Immersion imaging system and method for flow cell

Abstract

Systems and methods for soak imaging of a flow cell. In one example, an imaging system includes liquid ports positioned relative to front and rear ends of a scanning imaging objective. A front end liquid port distributes soak solution to the space between the distal end of the imaging objective and the cover of the flow cell, and a rear end liquid port collects soak solution. In another example, an imaging system includes a flow cell having a cover movable relative to a substrate. The system is configured to move the imaging objective and the flow cell cover as a unit relative to the flow cell substrate.

Description

Cross - Reference to Related Applications

[0001] This patent claims the priority of, and the benefit of the filing date of, the U.S. Provisional Patent Application Serial No. 63 / 382,738, filed on November 8, 2022, titled "Immersion Imaging Systems and Methods for Flow Cells", the entire content of which is incorporated herein by reference. Technical Field

[0002] Flow cell imaging systems and methods, such as imaging systems and methods for performing air - gap - free imaging of biochemical reactions in a flow cell during nucleic acid sequencing or other biochemical analysis processes. Background Art

[0003] Obtaining useful data from the optical imaging of biochemical reactions in a flow cell may require high spatial resolution, precision, and speed. Images may need to be acquired at a high enough magnification to clearly resolve the positions of individual samples (in some cases, down to the micron or nanoscale). At the same time, the images may need to cover a large enough field of view to correctly identify the samples. For large - scale studies, the speed of imaging and image processing should ideally be fast enough to be commercially viable. The numerical aperture (NA) of an optical system for sequencing has several implications: First, the NA determines the limit of optical resolution, or equivalently, the density of DNA samples that can be arranged on the sample. For first - order, the density of DNA samples will increase with the NA 2 and increase. Second, the NA determines the maximum fraction of light that the optical system can collect. For first - order, the collection efficiency of the optical system also increases with the NA 2 and increase. For example, a 10% increase in NA can result in a 21% increase in the optical signal. In addition, for a given density, a higher numerical aperture and higher optical resolution can significantly reduce optical crosstalk and associated noise levels.

[0004] Step - and - repeat imagers and time - delay integration (TDI) imagers are two major classes of imaging systems that can be used to image biochemical reactions in a flow cell. A step - and - repeat imaging system can acquire image data of approximately 10 million pixels with an alignment accuracy of about 5 microns per second. A TDI system can acquire image data of approximately 30 million pixels with an alignment accuracy of about 50 nanometers per second. For some applications, the performance of these two types of systems may be quite good, but for other applications, the existing iterations of these systems have some drawbacks in terms of structure and function that have an adverse impact on the overall throughput. For example, applications involving large - scale biochemical experimental studies (such as large - scale parallel whole - genome sequencing) would benefit from a higher overall throughput than many current step - and - repeat and TDI imaging systems can provide.

[0005] The numerical aperture of an optical system is limited by the lowest refractive index of the medium between the sample and the objective lens. The refractive index of most glasses is between 1.4 and 1.6, the refractive index of water is approximately 1.33, and the refractive index of air is 1.0. Therefore, an optical system that includes a layer of air or uses water instead of air can be considered to have a performance difference in numerical aperture of greater than 30%, which is significant in terms of optical resolution, DNA sample density, and optical collection efficiency. In addition, replacing the water layer with oil, which typically has an index of 1.5, also brings significant improvement. Summary of the Invention

[0006] The present disclosure presents improved systems and methods for flow cell imaging. These systems and methods can be used, for example, for sequencing template nucleic acid molecules disposed on a flow cell substrate. The substrate can include an array or other binding sites for receiving and retaining biochemical molecules. The systems and methods described herein can be configured to image the substrate by rapidly scanning an objective lens above the substrate. Compared with many traditional systems, the systems and methods described herein can provide improved performance, thereby increasing the density of biochemical molecules on the substrate and bringing other benefits.

[0007] In one example, a flow cell imaging system includes (a) a flow cell that includes a substrate, a lid, and a fluid channel between the substrate and the lid; (b) an imaging objective lens that includes a distal end spaced apart from the flow cell lid; (c) an actuator configured to translate the flow cell relative to the imaging objective lens; and (d) a first liquid port, wherein the system is configured to dispense liquid from the first liquid port into the space between the distal end of the imaging objective lens and the flow cell lid while the flow cell is translated relative to the imaging objective lens.

[0008] In some embodiments, the flow cell imaging system may further include a second liquid port, wherein the system is configured to collect liquid from the space between the distal end of the imaging objective lens and the flow cell lid into the second liquid port when the flow cell is translated relative to the imaging objective lens.

[0009] In some embodiments, the flow cell imaging system can be configured to maintain a moving liquid pit between the distal end of the imaging objective lens and the flow cell lid when the flow cell is translated relative to the imaging objective lens.

[0010] In some embodiments, the flow cell imaging system can be configured to maintain a moving liquid pit that fills the space between the distal end of the imaging objective lens and the flow cell lid when the flow cell is translated relative to the imaging objective lens.

