System and method for optical scanning and imaging through 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

[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 generally for use in nucleic acid sequencing and biochemical experiments.

Background Art

[0003] To obtain useful data from images of biochemical experiments, high spatial resolution, accuracy, and speed are required. Such images typically need to be acquired at a high magnification sufficient to clearly resolve individual samples. At the same time, to correctly identify the samples, the images need to cover a sufficiently wide field of view. In 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 in biochemical experiments. Step-and-repeat systems can acquire image data of approximately 10 megapixels per second with an alignment accuracy of about 5 μm. TDI systems can acquire image data of approximately 30 megapixels per second with an alignment accuracy of about 50 nm. These two types of systems function well for some applications, but for other applications, they have structural and functional disadvantages that negatively impact the overall throughput. For example, for applications involving large-scale biochemical experiment research (such as ultra-parallel whole-genome sequencing), typically, a higher overall throughput than what current step-and-repeat and TDI imaging systems can provide is required.

SUMMARY OF THE INVENTION

[0005] The present 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 disposed on a substrate. The substrate may have an array of spots having biochemical molecules. The present disclosure relates to an optical imaging system configured to image a substrate by rapidly scanning an objective lens over the substrate. According to the disclosed optical imaging system, the resolution is improved over conventional systems, thereby increasing the spot density on the substrate and as a result, significant cost savings can be achieved as described herein.

[0006] In some embodiments, the 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, wherein the substrate includes one or more nucleic acid sequences, an outer surface of the substrate is configured to receive a droplet, the outer surface of the substrate includes a first material, 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 the outer surface of the substrate, the distal end of the objective lens includes 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 moving 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 of optically imaging a substrate for nucleic acid sequencing may include disposing a droplet on an outer surface of the substrate, wherein the substrate has one or more nucleic acid sequences and the outer surface of the substrate includes a first material; positioning the objective lens at a first location on the outer surface of the substrate such that the distal end of the objective lens contacts the droplet, wherein the distal end of the objective lens includes 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, moving the objective lens being 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 include an actuator, a mounting element configured to receive a substrate, and an objective lens having a proximal end and a distal end, wherein the substrate includes vertical walls that define a region of the substrate, the vertical walls being configured to hold fluid within the region, the substrate having one or more nucleic acid sequences, the proximal end of the objective lens being coupled to the actuator, the distal end of the objective lens being configured to be positioned near an 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 of optically imaging a substrate for nucleic acid sequencing may include disposing 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 a simplified form of different embodiments of the present disclosure that will be described in more detail below. This summary is not intended 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

Figure 1B

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Figure 5A

Figure 5B

Figure 5C

Figure 5D

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Figure 7

[0012] In accordance with common practice, the described 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 INVENTION

[0013] The present disclosure describes an optical imaging system that can be used for imaging biochemical reactions. For example, the disclosed optical imaging system can be used for sequencing template nucleic acid molecules (e.g., DNA molecules, RNA molecules). In some embodiments, the template nucleic acid molecule may be bound to or disposed 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 positions (spots) on an array on the planar inner surface of a substrate (e.g., a flow cell) that exceed 10e7. 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) can flow across the substrate and be incorporated (into the growing strand) at each site where a complementary nucleotide base is present. In one approach, each of the four different nucleotides can be labeled with a fluorescent dye of a different color or bound with a dye-labeled antibody. In each sequencing cycle, a light source (e.g., a laser) irradiates the spots (e.g., in series), whereby the dye may emit light corresponding to each color. The color emitted from one of the four dyes at each spot may be detected by a camera (e.g., a time delay integration charge coupled device (TDI-CCD) camera or a similar camera), and the imaging system can thereby record the detection of the nucleotide corresponding to the detected color for each spot. Those skilled in the art will be aware of variations of the sequencing method, including variations in the template type (see, e.g., Huang et al., 2017, Gigascience 6:1-9; Mardis et al., 2013, Annu Rev Anal Chem 6:287-303), the labeling system (see, e.g., WO2018129214), and the labeling strategy (see, e.g., US 9,523,125). In conventional systems, the emitted light travels from the spots on the substrate, through a glass coverslip (e.g., on top of the substrate), through an air gap, into the objective lens of a microscope, and reaches a camera that captures (acquires) one or more images.In some embodiments, the objective lens comprises a housing configured to collect and focus light rays from a substrate, and one or more lenses, and focuses the light rays 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 nucleic acid sequencing, the present disclosure contemplates using the disclosed optical imaging system to image any suitable biochemical experiment.

