A] FIG. 1 is a schematic diagram of an optical imaging system. [Figure 1B] FIG. 1 is a schematic diagram of an optical imaging system. [Diagram 2]FIG. 2 shows the substrate shown in FIG. 1 divided into an array of subregions. [Figure 3A] FIG. 1 illustrates an exemplary embodiment of an optical imaging system in which a droplet is placed and held between an objective lens and a substrate. [Figure 3B] FIG. 1 illustrates an exemplary embodiment of an optical imaging system in which a droplet is placed and held between an objective lens and a substrate. [Figure 3C] FIG. 2 illustrates a cross section of an exemplary embodiment of an objective lens having a concave distal end. [Figure 4] FIG. 2 is an exemplary schematic diagram of an optical imaging system including a droplet monitoring subsystem and a droplet delivery subsystem. [Figure 5A] FIG. 13 illustrates an alternative embodiment of an optical imaging system in which the distal end of the objective lens is immersed in a fluid disposed on the outer surface of the substrate. [Figure 5B] FIG. 13 illustrates an alternative embodiment of an optical imaging system in which the distal end of the objective lens is immersed in a fluid disposed on the outer surface of the substrate. [Figure 5C] FIG. 1 illustrates 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] FIG. 1 illustrates an exemplary embodiment having curved vertical walls. [Figure 6] FIG. 1 illustrates an exemplary method for optically imaging a substrate for sequencing a nucleic acid sequence on the substrate. [Figure 7] FIG. 2 shows another exemplary method for optically imaging a substrate for sequencing a nucleic acid sequence on the substrate.

[0012] According to common practice, the depicted features and elements are not drawn to scale but are drawn to emphasize features and elements relevant to the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The present disclosure describes an optical imaging system that can be used to image biochemical reactions. For example, the disclosed optical imaging system can be used to sequence template nucleic acid molecules (e.g., DNA molecules, RNA molecules). In some embodiments, the template nucleic acid molecules can be attached to or disposed on a surface of a substrate (e.g., the inner surface of a flow cell) that can be imaged by the optical imaging system. For example, DNA templates can be immobilized at over 10e7 positions (spots) on an array on the planar inner surface of the substrate (e.g., a flow cell). In this example, the nucleic acid sequencing method can include performing over 400 sequencing cycles. In each cycle, one nucleotide (e.g., adenine, guanine, thymine, and cytosine) can flow across the substrate and be incorporated (into the growing strand) at each site where a complementary nucleotide base exists. In one approach, each of the four different nucleotides can be labeled with a different color fluorescent dye or bound with a dye-labeled antibody. In each sequencing cycle, a light source (e.g., a laser) may illuminate the spots (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 the imaging system may thereby record the detection of the nucleotide corresponding to the detected color for each spot. Those familiar with the art will be aware of variations in sequencing methods, including variations in template type (see, e.g., Huang et al., 2017, Gigascience 6:1-9; Mardis et al., 2013, Annu Rev Anal Chem 6:287-303), labeling system (see, e.g., WO2018129214), and labeling strategy (see, e.g., US 9,523,125). In conventional systems, emitted light travels from a spot on the substrate, through a glass coverslip (e.g., on top of the substrate), through an air gap, into a microscope objective, and onto a camera that captures one or more images.In some embodiments, the objective lens comprises a housing configured to collect and focus light beams from the substrate and one or more lenses to focus the light beams to generate 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 the substrate such that the entire array is imaged during each cycle. Although the present disclosure focuses on sequencing of nucleic acid molecules, the present disclosure contemplates using the disclosed optical imaging system to image any suitable biochemical experiment.

