Flow cell imaging systems and methods, and flow cells and other substrates used therein

Abstract

Dual-sided flow cell and other substrate imaging systems, such as imaging systems used in nucleic acid sequencing and similar processes. In one example, the imaging system includes a flipper that facilitates imaging of different surfaces of the flow cell or other substrate. In another example, the imaging system includes two optical systems for imaging different surfaces of the flow cell or other substrate. In another example, the imaging system is an immersion system. In these and other examples, the system may include an autofocus subsystem configured to precisely focus the optics on one surface of the dual-sided flow cell without interference from the other surface of the flow cell.

Description

[Technical field]

[0001] (Related Applications) This application claims priority to and benefit of the filing date of U.S. Provisional Patent Application No. 63 / 231,488 (filed August 10, 2021), the entire contents of which are incorporated herein by reference.

[0002] (Related Fields) This patent relates to flow cells and other substrate imaging systems, such as imaging systems used in nucleic acid sequencing and similar processes. [Background technology]

[0003] Many of the current nucleic acid sequencing systems and processes are resource intensive, requiring, among other things, significant amounts of reagents and significant amounts of time. Although massively parallel systems and processes have been developed to use resources more efficiently, there is room for improvement. Summary of the Invention [Means for solving the problem]

[0004] This patent describes several examples of flow cells and other substrates, and systems and methods for imaging these flow cells and other substrates, both sides of which have analytes for analysis. The flow cells, other substrates, and systems and methods for imaging these flow cells and other substrates described in this patent can promote more efficient use of resources, for example, minimizing the use of reagents and improving the speed at which samples can be processed.

[0005] In one example, the imaging system includes: a stage configured to hold a carrier configured to receive the specimen; a light source configured to illuminate the specimen with a light beam, the light beam being characterized by an optical path; a detector configured to detect light; a lens system configured to focus light from the specimen onto a detector after the light beam illuminates the specimen; A lens system is characterized by its optical axis, the light beam is configured to propagate through the lens system and illuminate the specimen; The optical path of the light beam incident on the carrier is not parallel to the optical axis of the lens system.

[0006] The imaging system may also include a controller configured to vary the distance between the carrier and the lens system to focus light from the specimen onto the detector.

[0007] The imaging system may also include one or more processors configured to perform the following operations. calculating a first light intensity based on the light detected by the first pixel block of the detector; calculating a second light intensity based on the light detected by a second block of pixels of the detector; comparing the first light intensity to the second light intensity to generate a focus error signal; Based on the focus error signal, movement of the lens system relative to the carrier is controlled.

[0008] The detector may be an array detector configured to focus a spot of light incident on the detector.

[0009] The array detector may be a one-dimensional array.

[0010] The carrier may be a flow cell, the flow cell comprising a first sample receiving surface and a second sample receiving surface separated by a width.

[0011] The depth of focus of the lens system may be less than the width.

[0012] The stage may be configured to move such that the lens system focuses light from a first sample receiving surface onto the detector and, but not simultaneously, focuses light from a second sample receiving surface onto the detector.

[0013] The light beam may be defined by a beam width, which may be measured at the carrier, and which may be 2 mm or less and / or 10 microns or more.

[0014] The lens system may include an immersion objective with a distal lens surface configured to be immersed in a fluid.

[0015] The imaging system may be configured as follows. (a) in a first configuration, the system is configured to image radiation emitted from a specimen attached to a first specimen receiving surface; the distal lens surface is spaced a first perpendicular distance from the first sample receiving surface; the first vertical distance includes the fluid segment and the substrate segment; (b) in a second configuration, the system is configured to image radiation emitted from a specimen attached to the second specimen receiving surface; the distal lens surface is spaced a second perpendicular distance from the second sample receiving surface; the second vertical distance includes the fluid segment and the substrate segment; (c) the first perpendicular distance is substantially the same as the second perpendicular distance; The fluid segment at the first perpendicular distance is substantially the same as the fluid segment at the second perpendicular distance.

[0016] The immersion objective lens may be at least partially immersed in a reservoir above the first sample receiving surface such that no air gap exists between the distal lens surface and the first sample receiving surface.

[0017] The imaging system may also include an xy translation stage configured to translate one of the immersion objective or the flow cell relative to the other of the immersion objective or the flow cell while the immersion objective is at least partially immersed in the reservoir during imaging.

[0018] The fluid in the reservoir may have substantially the same refractive index as the fluid in the fluid passage of the flow cell between said first and second sample receiving surfaces.