[0011] In some embodiments, the imaging objective lens of the flow cell imaging system can include a first side and a second side opposite the first side, wherein the first liquid port is disposed on the first side of the imaging objective lens and the second liquid port is disposed on the second side of the imaging objective lens.

[0012] In some embodiments, the flow cell imaging system can be configured to dispense liquid from a first liquid port and collect the liquid into a second liquid port while the system images the flow cell.

[0013] In some embodiments, the actuator of the flow cell imaging system can be a translation stage configured to move the flow cell.

[0014] In some embodiments, the flow cell imaging system can be operated such that when the actuator translates the flow cell relative to the imaging objective along a first direction, the first liquid port dispenses liquid into the space between the distal end of the imaging objective and the flow cell, and the second liquid port collects liquid from the space between the distal end of the imaging objective and the flow cell.

[0015] In some embodiments, the flow cell imaging system can be operated such that when the actuator translates the flow cell relative to the imaging objective along a first direction, the translation of the first liquid port relative to the imaging objective is in a leading position, and the translation of the second liquid port relative to the imaging objective is in a trailing position.

[0016] In some embodiments, the flow cell imaging system can be operated such that the system is configured to alternately dispense and collect liquid from the first liquid port, and is configured to alternately dispense and collect liquid from the second liquid port.

[0017] In some embodiments, the flow cell imaging system can be configured such that the first liquid port and the second liquid port are fluidly connected to a pumping subsystem that operates in a first state to dispense liquid from the first liquid port and collect the liquid into the second liquid port, and the pumping subsystem operates in a second state to dispense liquid from the second liquid port and collect the liquid into the first liquid port.

[0018] In some embodiments, the flow cell imaging system can be configured such that when the actuator translates the flow cell relative to the imaging objective along a second direction different from the first direction, the second liquid port dispenses liquid into the space between the distal end of the imaging objective and the flow cell, and the first liquid port collects liquid from the space between the distal end of the imaging objective and the flow cell.

[0019] In certain embodiments, the flow cell imaging system may further include a third liquid port and a fourth liquid port.

[0020] In some embodiments, the flow cell imaging system can be configured to dispense liquid from the first liquid port and collect the liquid into the second liquid port while the actuator translates the flow cell relative to the imaging objective along a first direction, and wherein the system is configured to dispense liquid from the third liquid port and collect the liquid into the fourth liquid port while the actuator translates the flow cell relative to the imaging objective along a second direction different from the first direction.

[0021] In some embodiments, the flow cell imaging system may be configured such that the second direction is opposite to the first direction.

[0022] In some embodiments, the flow cell imaging system may be configured to dispense liquid from a first liquid port and collect the liquid into a second liquid port while an actuator translates the flow cell relative to the imaging objective along a first direction; and may also be configured to dispense liquid from the second liquid port and collect the liquid into the first liquid port while the actuator translates the flow cell relative to the imaging objective along a second direction different from the first direction; and may further be configured to dispense liquid from a third liquid port and collect the liquid into a fourth liquid port while the actuator translates the flow cell relative to the imaging objective along a third direction different from the first and second directions.

[0023] In some embodiments, the lid of the flow cell may be the second substrate of the flow cell.

[0024] In some embodiments, the actuator is configured to translate the flow cell relative to the imaging objective and the first and second liquid ports.

[0025] In another example, a flow cell imaging method includes: (a) scanning an imaging objective relative to a flow cell, wherein: (i) the flow cell includes a substrate, a lid, and a fluid channel between the substrate and the lid, and (ii) the imaging objective includes a distal end spaced apart from the flow cell lid; (b) dispensing liquid from a liquid port that is in a leading position relative to the imaging objective when scanning the imaging objective relative to the flow cell, the liquid being dispensed into the space between the distal end of the imaging objective and the flow cell lid; and (c) collecting the liquid into a liquid port that is in a trailing position relative to the imaging objective when scanning the imaging objective relative to the flow cell, the liquid being collected from the space between the distal end of the imaging objective and the flow cell lid.

[0026] In some embodiments, scanning the imaging objective relative to the flow cell includes changing the translation direction of the flow cell relative to the imaging objective, and changing the translation direction changes which liquid port is in the leading position and which liquid port is in the trailing position.

[0027] In another example, a flow cell imaging system includes (a) a flow cell that includes a substrate, a lid, and a fluid channel between the substrate and the lid; (b) an imaging objective; and (c) an actuator configured to translate the flow cell substrate relative to the imaging objective and the flow cell lid; the system is configured to translate the imaging objective and the lid as a unit relative to the flow cell substrate.

[0028] In certain embodiments, the imaging objective is attached to the flow cell lid.

[0029] In some embodiments, the distal portion of the imaging objective is embedded in the flow cell lid.

[0030] In some embodiments, the distal portion of the imaging objective extends through the flow cell lid.

[0031] In some embodiments, the flow cell further includes a fluid seal between the flow cell substrate and the flow cell lid that facilitates retaining liquid within the fluid channels when the flow cell substrate is translated relative to the flow cell lid.