[0014] A large, or at least a majority, of the costs associated with nucleic acid sequencing per gigabase is the amount of reagents consumed in the sequencing process. Therefore, increasing the density of samples on the substrate will significantly reduce the cost of nucleic acid sequencing. In systems based on optical detection, the optical numerical aperture (NA) of the detection system determines in part the optical resolution of the system, which in turn determines the maximum density of samples. 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 NA may be limited by the lowest refractive index in the image chain. The reason for the NA limitation is the critical angle at each interface between materials. The critical angle defines the maximum ray angle at which there is no total internal reflection at the interface. If the refractive indices of each interface in the image chain are close, the critical angle will be large. If the difference in refractive indices is large, the critical angle will be small, and less light will be transmitted through the interface. The objective lens of the optical system may have a relatively high refractive index, and a segment of the image chain with a relatively low refractive index may form a relatively small critical angle, thereby reducing the amount of light transmitted to the objective lens. Thus, one way to increase the NA of such systems without resorting to the more expensive, larger, and more complex equipment of high NA systems may be to increase the refractive index of one or more segments of the image chain that tend to have a lower refractive index. Methods and systems for this are disclosed herein.

[0016] In conventional optical systems used for nucleic acid sequencing, the segment of the imaging chain with the lowest refractive index is often an air gap, which may be present, for example, between the objective lens and the substrate of the optical system. Air gaps generally have a refractive index of about 1.00. In this example, the NA of the exemplary optical imaging system may be about 0.8. Replacing the air with a material having a higher refractive index may increase the overall NA of the optical system. For example, replacing the air gap with water may increase the lowest refractive index of the imaging chain to 1.33. In this example, the NA of a similar optical imaging system using water instead of the air gap may be about 1.0. As another example, replacing the air gap with standard oil may increase the lowest refractive index of the imaging chain to 1.51. In this example, the NA of a similar optical imaging system using water instead of the air gap may be about 1.2. As another example, replacing the air gap with a high refractive index oil may further increase the NA to 1.4. As another example, any suitable aqueous or oil-based solution may be used to suitably produce the desired change in the lowest refractive index. Essentially, this disclosure proposes using a fluid with a higher refractive index than air as a medium between the objective lens and the substrate. Increasing the lowest refractive index of the imaging chain has a direct and measurable impact on the NA of the optical imaging system, which allows for improved resolution and a corresponding increase in the density of spots on the substrate. The increased density reduces costs by reducing the amount of reagent required. This can be illustrated by benchmarking an optical imaging system with an air gap, setting its density at 1.00 and its relative cost at 1.00. With this benchmark in mind, replacing the air gap with water may increase the density to about 1.56 with a corresponding decrease in the relative cost to about 0.64. Replacing the air gap with standard oil may increase the density to about 2.25 with a corresponding decrease in the relative cost to about 0.44. Replacing the air gap with high refractive index oil may increase the density to about 3.06 with a corresponding decrease in the relative cost to about 0.33.

[0017] The use of a fluid medium with a higher refractive index than air is well known in standard microscopy applications using immersion objectives, but such applications involve static imaging. For dynamic imaging processes, e.g., scanning optics as contemplated herein for imaging a substrate while the objective is moving rapidly over the substrate, conventional immersion objectives are insufficient. An exemplary optical scanning system can move the objective at speeds between 10 mm / s and 60 mm / s. In some cases, next-generation sequencers may be equipped that can move the objective at speeds as fast as 300 mm / s. Moving the objective of a conventional system at such high speeds in such a fluid medium tends to cause excessive turbulence (and bubble generation), which can affect image quality as well as cause loss of the fluid medium. In this disclosure, two approaches for a solution to replace the air gap with a fluid medium of higher refractive index while addressing potential problems that typically appear when moving the objective rapidly. In some embodiments, the approaches disclosed herein can be used to move the objective lens at speeds between 10 mm / sec and 60 mm / sec, or between 10 mm / sec and 300 mm / sec, while maintaining good image quality with a fluid medium. In some embodiments, very high speeds can be achieved, allowing speeds between 10 mm / sec and 3750 mm / sec, or between 30 mm / sec and 3750 mm / sec. This allows for high speed scanning and imaging, for example with camera line rates of about 1 M / sec.