[0014] Most, or at least a majority, of the cost associated with nucleic acid sequencing per gigabase is the amount of reagent consumed in the sequencing process. Thus, increasing the density of samples on the substrate can significantly reduce the cost of nucleic acid sequencing. In systems based on optical detection, the numerical aperture (NA) of the detection system partially determines the optical resolution of the system, and thereby the maximum density of samples that can be determined. High-NA optical systems tend to be more expensive, larger, and more difficult to align and maintain than low-NA optical systems.

[0015] In certain optical systems (e.g., confocal optical systems), the NA can be limited by the lowest refractive index within the image chain. The reason the NA is limited is the critical angle at each interface between materials. The critical angle defines the maximum ray angle at which total internal reflection does not occur at the interface. When the refractive indices of each interface within 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 through the objective lens decreases. Thus, one way to increase the NA of such a system without relying on more expensive, larger, and more complex devices of high-NA systems can be to increase the refractive index of one or more segments of the image chain that tend to have a low refractive index. Methods and systems therefor 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 that may exist, for example, between the objective lens of the optical system and the substrate. The air gap generally has a refractive index of about 1.00. In this example, the NA of an exemplary optical imaging system can be about 0.8. Replacing air with a substance having a higher refractive index can increase the overall NA of the optical system. For example, by replacing the air gap with water, the lowest refractive index of the image chain can be increased to 1.33. In this example, the NA of a similar optical imaging system using water instead of an air gap can be about 1.0. As another example, by replacing the air gap with standard oil, the lowest refractive index of the image chain can be increased to 1.51. In this example, the NA of a similar optical imaging system using water instead of an air gap can be about 1.2. As another example, by replacing the air gap with high refractive index oil, the NA can be further increased to 1.4. As another example, any suitable aqueous or oily solution can be used to appropriately cause a desired change in the lowest refractive index. Essentially, the present disclosure proposes using a fluid having a refractive index higher than that of air as the medium between the objective lens and the substrate. Increasing the lowest refractive index of the image chain has a direct and measurable effect on the NA of the optical imaging system, which can improve the resolution and accordingly increase the density of spots on the substrate. The increase in density reduces the required amount of reagent, leading to cost reduction. This can be explained by taking an optical imaging system with an air gap as a benchmark and setting its density to 1.00 and relative cost to 1.00. With this benchmark in mind, when the air gap is replaced with water, the density can increase to about 1.56, and correspondingly the relative cost can decrease to about 0.64. When the air gap is replaced with standard oil, the density can increase to about 2.25, and correspondingly the relative cost can decrease to about 0.44. When the air gap is replaced with high refractive index oil, the density can increase to about 3.06, and correspondingly the relative cost can decrease to about 0.33.