[0032] In some embodiments, the area of the flow cell lid is greater than the area of the flow cell substrate such that the system is configured to scan the imaging objective across the imaging area of the substrate while the lid continues to cover the flow cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 An example of a flow cell imaging system is schematically illustrated.

[0034] Figure 2 and Figure 3 shows Figure 1 an example operating state of the flow cell imaging system shown.

[0035] Figures 4A - 4H An example of the imaging objective and associated liquid ports relative to the flow cell scan is schematically shown.

[0036] Figure 5 Another example of the imaging objective and associated liquid ports relative to the flow cell scan is schematically shown.

[0037] Figures 6 - 7 Another example of a flow cell imaging system is schematically illustrated.

[0038] Figure 8 Another example of a flow cell imaging system is schematically illustrated.

[0039] Figure 9 shows Figure 8 a top view of the flow cell imaging system shown, where the dashed lines show the imaging objective and the flow cell lid.

[0040] Figures 10A - 10D shows Figure 8 a top view of the flow cell imaging system shown, showing the relative positions of the imaging objective and the flow cell lid relative to the flow cell substrate during the scan. DETAILED DESCRIPTION

[0041] The present disclosure describes optical imaging systems and methods that can be used for biochemical reaction imaging. For example, the disclosed optical imaging systems and methods can be used for sequencing template nucleic acid molecules (such as DNA molecules, RNA molecules). In some embodiments, the template nucleic acid molecule can be bound to or otherwise disposed on the surface of a substrate (such as the inner surface of a flow cell), and the substrate can be imaged by the optical imaging system. For example, DNA templates can be immobilized at greater than 10e7 positions (spots) in an array on a substrate (such as a flow cell). In this example, a nucleic acid sequencing method may require more than 400 sequencing cycles. In each cycle, individual nucleotides (such as adenine, guanine, thymine, and cytosine) can flow across the substrate and be incorporated (into the growing strand) at each site having a complementary nucleotide base. In one method, 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 (such as a laser) can illuminate the spots (such as in clusters), causing the dye to emit light corresponding to the respective color. The color emitted by one of the four dyes at each spot can be detected by a camera (such as a time delay integration charge coupled device (TDI-CCD) camera or a similar camera), and the imaging system can thus record the detection of the nucleotide corresponding to the detected color for each spot. Those skilled in the art will recognize variations of the sequencing method, including variations in the template type (e.g., see Huang et al., 2017, Gigascience 6:1–9; Mardis et al., 2013, Annu Rev Anal Chem 6:287–303), the labeling system (e.g., see WO2018129214), and the labeling strategy (e.g., see US9,523,125).

[0042] In a conventional system, the emitted light can originate from the spots on the substrate, pass through a glass coverslip, through an air gap, into a microscope objective, and reach a camera that captures one or more images. In certain embodiments, the objective can include a housing and one or more lenses disposed within the housing that are configured to collect and focus light from the substrate and focus the light to produce a magnified image that can be captured by the camera. The system can be configured such that the objective moves in a pattern over the array of spots on the substrate in order to image the entire array within each cycle. Although the present disclosure focuses primarily on the sequencing of nucleic acid molecules, the present disclosure also contemplates using the disclosed optical imaging system to image other biochemical reactions and perform other biochemical analyses.

[0043] One of the significant costs per gigabase associated with nucleic acid sequencing is the amount of reagent consumed during the sequencing process. Thus, increasing the sample density on a substrate can significantly reduce the cost of nucleic acid sequencing. The optical numerical aperture (NA) of a detection system can, to some extent, determine the optical resolution of the system and thus the maximum density of the sample. Optical systems with high NA values tend to be more expensive, larger, and more difficult to calibrate and maintain than those with low NA values.

[0044] In a particular optical system, such as a confocal optical system, the NA may be limited by the lowest refractive index in the image chain. The cause of the NA limitation is the critical angle at each interface between materials. The critical angle defines the maximum ray angle that will not be totally reflected at the interface. When the refractive indices at each interface in the image chain are similar, the critical angle is large. As the refractive index difference increases, the critical angle becomes smaller, reducing the amount of light transmitted through the interface. The objective lens of an optical system has a relatively high refractive index, while the part of the image chain with a relatively low refractive index may produce a relatively small critical angle, reducing the amount of light transmitted to the objective lens. Thus, one way to increase the NA value of such a system without using the more expensive, larger, and more complex equipment in high NA systems is to increase the refractive index of one or more parts of the image chain with a lower refractive index. Methods and systems for doing so are disclosed herein.