[0018] FIG. 1A is an exemplary schematic diagram of an optical imaging system 100. For example, referring to FIG. 1A, the optical imaging system 100 includes an objective lens 110. The objective lens 110 may be configured for use in capturing an image of a substrate 120. Referring to FIG. 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 an outer surface 120-1 of the substrate 120. In some embodiments, the biochemical molecules may be attached to or disposed on the interior of the substrate (e.g., the inner surface of the substrate). For example, the inner surface of the substrate may include one or more target nucleic acid sequences. In some embodiments, the outer surface of the substrate may be a surface of a separate element that may be removably positioned on the remainder of the substrate. For example, with reference to FIG. 1A, the outer surface 120-1 of the substrate 120 may be the surface of a cover slip that is positioned on top of the remainder of the substrate (e.g., with reference to FIG. 1, the bottom of the substrate, which may be a portion other than the top wall) (e.g., a flow cell where the substrate is not covered on top with anything other than a cover slip). In this example, biochemical molecules such as polynucleotides may be disposed on the inner surface of the bottom of the substrate, and an element such as a cover slip may be located on top of the bottom of the substrate. In other embodiments, the substrate may be a more integral structure with the outer surface integrated into the substrate. For example, with reference to FIG. 1A, the outer surface 120-1 may be an integral, non-removable part 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 disposed on the inner surface 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 across multiple locations on the outer surface 120-1 of the substrate 120. For example, with reference to FIG. 1A, the objective lens 110 is coupled to an actuator 130. Although the actuator is illustrated in FIG. 1A as a single block, it will be understood that the actuator may be a multi-component subsystem that can move the objective lens across the outer surface 120-1 of the substrate 120.

[0020] FIG. 1B is another exemplary schematic diagram of an 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 shown diagrammatically in FIG. 1B. As shown, the flow cell includes a first substrate 120, a second substrate 140, and a flow space 150. In one approach, nucleic acid template molecules (e.g., DNBs) are immobilized at positions on an inner surface of the substrate (e.g., surface 120-2 or 140-1), and reagents and wash buffers are flowed through the space 150. Thus, the space 150 is generally an aqueous environment, which may be necessary to preserve the nucleic acid templates disposed in the space 150. The positions, or spots, may be organized as regular ordered arrays on the surface 120-2 or 140-1, and are adapted to contain the nucleic acid template molecules. For example, the locations may be regions of a substrate surface that have been derivatized to bind nucleic acid molecules (e.g., DNBs, 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, e.g., a fluorescent or luminescent signal, is emitted by a dye associated with the template molecule. For example, in sequencing by synthesis, the dye may be attached to the nucleotide incorporated into the growing chain at the respective location, or may be associated with an affinity reagent that binds to the incorporated nucleotide. The emitted signal (hereafter referred to as a "fluorescent" signal) travels from the template immobilized on surface 140-1 or 120-2, through substrate (e.g., glass cover slip) 120, and gap 160 to the distal end 110-2 of the objective lens. In this approach, substrate 120 is transparent to the optical signal, although substrate 140 may be opaque. As described elsewhere herein, gap 160 may contain water or oil. In one approach, a nucleic acid template is immobilized on surface 120-2 of substrate 120.In some approaches, the nucleic acid templates are positioned on a patterned array on the surface 120-2. In these approaches (e.g., when the nucleic acid templates are positioned on the surface 120-2), the fluorescent signal does not need to travel through the aqueous environment of the space 150. This can be advantageous, especially when the gap 160 includes oil or another material with a relatively high refractive index. As explained below, image quality and resolution are limited in part by the lowest refractive index medium through which the light beam passes. Thus, eliminating the aqueous medium from the path of the light beam (by placing the nucleic acid template on the surface 120-2) can improve image quality and resolution by eliminating the relatively low refractive index aqueous medium in the space 150. For example, when the nucleic acid template is located on the surface 120-2, the light beam from the nucleic acid template only needs to pass through the substrate 120, the oil or other high refractive index material, and the objective lens before being imaged. That is, the light beam does not need to pass through an aqueous medium that may need to be present in the space 150. In some embodiments, the nucleic acid template may be positioned on the surface 140-1. For the avoidance of doubt, the description of a particular flow cell is not intended to limit the present invention.