[0017] The use of a fluid medium having a refractive index higher than that of 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. Optionally, a next-generation sequencer capable of moving the objective lens at about 300 mm / second may be provided. 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 has the potential to 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. For example, referring to Figure 1A, the optical imaging system 100 includes an objective lens 110. The objective lens 110 may be configured to be used when capturing an image of a 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 (directly or indirectly) coupled to an actuator, and the distal end 110-2 of the objective lens 110 may be configured to be positioned near an outer surface 120-1 of the substrate 120. In some embodiments, biochemical molecules may be bound to the interior (e.g., the inner surface of the substrate) or disposed thereon. 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 the surface of a separate element removably positioned over the remainder 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 over the remainder of the substrate (e.g., the bottom of the substrate, referring to Figure 1. This may be the portion other than the upper wall). 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 coverslip may be positioned over the bottom of the substrate. In other embodiments, the substrate may have a more integrated structure where 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, one or more chambers and / or conduits may be present within the substrate 120, and biochemical molecules such as nucleic acid sequences may be disposed 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 across multiple 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 across 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 to be used when capturing one or more images of a substrate (e.g., a flow cell). One embodiment of the flow cell is schematically shown in Figure 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 the 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 a regular ordered array on the surface 120-2 or 140-1 and are adapted to contain nucleic acid template molecules. For example, the above positions may be regions of the substrate surface 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, such as a fluorescence or luminescence signal, is emitted by a dye associated with the template molecule. For example, in the synthesis-based sequencing method, the dye may be linked to the nucleotide incorporated into the growing strand at each position, or may be linked in relation to an affinity reagent bound to the incorporated nucleotide. The emitted signal (hereinafter referred to as the "fluorescence" signal) travels from the template immobilized on the surface 140-1 or 120-2 through the substrate (e.g., a glass coverslip) 120 and the gap 160 to the distal end 110-2 of the objective lens. In this approach, the substrate 120 may be transparent to the optical signal, while the substrate 140 may be opaque. As described elsewhere herein, the gap 160 may contain water or oil. In one approach, the nucleic acid template is immobilized on the surface 120-2 of the substrate 120.In one approach, the nucleic acid template is positioned on the patterned array of surface 120-2. In these approaches (e.g., when the nucleic acid template is positioned on surface 120-2), the fluorescent signal need not travel through the aqueous environment of space 150. This can be advantageous, particularly when gap 160 contains oil or another material having a relatively high refractive index. As will be explained below, image quality and resolution are partially limited by the medium with the lowest refractive index through which the light passes. Thus, by eliminating the aqueous medium from the path of the light (by placing the nucleic acid template on surface 120-2), the image quality and resolution can be improved by eliminating the relatively low refractive index aqueous medium within space 150. For example, when the nucleic acid template is positioned on surface 120-2, the light from the nucleic acid template need only pass through substrate 120, oil or other high refractive index material, and the objective lens before being imaged. That is, the light need not pass through the aqueous medium that may be present in space 150. In some embodiments, the nucleic acid template may be disposed on surface 140-1. To avoid doubt, the description of a particular flow cell is not intended to limit the invention.

[0021] FIG. 2 is a diagram showing the substrate 120 shown in FIG. 1, which is divided into an array of sub-regions. In some embodiments, the substrate may be divided into a plurality of sub-regions. For example, referring to FIG. 2, the substrate 120 may have a plurality of sub-regions such as a first sub-region 210 and a second sub-region 220. In one approach, the substrate has a patterned array of derivatized regions ( "spots") where DNA is templated (e.g., DNBs are immobilized). In some embodiments, there may be a physical barrier between the sub-regions. In other embodiments, the sub-regions may not be separated by a physical barrier. That is, the division between the regions may be a virtual division that is not bounded by a structural element. Each of the sub-regions may correspond to a spot, and each of the spots 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 sub-regions of the substrate. For example, referring to FIG. 2, the actuator may be configured to move the objective lens from a first location (e.g., corresponding to sub-region 210) on the outer surface 120-1 of the substrate 120 to a second location (e.g., corresponding to sub-region 220) on the outer surface 120-1 of the substrate 120. Although FIG. 2 illustrates the substrate 120 divided into discrete sub-regions (e.g., a first sub-region 210 and a second sub-region 220), the present disclosure contemplates that the substrate may not 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 - 3B are diagrams showing an exemplary embodiment of an optical imaging system 100 that disposes and holds a droplet 310 between an objective lens 110 and a substrate 120. In some embodiments, in the optical imaging system 100, the conventionally existing air gap in the optical scanning system can be replaced with a droplet of fluid having a refractive index greater than that of air. For example, referring to FIG. 3A, in the optical system 100, the droplet 310 is disposed 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. Thereby, the fluid of the droplet functions 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 water droplet, an oil droplet, a droplet of an aqueous solution, or a droplet of an oily solution.