[0045] In traditional optical systems for nucleic acid sequencing, the part of the image chain with the lowest refractive index is typically an air gap that may exist, for example, between the objective lens of the optical system and the flow cell lid. The refractive index of the air gap is typically about 1.00. In this example, the NA value of the exemplary optical imaging system is about 0.8. Replacing air with a substance having a higher refractive index can increase the overall NA value of the optical system. For example, water can be used to replace the air gap, which can increase the lowest refractive index of the image chain to 1.33. In this example, the NA value of a similar optical imaging system with water replacing the air gap may be about 1.0. Another example is that replacing the air gap with standard oil can increase the lowest refractive index of the image chain to 1.51. In this example, the NA value of a similar optical imaging system with water replacing the air gap may be about 1.2. Another example is that replacing the air gap with high refractive index oil can further increase the NA to 1.4. Another example is that any suitable water-based or oil-based solution can be used to appropriately cause the desired change in the lowest refractive index. In essence, the present disclosure proposes using a fluid with a refractive index higher than 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 impact on the NA of the optical imaging system, thereby improving the resolution and further increasing the density of the light spots on the substrate. Since the demand for reagents is reduced, the increase in density is also a reduction in cost. This can be illustrated by taking the optical imaging system with an air gap as a reference. For example, setting the density of this system to 1.00 and the relative cost to 1.00. Considering this reference, replacing the air gap with water can increase the density to about 1.56 and can correspondingly reduce the relative cost to about 0.64; replacing the air gap with standard oil can increase the density to about 2.25 and can correspondingly reduce the relative cost to about 0.44; replacing the air gap with high refractive index oil can increase the density to about 3.06 and can correspondingly reduce the relative cost to about 0.33.

[0046] Although it is known to use a fluid medium with a refractive index higher than air in standard microscope applications using an immersion objective lens optical system, such applications involve a static imaging process. In a dynamic imaging process, such as the scanning optical system considered herein for imaging a substrate when the objective lens moves rapidly above the substrate, the traditional immersion objective lens optical system is not ideal. The exemplary optical scanning system can move the objective lens at a speed between 10 mm / sec and 60 mm / sec. In some cases, next-generation sequencing systems can be equipped with an objective lens that moves at a speed of about 300 mm / sec. Moving the objective lens of a traditional system at such a high speed in such a fluid medium often results in excessive turbulence (and the generation of bubbles), which affects the image quality and causes loss of the fluid medium.

[0047] The present disclosure describes several methods for eliminating air gaps while addressing potential problems that typically occur when the objective lens moves rapidly in an immersion liquid. In certain embodiments, the methods disclosed herein can be used to move the objective lens at speeds between 10 mm / s and 60 mm / s or between 10 mm / s and 300 mm / s while maintaining good image quality using a liquid medium. In certain embodiments, very high speeds can be achieved, with speeds between 10 millimeters per second and 3750 millimeters per second, or between 30 millimeters per second and 3750 millimeters per second. This can allow for rapid scanning and imaging at a line speed of approximately 1 meter per second for the camera. Multi-nozzle system

[0048] Figure 1 Shows an example of a flow cell imaging system. Figure 1 The system shown includes a flow cell 100, an imaging objective lens 200, an actuator 300, and a first liquid port 400 and a second liquid port 402.

[0049] Figure 1 The flow cell 100 shown in includes a substrate 102, a lid 104, and a fluid channel 106 between the substrate 102 and the lid 104. Biochemical molecules 108 can be bound to or otherwise disposed on the inner surface of the substrate 102 in the fluid channel 106, and the fluid channel 106 can be filled with a water-based or oil-based liquid or other liquid suitable for a particular analysis using the flow cell 100. For ease of illustration, Figure 1 only a single row of several biochemical molecules 108 fixed to the substrate 102 is shown. In some embodiments, more than 10e7 biochemical molecules can be fixed in a two-dimensional array across the entire substrate 102. In some embodiments, alternatively or additionally, the lid 102 can be a substrate to which biochemical molecules are bound. The lid 102 and other components of the flow cell 100 can be optionally optically transparent to facilitate imaging therethrough. Although Figure 1 not shown, the flow cell 100 can also include fluid inputs and outputs for flowing sequencing reagents and other liquids through the fluid channel.

[0050] In Figure 1 the example of, the flow cell 100 is located within a container 110 that is configured to capture and retain excess liquid from the first liquid port 402 and / or the second liquid port 404, as described below. A liquid sensor 112 can be used to monitor the liquid level in the container 110 and interrupt system operation and / or trigger an alarm if the liquid level in the container 110 reaches an undesirable level, which will be discussed in further detail below. In certain cases, the container may include a drain.

[0051] The flow cell 100 and the container 110 are located on an actuator 300 (in this example, an X-Y translation stage), which is configured to translate the flow cell 100 and the container 110 relative to the imaging objective 200. Thus, the imaging objective 200 can scan or otherwise translate relative to the entire area to be imaged in the substrate 102. In other embodiments, the flow cell 100 can remain stationary, and the imaging objective 200 can be associated with an actuator configured to translate the imaging objective 200, the first liquid port 402, and the second liquid port 404 relative to the flow cell 100.