[0021] FIG. 2 illustrates the substrate 120 shown in FIG. 1 divided into an array of subregions. In some embodiments, the substrate may be divided into multiple subregions. For example, referring to FIG. 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") where DNA is templated (e.g., where DNBs are immobilized). In some embodiments, there may be physical barriers between the subregions. In other embodiments, the subregions may not be separated by physical barriers. That is, the division between the regions may be a virtual division that is not bounded by a structural element. Each of the subregions may correspond to a spot, and each of the spots may include a specific subset of molecules (e.g., a specific 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, with reference to Figure 2, an actuator may be configured to move an objective lens from a first location on outer surface 120-1 of substrate 120 (e.g., corresponding to subregion 210) to a second location on outer surface 120-1 of substrate 120 (e.g., corresponding to subregion 220). Although Figure 2 illustrates substrate 120 divided into discrete subregions (e.g., first subregion 210 and second subregion 220), the present disclosure contemplates that the substrate may not be divided into discrete subregions at all, and the actuator may simply move continuously from one location to another on outer surface 120-1 of substrate 120 while imaging substrate 120.

[0022] 3A-3B are diagrams illustrating an exemplary embodiment of an optical imaging system 100 in which a droplet 310 is positioned and held between the objective lens 110 and the substrate 120. In some embodiments, the optical imaging system 100 may replace the air gap traditionally present in optical scanning systems with a droplet of a fluid having a refractive index greater than that of air. For example, referring to FIG. 3A, the optical system 100 may include a droplet 310 positioned to contact both the distal end 110-2 of the objective lens 110 and the outer surface 120-1 of the substrate 120. The fluid of the droplet thereby serves as a medium through which a light beam 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, a droplet of oil, a droplet of an aqueous solution, or a droplet of an oil-based solution.

[0023] In some embodiments, the droplets 310 may be disposed by a droplet delivery subsystem. The droplet delivery subsystem may comprise, for example, a conduit (e.g., a pipe or other suitable flow path) coupled to a fluid reservoir. The droplet delivery subsystem may comprise any suitable microfluidic droplet generator configured to generate droplets from a fluid in the fluid reservoir. The droplet generator may be configured to generate droplets at a suitable predetermined size. The droplets may be directed 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-dielectric (EWOD) or pressure differential.

[0024] 3A-3B illustrate how the objective lens 110 may be moved from a first location (FIG. 3A) to a second location (FIG. 3B) over the substrate 120 as one or more images of the substrate 120 are captured. As discussed above, this movement may occur at high speeds (e.g., 15 mm / sec, 300 mm / sec). Despite this high speed movement, the optical imaging system 100 may hold the droplet 310 (either entirely, or nearly entirely with little loss of fluid) in a position 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. As the objective lens 110 is moved, both the distal end 110-2 and the outer surface 120-1 of the substrate may maintain contact with the droplet during and at the end of the movement. The optical imaging system 100 can image the substrate 120 over multiple such movements without an air gap between the objective lens 110 and the substrate 120, with the droplet 310 acting as the fluid medium.

[0025] It should be noted that while Figures 3A-3B illustrate the above concepts with respect to the single substrate design of the simplified schematic of Figure 1A (e.g., the nucleic acid sequence to be imaged may be located on or within substrate 120), the present disclosure contemplates that the same concepts also apply to the schematic of Figure 1B where there are two substrates. For example, with reference to Figure 1B, the nucleic acid sequence to be imaged according to Figures 3A-3B may be located within space 150 (e.g., may be attached to surface 120-2 or surface 140-1).