[0023] In some embodiments, the droplet 310 may be disposed by a droplet delivery subsystem. The droplet delivery subsystem may include, for example, a conduit (e.g., a pipe or other suitable flow path) connected to a fluid reservoir. The droplet delivery subsystem may include 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 dielectrophoretic electro-wetting (EWOD) or a pressure difference.

[0024] Figures 3A-3B illustrate a method of moving the objective lens 110 from a first location (FIG. 3A) to a second location (FIG. 3B) on the substrate 120 when taking 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 hold the droplet 310 (either in its entirety or substantially in its entirety with little fluid loss) in a position below the distal end 110-2 of the objective lens 110 such that a 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 at the end of the movement. The optical imaging system 100 can image the substrate 120 over such multiple movements while causing the droplet 310 to function as a fluid medium without an air gap existing between the objective lens 110 and the substrate 120.

[0025] Although FIGS. 3A-3B illustrate the above concepts for a single substrate design of the simplified schematic of FIG. 1A (e.g., the nucleic acid sequences to be imaged may be disposed on or within the substrate 120), it should be noted that the present disclosure contemplates that the same concepts apply to the schematic of FIG. 1B where two substrates are present. For example, referring to FIG. 1B, the nucleic acid sequences imaged according to FIGS. 3A-3B may be disposed within the space 150 (e.g., may be bound to the surface 120-2 or the surface 140-1).

[0026] In some embodiments, the optical imaging system 100 may be enabled to hold 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 the purposes of the present disclosure, friction (frictional force) may be characterized as the force that resists the relative movement 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, and the second material 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, 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 across the outer surface 120-1 without adhering to the outer surface 120-1. In other words, the surface chemistry may be selected such that the distal end 110-2 of the objective lens is configured to attract the droplet 310 and the outer surface 120-1 of the substrate is configured to repel (repulse) 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 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 as it results in a greater repulsive force by the droplet surface.As another example, the contact angle may be between 11 degrees 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 the fluid medium placed on a flat surface made of that material has a contact angle between 20 degrees and 80 degrees.

[0027] The degree of friction between various surfaces and a fluid can be determined and compared by well-known means. Surfaces with different material compositions can have different degrees of friction with respect to a particular fluid. Based on the relative friction with respect to a particular fluid medium, an appropriate material may be selected to achieve the attracting / repelling effect described above. 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, perfluoroalkoxy alkane), 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, when the droplet 310 is, for example, oil or oily, the first material of the outer surface 120-1 of the substrate may include a lipophilic 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 a lipophobic material (e.g., Teflon and Teflon derivatives, hexadecane, polytetrafluoroethylene).

[0028] FIG. 3C is a diagram showing 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 so as to easily maintain contact between the objective lens 110 and the droplet 310 when the objective lens 110 moves. In these embodiments, the curvature of the distal end 110-2 can serve to increase the attracting force and assist 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 helping to hold the droplet when the objective lens moves. In some embodiments, the distal end 110-2 may be concave and may also have surface chemistry as described above (e.g., the distal end 110-2 includes a material having a higher frictional force and attracting force with respect to the droplet). Thereby, the ability of the distal end 110-2 to move the droplet 310 together with the objective lens 110 is enhanced. In these embodiments, the recess of the distal end 110-2 provides a certain level of synergistic effect to achieve this purpose because the surface area in contact with the droplet 310 is larger than when the distal end 110-2 is a flat surface (this can impart frictional force and attracting force to the droplet 310).

[0029] FIG. 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 FIG. 4, the optical imaging system 100 includes an optical scanner (comprising hardware for imaging a substrate 120 such as an objective lens 110 and an actuator 130) to continuously or periodically monitor whether the outer surface 120-1 of the objective lens 110 and the substrate 120 is 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 be analyzed to confirm that the light rays are refracted appropriately, indicating that the droplet 310 is still present. If it is determined that neither the outer surface 120-1 of the objective lens 110 nor the substrate 120 is in contact with the droplet 310, the droplet delivery subsystem may be triggered to deliver an additional droplet to an appropriate location (e.g., under the objective lens 110) on the outer surface 120-1 of the substrate 120 to the optical scanner. In this way, the droplet monitoring subsystem and the droplet delivery subsystem may cooperate with the optical scanner to form a feedback control loop that operates to hold the droplet 310 over a long period of time and / or correct system errors that cause the droplet 310 to deviate from its position under the objective lens 110.