[0052] In Figure 1 the example of, the imaging objective 200 is part of an optical subsystem that includes a light source 202 (such as a laser), an autofocus assembly 204, a Z-direction actuator 206, additional optical elements (such as optical element 208), and a camera 210 (such as a time-delay integration charge-coupled device (TDI-CCD) camera or a similar camera). A controller 500 (such as a digital microprocessor) can monitor and control the various components of the optical subsystem as well as Figure 1 the other subsystems of the flow cell imaging system.

[0053] The imaging objective 200 can be an optical objective configured to be immersed in an oil-based, water-based, or other immersion liquid having a refractive index greater than that of air. As described below, the immersion liquid can be stored in a reservoir 412.

[0054] In Figure 1 the example of, the first liquid port 402 and the second liquid port 402 are part of a liquid delivery and collection subsystem that also includes pumps 408, 410 fluidly connected to the first liquid port 402 and the second liquid port 402, respectively, and a reservoir 412 fluidly connected to the pumps 408, 410. The liquid delivery and collection subsystem can be configured such that during imaging, there is no air gap or bubble in the space between the distal end of the imaging objective 200 and the lid 104, and this space is filled with a liquid 406. The liquid 406 can be an oil-based or water-based immersion liquid having a refractive index higher than that of air, which is commonly used for immersion microscopy, or any other liquid suitable for use in the system.

[0055] Figure 1 The liquid delivery and collection subsystem in can be configured to distribute liquid into and collect liquid from the space between the distal end 212 of the imaging objective 200 while the flow cell 100 is translated relative to the imaging objective 200 and the first liquid port 402 and the second liquid port 404 by the actuator 300. In this particular example, depending on the translation direction, the two liquid ports 402, 404 can alternately distribute or collect liquid from the space between the objective 200 and the flow cell 100.

[0056] Figure 2 shows Figure 1 the operating state of the system, in which the actuator 300 translates the flow cell 100 in the direction 302, causing the imaging objective 200 and the first liquid port 402 and the second liquid port 404 to translate relative to each other in the direction 304. In this example, the pumps 408, 410 are reversible pumps. The system operates the pump 408 to pump liquid from the reservoir 412 to the first liquid port 402, thereby dispensing the liquid 406 into the space between the distal end 212 of the imaging objective 200 and the lid 104. The system operates the pump 410 to pump the liquid in the space between the distal end 212 of the imaging objective 200 and the lid 104 into the reservoir 412 via the second liquid port 404.

[0057] In this way, when the imaging objective 200 translates relative to each other in the direction 304, a moving "pit" or other discrete volume of liquid is maintained around the distal end 212 of the imaging objective 200, and the liquid 406 is shown to be sufficient to prevent any air gap or bubble from existing between the distal end 212 of the imaging objective 200 and the lid 104 of the flow cell 100. Although Figure 2 the volume of the liquid 406 is shown to start and end at the liquid ports 402, 404, in other embodiments, the liquid 406 may extend beyond the liquid ports 402, 404.

[0058] Figure 3 shows Figure 1 a second operating state of the system, in which the actuator 300 translates the flow cell 100 in the direction 306 opposite to the direction 302 in Figure 2 above, causing the imaging objective 200 and the first liquid port 402 and the second liquid port 404 to translate relative to each other in the direction 308. In the second state, the system operates the pump 410 to pump liquid from the reservoir 412 to the second liquid port 404, thereby dispensing the liquid 406 into the space between the distal end 212 of the imaging objective 200 and the lid 104. The system operates the pump 408 to pump the liquid in the space between the distal end 212 of the imaging objective 200 and the lid 104 into the reservoir 412 through the first liquid port 402. In this way, when the imaging objective 200 translates relative to each other in the direction 308, a "pit" or other discrete volume of liquid is maintained around the distal end 212 of the imaging objective 200.

[0059] The system can be configured to monitor the performance of the liquid delivery and collection subsystem during operation to ensure that the liquid is neither too much nor too little. As described above, when too much liquid is dispensed and / or too little liquid is collected, the liquid sensor 112 can be triggered. Defects in the images collected by the imaging subsystem can also be detected, indicating the presence of an air gap or bubble between the objective 200 and the lid 104, which in turn may indicate insufficient liquid dispensed from the liquid ports 402, 404 and / or too much liquid collected.

[0060] Figures 4A - 4H shows the scanning process using Figures 1 - 3 the system. In Figures 4A - 4H , the imaging objective lens 200 and the first liquid port 402 and the second liquid port 404 are sequentially scanned relative to the flow cell 100 in directions 310( Figures 4A - 4B ), 312( Figures 4B - 4C ), 314( Figures 4C - 4D ), 316( Figures 4D - 4E ), 318( Figures 4E - 4F ), 320( Figures 4F - 4G ) and 322( Figures 4G - 4H ). The directions 310, 314, 318 and 322 are oriented along the X-axis, and the directions 312, 316 and 318 are oriented along the Y-axis. The X-axis directions 310 and 318 are opposite to the X-axis directions 314 and 322.