[0026] In some embodiments, the optical imaging system 100 may be enabled to retain the droplet 310 as described above in part 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 such that 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 is optimized to enhance droplet retention. For purposes of this disclosure, friction (frictional force) may be characterized as the force that resists 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, which may be configured to provide a higher frictional force against the droplet than the first material. That is, the surface chemistry may be configured such that the droplet 310 experiences 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, as the objective lens 110 moves, the portion of the droplet 310 in contact with the distal end 110-2 of the objective lens is attracted to the distal end, thereby moving the droplet with the objective lens 110. In contrast, the portion of the droplet 310 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 such that the droplet 310 is free to move across the outer surface 120-1 without adhering to the outer surface 120-1. Stated another way, the surface chemistry may be selected such that the objective lens distal end 110-2 is configured to attract the droplet 310, while the substrate outer surface 120-1 repels the droplet 310. In some embodiments, materials may be selected such that when a droplet of a particular fluid medium (e.g., an oil or aqueous solution) is placed on the surface, the droplet will rest at a desired contact angle. For example, a droplet of the fluid medium placed on the surface 120-1 may have a contact angle between 93 degrees and 160 degrees. A larger contact angle may be desirable since it will be more repelled by the droplet surface.As another example, the contact angle may be between 11 and 120 degrees. Similarly, the material of distal end 110-2 of objective lens 110 may be selected such that a drop of 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 having different material compositions may have different degrees of friction with a particular fluid. Based on the relative friction with a particular fluid medium, appropriate materials may be selected to achieve the above-mentioned attractive / repulsive effects. In some embodiments, when the droplet 310 is, for example, water or aqueous, 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, where the droplet 310 is, for example, oil or oil-based, a first material at the substrate outer surface 120-1 may comprise an oleophilic material (e.g., barium sulfate, polyethylene terephthalate (PET), polypropylene, carbon-based sponge, functional polymer sponge, kapok / milkweed seed hair) and a second material at the objective lens distal end 110-2 may comprise an oleophobic material (e.g., Teflon and Teflon derivatives, hexadecane, polytetrafluoroethylene).

[0028] FIG. 3C is a diagram illustrating a cross section of an example 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 help maintain 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 serve to increase the attractive force and aid in pulling the droplet 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 droplets 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 helping to retain the droplet as the objective lens moves. In some embodiments, the distal end 110-2 may be concave and may also have a surface chemistry as described above (e.g., the distal end 110-2 includes a material that has higher friction and attraction to the droplet), thereby enhancing the ability of the distal end 110-2 to move the droplet 310 with the objective lens 110. In these embodiments, the concave portion of the distal end 110-2 may provide a level of synergy to achieve this goal, as it provides a larger surface area in contact with the droplet 310 (which may provide friction and attraction to the droplet 310) than if the distal end 110-2 were a flat surface.

[0029] FIG. 4 is an exemplary schematic diagram of an optical imaging system 100 including a droplet monitoring subsystem and a droplet delivery subsystem. In some embodiments, as shown in FIG. 4, the optical imaging system 100 may include a droplet monitoring subsystem that monitors (continuously or periodically) an optical scanner (including hardware for imaging the objective lens 110 and the substrate 120, such as the 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, it may use a resistive or capacitive sensor to determine the presence of the droplet 310 or an optical sensor to detect the presence of the droplet 310. Alternatively, it may simply analyze the image quality captured through the objective lens 110 to ensure that the light beam is properly refracted (thereby indicating that the droplet 310 is still present). If it is determined that both objective lens 110 and outer surface 120-1 of substrate 120 are not in contact with droplet 310, then a droplet delivery subsystem may be triggered to deliver an additional droplet to the optical scanner at an appropriate location on outer surface 120-1 of substrate 120 (e.g., below objective lens 110). In this manner, the droplet monitoring subsystem and droplet delivery subsystem, in conjunction with the optical scanner, may form a feedback control loop that operates to retain droplet 310 over an extended period of time and / or correct for systematic errors that cause droplet 310 to move out of position beneath objective lens 110.