[0030] Figures 5A-5B are diagrams showing alternative embodiments of the optical imaging system 100, where 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 comprises a vertical wall 525 that bounds (surrounds) 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 at a desired depth. The fluid may be any suitable fluid, including the fluids described above with respect to the droplet 310. In some embodiments, the vertical wall 525 may be part of the substrate (e.g., may be molded into the substrate). In an alternative embodiment, the vertical wall 525 may not be part of the substrate and instead may be part of the housing of the optical imaging system 100 that extends over the substrate, thereby achieving the same or a similar effect.

[0031] In these embodiments, when the actuator is moved from a first location (FIG. 5A) to a second location (FIG. 5B) on the substrate 120, the distal end 110-2 may remain immersed in the fluid. Thus, the light beam 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 in order to reduce the turbulent flow when the objective lens moves from the first location to the second location. As described above, in high-throughput imaging applications, a high-speed movement of the objective lens may be required, which may amplify the turbulent flow. Also, as described above, the turbulent flow can affect the image quality (e.g., by forming bubbles or flow aberrations that can cause undesirable image artifacts) and / or cause loss of the fluid 510 over time. By reducing this turbulent flow, these problems are addressed. Unlike embodiments of the optical imaging system 100 that use droplets, in embodiments where the objective lens is moved while immersed in the fluid, a concave distal end 110-2 can cause problems. This is because the fluid 510 around the objective lens 110 having the concave distal end 110-2 flows at different speeds along points of different curvatures where the fluid 510 contacting the concave distal end 110-2 flows, having a flow profile that flows in a non-laminar state. This flow pattern propagates throughout the region as the objective lens 110 moves, thereby causing excessive turbulent flow. In contrast, with a flat distal end 110-2, a more laminar flow can be obtained, which can reduce such turbulent flow.

[0032] FIG. 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 an embodiment, 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 makes it easier to generate a laminar flow when the objective lens 110 moves, and the turbulent flow can be reduced.

[0033] FIG. 5D shows 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, like the vertical wall 525 in 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 and may be arranged obliquely. In some embodiments, the interior of the vertical wall may be flat, like the vertical wall 525 in FIGS. 5A-5C. In other embodiments, the interior of the vertical wall may be curved, like the vertical wall 526 in 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 is optimized to a height sufficient to hold the fluid 510 within the region bounded (enclosed) by the vertical wall 525 and prevent the fluid 510 from splashing or otherwise flowing out of that 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., the embodiments 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 each be replaced by a fluid monitoring subsystem and a fluid delivery subsystem. In some embodiments, the fluid monitoring subsystem may continuously or periodically determine that the level of the fluid 510 within 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 beneath 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 light rays are properly refracted (thereby 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 air bubbles in the fluid, and since the air bubbles may indicate evaporation of the fluid, determine whether a certain amount of fluid has evaporated or flowed out of the region. The fluid monitoring subsystem can correlate the amount of air bubbles with the amount of evaporated fluid. In some embodiments, if the fluid monitoring subsystem determines that a threshold amount of fluid has evaporated or flowed out (e.g., based on the detection of a threshold number of air bubbles, or based on the electrical or optical detection that the fluid level line (liquid surface) 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 transported to the region by any suitable means (e.g., EWOD, pressure difference generated by a pump).

[0036] Figures 5A-5D illustrate the above concepts with respect to the single substrate design of the simplified schematic of FIG. 1A (e.g., the imaged nucleic acid sequences may be disposed on or within substrate 120), it should be noted that the present disclosure contemplates that the same concepts apply to the schematic of FIG. 1B where two substrates are present. For example, referring to FIG. 1B, the nucleic acid sequences to be imaged in accordance with FIGS. 5A-5D may be disposed (e.g., bound to surface 120-2 or surface 140-1) within space 150.