[0061] In this example, when the imaging objective lens 200 is scanned relative to the flow cell 100 along the X-axis directions 310 and 318 (as shown in Figures 4A - 4B and Figures 4E - 4F ), liquid is dispensed from the liquid port 402 and collected at the liquid port 404, where the liquid port 402 is in the leading position relative to the imaging objective lens 200 and the liquid port 404 is in the trailing position relative to the imaging objective lens 200. When the imaging objective lens 200 is scanned relative to the flow cell 100 along the x-axis directions 314 and 322 (as shown in Figures 4C - 4D and Figures 4G - 4H ), liquid is dispensed from the liquid port 404 and collected at the liquid port 402, with the liquid port 404 in the leading position and the liquid port 402 in the trailing position. As shown in Figures 4A - 4H , changing the translation direction of the imaging objective lens relative to the flow cell also changes which liquid port dispenses liquid in the leading position relative to the imaging objective lens 200 and which liquid port collects liquid in the trailing position relative to the imaging objective lens 200. As shown in Figures 4A - 4H , this forms a moving liquid pit 406 that moves with the imaging objective lens 200.

[0062] Figure 5 shows an example of the scanning process using a system with a system configuration different from that shown in Figures 1 - 3 . Figure 5The system shown includes four liquid ports 402, 404, 432, and 434 located on four sides of the imaging objective 200, where the liquid ports 402 and 404 are opposite each other and the liquid ports 432 and 434 are opposite each other. In this example, when the imaging objective 200 is scanned relative to the flow cell 100 in the x-axis directions 310, 314, 318, and 322, and when the imaging objective 200 is scanned relative to the flow cell 100 in the y-axis directions 312, 316, and 320, the system is configured to dispense and collect liquid.

[0063] In this example, the liquid port that is in the leading position relative to the translation direction will operate to dispense liquid, while the liquid port that is in the trailing position relative to the translation direction will operate to collect liquid.

[0064] Specifically, when the imaging objective 200 is initially translated relative to the flow cell 100 in the 310 direction, the liquid port 402 will dispense liquid, the liquid port 404 will collect liquid, and the liquid ports 432 and 434 will not dispense or collect liquid. When the imaging objective 200 is next translated relative to the flow cell in the 312 direction, the liquid port 432 will dispense liquid, the liquid port 434 will collect liquid, and the liquid ports 402 and 404 will not dispense or collect liquid. When the imaging objective 200 is next translated relative to the flow cell in the 314 direction, the liquid port 404 will dispense liquid, the liquid port 402 will collect liquid, and the liquid ports 432 and 434 will not dispense or collect liquid. When the imaging objective 200 is next translated relative to the flow cell in the 316 direction, the liquid port 432 will dispense liquid, the liquid port 434 will collect liquid, and the liquid ports 402 and 404 will not dispense or collect liquid. When the imaging objective 200 is next translated relative to the flow cell in the 318 direction, the liquid port 402 will dispense liquid, the liquid port 404 will collect liquid, and the liquid ports 432 and 434 will not dispense or collect liquid. When the imaging objective 200 is next translated relative to the flow cell in the 320 direction, the liquid port 432 will dispense liquid, the liquid port 434 will collect liquid, and the liquid ports 402 and 404 will not dispense or collect liquid. Finally, when the imaging objective 200 is moved relative to the flow cell in the 322 direction, the liquid port 404 will dispense liquid, the liquid port 402 will collect liquid, and the liquid ports 432 and 434 will not dispense or collect liquid.

[0065] In Figure 5 the example, the liquid ports that are not aligned with the translation direction do not operate to dispense or collect liquid. In other embodiments, the liquid ports that are not aligned with the translation direction may also be assigned to collect liquid.

[0066] In other embodiments, the liquid ports need not be aligned with the translation direction but can still be operated in a way that distributes and collects liquid such that, during scanning relative to the flow cell, the moving liquid pits move with the imaging objective.

[0067] The liquid delivery and collection subsystem may be configured differently than described above. For example, Figures 1 - 3 it is shown that pumps 408, 410 are reversible; however, in other configurations, each liquid port 402, 404 may be associated with two pumps, one configured to dispense liquid from its associated port and the other configured to collect liquid from its associated port. Alternatively or additionally, the liquid delivery and collection subsystem may also include multiple fluid reservoirs, one configured to dispense fresh liquid and another configured to collect used liquid.

[0068] In Figures 1 - 3 the example shown, fluid ports 402, 404 are positioned and oriented to dispense and collect fluid directly in the space between the distal end 212 of the objective and the lid 104. In other configurations, fluid ports 402, 404 may still dispense and collect fluid to the space between the distal end 212 of the objective and the lid 104, but their positions are further "upstream" and "downstream" away from the objective 200.