[0030] 5A-5B are diagrams illustrating 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 disposed on the outer surface 120-1 of the substrate 120. In some embodiments, referring to FIG. 5A, the substrate includes a vertical wall 525 that bounds (encloses) a region of the substrate. The vertical wall 525 may be configured to retain 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 at a desired depth. The fluid may be any suitable fluid, including those fluids 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 may instead be part of the housing of the optical imaging system 100 that extends over the substrate, thereby achieving the same or similar effects.

[0031] In these embodiments, the distal end 110-2 may remain submerged in the fluid as the actuator is moved from a first location (FIG. 5A) to a second location (FIG. 5B) on the substrate 120. Thus, the light beam may 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 discussed above, high throughput imaging applications may require fast movement of the objective lens, which may amplify turbulence. Also, as discussed above, turbulence may adversely affect image quality (e.g., by forming bubbles or flow aberrations that may result in undesirable image artifacts) and / or cause loss of fluid 510 over time. Reducing this turbulence addresses these issues. Unlike the droplet-based optical imaging system 100 embodiment, in an embodiment where the objective is moved while immersed in a fluid, the concave distal end 110-2 can cause problems because the fluid 510 around the objective 110 with the concave distal end 110-2 has a flow profile such that the fluid 510 contacting the concave distal end 110-2 flows at different speeds along different points of curvature and in a non-laminar manner. This flow pattern propagates throughout the area as the objective 110 moves, thereby creating excessive turbulence. In contrast, a flat distal end 110-2 can reduce such turbulence by providing a more laminar flow.

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

[0033] FIG. 5D illustrates an exemplary embodiment with 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, such as the vertical wall 525 of FIGS. 5A-5C. In other embodiments, the interior of the vertical wall may not be perpendicular to the outer surface 120-1 of the substrate 120, but may be disposed at an angle. In some embodiments, the interior of the vertical wall may be flat, such as the vertical wall 525 of FIGS. 5A-5C. In other embodiments, the interior of the vertical wall may be curved, such as the vertical wall 526 of FIG. 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 high enough to retain the fluid 510 within an area bounded (enclosed) by the vertical wall 525 and prevent the fluid 510 from splashing or otherwise flowing out of the area as the objective lens 110 moves within the area.

[0035] Embodiments in which the objective lens 110 is submerged in the fluid (e.g., the embodiment illustrated in FIGS. 5A-5D) may also have a feedback control loop similar to that illustrated in FIG. 4. In such cases, the droplet monitoring subsystem and the droplet delivery subsystem may be replaced with 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 the fluid 510 within the area bounded by the vertical wall 525 is sufficient to submerge 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 the substrate 120 and a scale below the substrate that measures the weight of the fluid 510), or simply analyze the image quality captured through the objective lens 110 to ensure that light rays are properly refracted (thereby indicating that the objective lens 110 is still submerged in the fluid 510). As another example, the fluid monitoring subsystem may use an optical sensor to detect the presence of air bubbles in the fluid to determine if an amount of fluid has evaporated or flowed out of the region, since air bubbles may indicate evaporation of the fluid. The fluid monitoring subsystem may correlate the amount of air bubbles with the amount of fluid that has evaporated. In some embodiments, if the fluid monitoring subsystem determines that a threshold amount of fluid has evaporated or flowed out (e.g., based on a threshold number of air bubbles being detected, based on an electrical or optical detection of a drop in the fluid level line by a threshold amount), a fluid delivery subsystem may be triggered to automatically replenish the lost fluid. For example, the fluid delivery subsystem may include a conduit coupled to a fluid reservoir, and an appropriate amount of replacement fluid may be transported to the region by any suitable means (e.g., EWOD, pressure differential created by a pump).