[0037] FIG. 6 shows an exemplary method 600 for optically imaging a substrate to determine a nucleic acid sequence. The method may include, in step 610, disposing droplets 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 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 along 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 of FIG. 6 may be repeated as necessary. Although the present disclosure has described and illustrated the specific steps of the method of FIG. 6 as occurring in a particular order, the present disclosure contemplates that any suitable steps of the method of FIG. 6 may occur in any suitable order. Further, although the present disclosure has described and illustrated an exemplary method for optically imaging a substrate to determine the nucleic acid sequences on the substrate, including specific steps of the method of FIG. 6, the present disclosure contemplates any suitable method for optically imaging a substrate to determine the nucleic acid sequences on the substrate, which may, as necessary, include all, some, or none of the steps of the method of FIG. 6. Further, although the present disclosure has described and illustrated specific components, devices, or systems for performing specific steps of the method of FIG. 6, the present disclosure contemplates any suitable combination of any suitable components, devices, or systems for performing any suitable steps of the method of FIG. 6.

[0039] FIG. 7 shows another exemplary method 700 for optically imaging a substrate to sequence the nucleic acid sequences on the substrate. The method may include, in step 710, disposing a fluid within a region of the substrate. The region is bounded by vertical walls and the substrate includes one or more nucleic acid sequences (e.g., at locations within the boundaries 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 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 necessary. Although the present disclosure has been described and illustrated as if the particular steps of the method of FIG. 7 occur in a particular order, the present disclosure contemplates that any suitable steps of the method of FIG. 7 may occur in any suitable order. Further, although the present disclosure has described and illustrated exemplary methods for optically imaging a substrate to determine a nucleic acid sequence on the substrate, including particular steps of the method of FIG. 7, the present disclosure contemplates any suitable method for optically imaging a substrate to determine a nucleic acid sequence on the substrate, which may, as necessary, include all, some, or none of the steps of the method of FIG. 7. Further, although the present disclosure has described and illustrated particular components, devices, or systems for performing particular steps of the method of FIG. 6, the present disclosure contemplates any suitable combination of any suitable components, devices, or systems for performing any suitable steps of the method of FIG. 7.

[0041] The examples and embodiments described herein are for illustrative purposes only, and various modifications or changes considered in light of them will be suggested to those skilled in the art and 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 cited herein are hereby incorporated by reference in their entirety for all purposes.

[0042] It will be understood that the above description is exemplary and not restrictive. Many embodiments will be apparent to those skilled in the art upon review of the above description. Accordingly, the scope of the invention should not be determined with reference to the above description, but instead should be determined with reference to the appended claims, along with the full scope of their equivalents.

[0043] Note that while the foregoing disclosure shows exemplary aspects of the present disclosure, 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 of the method claims according to the aspects of the present disclosure described herein need not be performed in any particular order. Further, elements of the present disclosure may be described or claimed in the singular, but the plural is also intended unless the limitation to the singular is explicitly stated.

Claims

1. 1. An optical imaging system for nucleic acid sequencing, comprising: An actuator; a mounting element configured to receive a substrate; an objective lens having a proximal end and a distal end; the substrate comprises vertical walls that bound an area of ​​the substrate, the vertical walls being configured to retain a fluid within the area, the substrate having 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 positioned near an outer surface of the substrate, and the distal end of the objective lens is 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.

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

3. The system of claim 2 , wherein the fluid comprises water.

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

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

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

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

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

10. a fluid monitoring subsystem for determining whether a portion of the fluid has evaporated from the region; The system of claim 9 , wherein the fluid delivery subsystem is configured to deliver additional fluid upon a positive determination by the fluid monitoring subsystem.

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

12. the fluid monitoring system comprising an optical sensor for detecting the presence of air bubbles in the fluid; The system of claim 10 , wherein determining whether a portion of the fluid has evaporated from the region comprises determining whether a threshold number of air bubbles are detected in the fluid.

13. 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 droplet; and 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.