[0069] Figures 6 - 7 Another example of the configuration of the liquid delivery and collection subsystem is shown. In this example, each pump and liquid port assembly is configured to move liquid in a single direction. In this example, there are four liquid ports 414, 416, 418, 420. Two of the liquid ports 414, 416 are located on one side of the imaging objective 200, and two liquid ports 418, 420 are located on the other side of the imaging objective 200. Each liquid port 414, 416, 418, 420 is fluidly connected to a pump 422, 424, 426, 428, respectively. In this example, each pump 422, 424, 426, 428 is configured to pump liquid in a single direction. In operation, pump 422 is configured to pump liquid from reservoir 412 to liquid port 414. In operation, pump 424 is configured to pump liquid from reservoir 412 to liquid port 416. In operation, pump 426 is configured to pump liquid from liquid port 418 to liquid reservoir 412. In operation, pump 428 is configured to pump liquid from liquid port 420 to liquid reservoir 412.

[0070] In Figure 6 the operating state shown, when the objective 200 is translated in the 304 direction relative to the flow cell 100, liquid pump 422 pumps liquid from reservoir 412 to liquid port 414, and liquid pump 428 pumps liquid from liquid port 420 to reservoir 412. InFigure 6 In the operation state shown, pumps 424 and 426 are not operating. In Figure 7 the operation state shown, when the objective lens 200 is translated relative to the flow cell 100 in the direction 308, the liquid pump 424 pumps liquid from the reservoir 412 to the liquid port 416, and the liquid pump 426 pumps liquid from the liquid port 418 to the reservoir 412. In Figure 7 the operation state shown, pumps 422 and 428 are not operating. Moving lid system

[0071] Figure 8 shows another example of a flow cell imaging system that avoids any air gap between the distal end of the imaging objective lens and the flow cell. The system in this example includes a flow cell 800, an imaging objective lens 900, and an actuator 1000.

[0072] The flow cell 800 includes a substrate 802, a lid 804, and a fluid channel 806 between the substrate 802 and the lid 804. In this example, the lid 804 and the substrate 802 are not connected and can move relative to each other. A seal 808 can be located at the position where the lid 804 contacts the wall of the substrate 802 to allow the lid 804 to move relative to the substrate 802 while retaining the liquid within the fluid channel 806.

[0073] As Figure 8 shown, the lid 804 is attached to the imaging objective lens 900, where the distal portion 902 of the imaging objective lens 900 is embedded in the lid 904 or otherwise extends through the lid 904. A clamping ring 810 can be attached or otherwise fixed to the lid 804 and can be clamped around the imaging objective lens 900 to fix the imaging objective lens 900 to the lid 804.

[0074] The actuator 1000 is configured to translate the flow cell substrate 802 relative to the flow cell lid 804, and since the imaging objective lens 900 is attached to the flow cell lid 804, the imaging objective lens and the lid will translate as a unit relative to the flow cell substrate 802.

[0075] As Figure 8 shown, the flow cell lid 804 can be larger than the flow cell substrate 802. Thus, the system can be configured to scan the imaging objective lens 900 over the imaging area of the flow cell substrate 802 while the lid 804 continues to cover the substrate 802. Figure 9 Shown from top to bottom is the Figure 8 system, Figures 10A - 10D illustrating how the lid 804 has a large enough area such that the imaging objective lens 900 can be scanned over the imaging area 812 of the substrate 802 while the lid 804 continues to cover the substrate 802. Conclusion

[0076] The above systems and methods are provided by way of example only. Additions, deletions, substitutions, modifications, and other changes may be made to the above examples without departing from the scope or spirit of the following claims.

Claims

1. A flow cell imaging system, comprising: (a) Flow cell, the flow cell comprising a substrate, a lid, and a fluid channel between the substrate and the lid; (b) Imaging objective, the imaging objective comprising a distal end spaced apart from the flow cell lid; (c) Actuator, which is configured to translate the flow cell relative to the imaging objective; And (d) First liquid port, wherein the system is configured to dispense liquid from the first liquid port into the space between the distal end of the imaging objective and the flow cell lid while the flow cell is translated relative to the imaging objective.

2. The flow cell imaging system according to claim 1, further comprising a second liquid port, wherein the system is configured to collect the liquid from the space between the distal end of the imaging objective and the flow cell lid into the second liquid port while the flow cell is translated relative to the imaging objective.

3. The flow cell imaging system according to claim 2, wherein while the flow cell is translated relative to the imaging objective, the system is configured to maintain a moving liquid pit between the distal end of the imaging objective and the lid of the flow cell.

4. The flow cell imaging system according to claim 2, wherein while the flow cell is translated relative to the imaging objective, the system is configured to maintain a moving liquid pit that fills the space between the distal end of the imaging objective and the lid of the flow cell.

5. The flow cell imaging system according to claim 2, wherein the imaging objective includes a first side and a second side opposite the first side, wherein the first liquid port is provided on the first side of the imaging objective and the second liquid port is provided on the second side of the imaging objective.

6. The flow cell imaging system according to claim 2, wherein the system is configured to dispense the liquid from the first liquid port and collect the liquid into the second liquid port while the system images the flow cell.

7. The flow cell imaging system according to claim 1, wherein the actuator includes a translation stage configured to move the flow cell.