[0036] It should be noted that while Figures 5A-5D illustrate the above concepts with respect to the single substrate design of the simplified schematic of Figure 1A (e.g., the imaged nucleic acid sequence may be disposed on or within substrate 120), the present disclosure contemplates that the same concepts apply to the schematic of Figure 1B where there are two substrates. For example, with reference to Figure 1B, the nucleic acid sequence imaged according to Figures 5A-5D may be disposed within space 150 (e.g., attached to surface 120-2 or surface 140-1).

[0037] FIG. 6 illustrates an exemplary method 600 for optically imaging a substrate for sequencing a nucleic acid sequence. The method may include, in step 610, disposing a droplet on an outer surface of the substrate. The substrate includes one or more nucleic acid sequences, and the outer surface of the substrate includes 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 a distal end of the objective lens contacts the droplet. The distal end of the objective lens includes a second material, the second material 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 with the distal end of the objective lens such that the objective lens and the outer surface of the substrate hold contact with the droplet at the second location.

[0038] In certain embodiments, one or more steps of the method of FIG. 6 may be repeated, as desired. Although the present disclosure describes and illustrates certain steps of the method of FIG. 6 as occurring in a particular order, the present disclosure contemplates any suitable steps of the method of FIG. 6 occurring in any suitable order. Additionally, although the present disclosure describes and illustrates an exemplary method for optically imaging a substrate to sequence a nucleic acid sequence thereon, including certain steps of the method of FIG. 6, the present disclosure contemplates any suitable method of optically imaging a substrate to sequence a nucleic acid sequence thereon, which may include all, some, or none of the steps of the method of FIG. 6, as desired. Additionally, although the present disclosure describes and illustrates certain components, devices, or systems that perform certain steps of the method of FIG. 6, the present disclosure contemplates any suitable combination of any suitable components, devices, or systems that perform any suitable steps of the method of FIG. 6.

[0039] 7 illustrates another exemplary method 700 for optically imaging a substrate to sequence nucleic acid sequences thereon. The method may include, in step 710, disposing a fluid within a region of the substrate, the region being bounded by vertical walls, the substrate including one or more nucleic acid sequences (e.g., at a location within the bounds 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 a distal end of the objective lens contacts 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 keeping the distal end of the objective lens immersed in the fluid.

[0040] In certain embodiments, one or more steps of the method of FIG. 7 may be repeated, as desired. Although the present disclosure describes and illustrates certain steps of the method of FIG. 7 as occurring in a particular order, the present disclosure contemplates any suitable steps of the method of FIG. 7 occurring in any suitable order. Additionally, although the present disclosure describes and illustrates an exemplary method for optically imaging a substrate to sequence a nucleic acid sequence thereon, including certain steps of the method of FIG. 7, the present disclosure contemplates any suitable method of optically imaging a substrate to sequence a nucleic acid sequence thereon, which may include all, some, or none of the steps of the method of FIG. 7, as desired. Additionally, although the present disclosure describes and illustrates certain components, devices, or systems that perform certain steps of the method of FIG. 6, the present disclosure contemplates any suitable combination of any suitable components, devices, or systems that perform any suitable steps of the method of FIG. 7.

[0041] It is understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes in light thereof that will be suggested to those skilled in the art are within the spirit and scope of this application and are to be included within the scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated 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 review of the above description. Therefore, the scope of the present invention should not be determined with reference to the above description, but should instead be determined with reference to the appended claims along with their full scope of equivalents.

[0043] While the foregoing disclosure illustrates exemplary aspects of the disclosure, it should be noted that various changes and modifications can be made without departing from the scope of the disclosure as defined by the appended claims. The functions, steps and / or actions of the method claims in accordance with the aspects of the disclosure described herein do not have to be performed in any particular order. Further, although elements of the disclosure may be described or claimed in the singular, the plural is contemplated unless limitation to the singular is expressly stated.