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

15. The method of claim 13 , wherein the fluid comprises water.

16. The method of claim 13 , 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.

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

18. The method of claim 13 , wherein the objective lens is moved from the first location to the second location at a speed of 15 mm / sec.

19. The method of claim 13 , wherein the inside of the vertical wall is flat.

20. The method of claim 13 , wherein the inside of the vertical wall is curved.

21. determining that a portion of the fluid has evaporated from the region; The method of claim 13 , further comprising: delivering additional fluid via a fluid delivery subsystem based on the determination.

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

23. Determining that a portion of the fluid has evaporated from the region includes: detecting the presence of air bubbles in the fluid; and determining that a threshold number of air bubbles are detected.

24. 1. An optical imaging system for nucleic acid sequencing, comprising: An actuator; a mounting element configured to receive a substrate; an objective lens having a proximal end and a distal end; the substrate comprises one or more nucleic acid sequences, an outer surface of the substrate is configured to receive a droplet, the outer surface of the substrate comprises a first material; the proximal end of the objective lens is coupled to the actuator, the distal end of the objective lens is configured to be positioned proximate the outer surface of the substrate, the distal end of the objective lens comprises a second material, the second material configured to provide a higher friction force against 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 moving the objective lens is configured to move the droplet along 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.

25. 25. The system of claim 24, wherein the droplet comprises oil, the first material being oleophobic and the second material being oleophilic.

26. 25. The system of claim 24, wherein the droplet comprises water, the first material is hydrophobic, and the second material is hydrophilic.

27. 27. The system of claim 26, wherein the first material comprises Teflon.

28. 27. The system of claim 26, wherein the second material comprises glass, silicon dioxide, sapphire, or calcium oxide.

29. 25. The system of claim 24, wherein the distal end of the objective lens is concave to facilitate maintaining contact between the objective lens and the droplet.

30. 30. The system of claim 29, wherein the distal end of the objective lens has a radius of curvature between 45 mm and 160 mm and a depth between 0.6 mm and 2.1 mm.

31. 25. The system of claim 24, wherein the actuator is configured to move the objective lens from the first location to the second location at a rate of 15 mm / sec.

32. 25. The system of claim 24, further comprising a droplet delivery subsystem for droplet delivery to the exterior surface of the substrate.

33. a droplet monitoring subsystem for determining whether the objective lens and the outer surface of the substrate are in contact with the droplet; 33. The system of claim 32, wherein the droplet delivery subsystem is configured to deliver additional droplets upon a negative determination by the droplet monitoring subsystem.

34. 33. The system of claim 32, wherein the droplet delivery subsystem comprises a conduit coupled to a fluid reservoir.

35. 1. A method for optically imaging a substrate for nucleic acid sequencing, the method comprising: disposing a droplet on an outer surface of the 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 above 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 friction force against the droplet than the first material; moving the objective lens to a second location on the outer surface of the substrate; moving the objective lens is configured to move the droplet along 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.

36. 36. The method of claim 35, wherein the droplets comprise oil, the first material being oleophobic and the second material being oleophilic.

37. 36. The method of claim 35, wherein the droplet comprises water, the first material is hydrophobic, and the second material is hydrophilic.

38. 38. The method of claim 37, wherein the first material comprises Teflon.

39. 38. The method of claim 37, wherein the second material comprises glass, silicon dioxide, sapphire, or calcium oxide.

40. 36. The method of claim 35, wherein the distal end of the objective lens is concave to facilitate maintaining contact between the objective lens and the droplet.

41. 41. The method of claim 40, wherein the distal end of the objective lens has a radius of curvature between 45 mm and 160 mm and a depth between 0.6 mm and 2.1 mm.

42. 38. The method of claim 37, wherein the objective lens is moved from the first location to the second location at a rate of 15 mm / sec.

43. determining that the objective lens and the outer surface of the substrate are not in contact with the droplet; delivering additional droplets via a droplet delivery subsystem based on said determining; and 38. The method of claim 37, further comprising:

44. 44. The method of claim 43, wherein the droplet delivery subsystem comprises a conduit coupled to a fluid reservoir.

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