8. The flow cell imaging system according to claim 2, wherein, While the actuator translates the flow cell relative to the imaging objective in a first direction, the first liquid port dispenses the liquid into the space between the distal end of the imaging objective and the flow cell and the second liquid port collects the liquid from the space between the distal end of the imaging objective and the flow cell.

9. The flow cell imaging system according to claim 8, wherein, While the actuator translates the flow cell relative to the imaging objective in the first direction, the first liquid port is in a leading position relative to the translation of the imaging objective, while the second liquid port is in a trailing position relative to the translation of the imaging objective.

10. The flow cell imaging system according to claim 8, wherein the system is configured to alternately dispense and collect the liquid from the first liquid port and is configured to alternately dispense and collect the liquid from the second liquid port.

11. The flow cell imaging system according to claim 10, wherein the first liquid port and the second liquid port are in fluid connection with a pumping subsystem, the pumping subsystem operating in a first state to dispense the liquid from the first liquid port and collect the liquid into the second liquid port, and operating in a second state to dispense the liquid from the second liquid port and collect the liquid into the first liquid port.

12. The flow cell imaging system according to claim 8, wherein while the actuator translates the flow cell relative to the imaging objective lens in a second direction different from the first direction, the second liquid port dispenses the liquid into the space between the distal end of the imaging objective lens and the flow cell and the first liquid port collects the liquid from the space between the distal end of the imaging objective lens and the flow cell.

13. The flow cell imaging system according to claim 2, further comprising a third liquid port and a fourth liquid port.

14. The flow cell imaging system according to claim 13, wherein the system is configured to dispense the liquid from the first liquid port and collect the liquid into the second liquid port while the actuator translates the flow cell relative to the imaging objective lens in a first direction, and wherein the system is configured to dispense the liquid from the third liquid port and collect the liquid into the fourth liquid port while the actuator translates the flow cell relative to the imaging objective lens in a second direction different from the first direction.

15. The flow cell imaging system according to claim 14, wherein the second direction is opposite to the first direction.

16. The flow cell imaging system according to claim 13, wherein the system is configured to dispense the liquid from the first liquid port and collect the liquid into the second liquid port while the actuator translates the flow cell relative to the imaging objective lens in the first direction; Wherein the system is configured to dispense the liquid from the second liquid port and collect the liquid into the first liquid port while the actuator translates the flow cell relative to the imaging objective in a second direction different from the first direction; And Wherein the system is configured to dispense the liquid from the third liquid port and collect the liquid into the fourth liquid port while the actuator translates the flow cell relative to the imaging objective in a third direction different from the first direction and the second direction.

17. The flow cell imaging system according to claim 1, wherein the lid includes a second substrate of the flow cell.

18. The flow cell imaging system according to claim 1, wherein the actuator is configured to translate the flow cell relative to the imaging objective lens and the first liquid port and the second liquid port.

19. A flow cell imaging method, comprising: (a) Scanning an imaging objective relative to a flow cell, wherein: (i) The flow cell comprises a substrate, a lid, and a liquid channel between the substrate and the lid, and (ii) The imaging objective comprises a distal end spaced apart from the flow cell lid; (b) While scanning the imaging objective relative to the flow cell, dispensing liquid from a liquid port in a leading position relative to the imaging objective, the liquid being dispensed into the space between the distal end of the imaging objective and the flow cell lid; and (c) While scanning the imaging objective relative to the flow cell, collecting the liquid into a liquid port in a trailing position relative to the imaging objective, the liquid being collected from the space between the distal end of the imaging objective and the flow cell lid.

20. The flow cell imaging method according to claim 19, wherein scanning the imaging objective relative to the flow cell includes changing the translation direction of the flow cell relative to the imaging objective, and wherein changing the translation direction changes which liquid port is in the leading position and which liquid port is in the trailing position.

21. A flow cell imaging system, comprising: (a) Flow cell, the flow cell comprising a substrate, a lid, and a liquid channel between the substrate and the lid; (b) Imaging objective; And (c) Actuator, which is configured to translate the flow cell substrate relative to the imaging objective and the flow cell lid; Wherein the system is configured to translate the imaging objective and the lid as a unit relative to the flow cell substrate.

22. The flow cell imaging system according to claim 21, wherein the imaging objective is attached to the flow cell lid.

23. The flow cell imaging system according to claim 21, wherein the distal portion of the imaging objective is embedded in the flow cell lid.

24. The flow cell imaging system according to claim 21, wherein the distal portion of the imaging objective extends through the flow cell lid.

25. The flow cell imaging system according to claim 21, further comprising a fluid seal between the flow cell substrate and the flow cell lid, the fluid seal assisting in retaining liquid in the fluid channels while the flow cell substrate translates relative to the flow cell lid.

26. The flow cell imaging system according to claim 21, wherein the area of the lid of the flow cell is greater than the area of the substrate of the flow cell, such that the system is configured to scan the imaging objective over the imaging area of the substrate while the lid continues to cover the flow cell.

Images