Claims

1. 1. An optical imaging system for nucleic acid sequencing, comprising: An actuator; a substrate having vertical walls bounding an area of ​​the substrate; an objective lens having a proximal end and a distal end; the vertical wall is configured to retain a fluid within the region; the substrate comprises one or more nucleic acid sequences; the proximal end of the objective lens is coupled to the actuator; the distal end of the objective lens is configured to be located near an outer surface of the substrate, the distal end of the objective lens being configured to be immersed in the fluid; the actuator is configured to move the objective lens from a first location above the outer surface of the substrate to a second location above the outer surface of the substrate while keeping the distal end of the objective lens submerged in the fluid; the optical imaging system further comprising a fluid monitoring subsystem for determining whether a portion of the fluid has evaporated from the region; the fluid monitoring subsystem comprising an optical sensor for detecting the presence of air bubbles in the fluid; The optical imaging system, wherein determining whether a portion of the fluid has evaporated from the region includes determining whether a threshold number of gas bubbles are detected in the fluid.

2. The optical imaging system of claim 1 , wherein the fluid comprises oil.

3. The optical imaging system of claim 1 , wherein the fluid comprises water.

4. The optical imaging system of claim 2 , wherein the distal end of the objective lens is flat to reduce turbulence when moving the objective lens from the first location to the second location.

5. The optical imaging system of claim 4 , wherein the objective lens has a proximal portion and a distal portion, the distal portion being non-tapered.

6. The optical imaging system of claim 2 , wherein the actuator is configured to move the objective lens from the first location to the second location at a speed of 15 mm / sec.

7. The optical imaging system of claim 2 , wherein the inside of the vertical wall is flat.

8. The optical imaging system of claim 2 , wherein the inside of the vertical wall is curved.

9. The optical imaging system of claim 2 , further comprising a fluid delivery subsystem for fluid delivery to the region.

10. The optical imaging system of claim 9, wherein the fluid delivery subsystem is configured to deliver additional fluid upon a positive determination by the fluid monitoring subsystem that a portion of the fluid has evaporated.

11. The optical imaging system of claim 10 , wherein the fluid delivery subsystem comprises a conduit coupled to a fluid reservoir.

12. 1. A method for optically imaging a substrate for nucleic acid sequencing, the method comprising: disposing a fluid within a region of the substrate, the region being bounded by vertical walls, the substrate having one or more nucleic acid sequences; positioning the objective lens at a first location above an outer surface of the substrate such that a distal end of the objective lens contacts the fluid; moving the objective lens to a second location above an outer surface of the substrate while keeping the distal end of the objective lens submerged in the fluid; determining that a portion of the fluid has evaporated from the region; Determining that a portion of the fluid has evaporated from the region includes: detecting the presence of air bubbles in the fluid; determining that a threshold number of air bubbles have been detected.

13. The method of claim 12 , wherein the fluid comprises oil.

14. The method of claim 12 , wherein the fluid comprises water.

15. The method of claim 12 , wherein the distal end of the objective lens is flat to reduce turbulence when moving the objective lens from the first location to the second location.

16. The method of claim 15 , wherein the objective lens has a proximal portion and a distal portion, the distal portion being non-tapered.

17. The method of claim 12 , wherein the objective lens is moved from the first location to the second location at a speed of 15 mm / sec.

18. The method of claim 12 , wherein the inside of the vertical wall is flat.

19. The method of claim 12 , wherein the inside of the vertical wall is curved.

20. The method of claim 12 , further comprising delivering additional fluid via a fluid delivery subsystem based on a determination that a portion of the fluid has evaporated from the region.

21. The method of claim 20 , wherein the fluid delivery subsystem comprises a conduit coupled to a fluid reservoir.

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