[0019] In another example, the imaging system includes: (a) a flow cell comprising a first substrate including a first surface, a second substrate including a second surface, and a fluid passage between the first surface and the second surface; (b) an imager including an immersion objective, the immersion objective including a distal lens surface, the immersion objective being at least partially immersed in a fluid; (c) in a first configuration, the system is configured to image radiation emitted from a specimen attached to the first surface; the distal lens surface is spaced a first perpendicular distance from the first surface; the first vertical distance includes the fluid segment and the substrate segment; (d) in a second configuration, the system is configured to image radiation emitted from a specimen attached to the second surface; the distal lens surface is spaced a second perpendicular distance from the second surface; the second vertical distance includes the fluid segment and the substrate segment; (e) the first perpendicular distance is substantially the same as the second perpendicular distance; The fluid segment at the first perpendicular distance is substantially the same as the fluid segment at the second perpendicular distance.

[0020] The system may further include a z-translation stage configured to translate one of the immersion objective or the flow cell vertically relative to the other of the immersion objective or the flow cell.

[0021] The system may be configured to change from the first configuration to the second configuration by vertically translating one of the immersion objective lens or the flow cell relative to the other of the immersion objective lens or the flow cell a distance substantially equal to the height of the fluid passage.

[0022] The system may further include an autofocus subsystem configured to focus on a first surface when the system is in the first configuration, and configured to focus on a second surface when the system is in the second configuration.

[0023] The first surface may be an inner surface of a first substrate, and the second surface may be an inner surface of a second substrate, and the first and second surfaces may face each other across a fluid passage.

[0024] The system may further include a radiation source configured to stimulate radiation emitted from the analytes attached to the first surface and the second surface.

[0025] The first substrate is substantially transparent to radiation from the radiation source and may be substantially transparent to radiation emitted from specimens attached to the first and second surfaces.

[0026] The immersion objective may be at least partially immersed in a reservoir on the first substrate such that there is no air gap between the distal lens surface and the first surface of the flow cell.

[0027] The system may further include an xy translation stage configured to translate one of the immersion objective or the flow cell relative to the other of the immersion objective or the flow cell while the immersion objective is at least partially immersed in the reservoir during imaging.

[0028] The fluid in the reservoir may have substantially the same refractive index as the fluid in the fluid passage.

[0029] In another example, an imaging system includes (a) a double-sided substrate including a first surface and a second surface; (b) an imager; and (c) a flipper configured to flip the substrate between a first orientation and a second orientation, where when the substrate is in the first orientation, the system is configured to image radiation emitted from a specimen attached to the first surface, and when the substrate is in the second orientation, the system is configured to image radiation emitted from a specimen attached to the second surface.

[0030] The double-sided substrate may be a flow cell with a fluid passageway located between the first surface and the second surface.

[0031] The flow cell may include a first substrate and a second substrate, the first surface being an inner surface of the first substrate and the second surface being an inner surface of the second substrate, the first surface and the second surface facing each other across a fluid passage.

[0032] The first substrate may include a first thickness and the second substrate may include a second thickness, the first thickness and the second thickness being substantially the same.

[0033] The system may further include a radiation source configured to stimulate radiation emitted from analytes attached to the first surface and the second surface.

[0034] The first and second substrates are substantially transparent to radiation from the radiation source and may be substantially transparent to radiation emitted from analytes attached to the first and second surfaces.

[0035] The double-sided board may include a first substrate bonded to a second substrate, the first surface and the second surface being exterior surfaces of the double-sided board.

[0036] The system may further include an autofocus subsystem configured to focus on a first surface when the double-sided substrate is in the first orientation, and configured to focus on a second surface when the double-sided substrate is in the second orientation.

[0037] The imaging system may further include an imaging station, at least one additional station, and a transport device configured to move the double-sided substrate between said stations.

[0038] The transport device may include a flipper.

[0039] In another example, an imaging system includes (a) a double-sided substrate including a first surface and a second surface, (b) a first imager configured to image radiation emitted from a specimen attached to the first surface, and (c) a second imager configured to image radiation emitted from a specimen attached to the second surface.

[0040] The double-sided substrate may be a flow cell.

[0041] The flow cell may have a first substrate and a second substrate, the first surface being an inner surface of the first substrate and the second surface being an inner surface of the second substrate, the first surface and the second surface facing each other across the fluid passage.

[0042] The imaging system may further include a radiation source configured to stimulate emission of radiation from a specimen attached to the first surface and the second surface.

[0043] The radiation source may be configured to simultaneously stimulate emission of radiation from analytes attached to the first surface and the second surface.

[0044] The radiation source may include a single laser beam configured to simultaneously stimulate emission of radiation from specimens attached to the first and second surfaces.

[0045] The imaging system may be configured to simultaneously image radiation emitted from a specimen attached to a first surface using a first imager and radiation emitted from a specimen attached to a second surface using a second imager.

[0046] The first imager may have a first objective lens with a first optical axis, the second imager may have a second objective lens with a second optical axis, and the system may be configured to position the flow cell between the first and second objective lenses.

[0047] The flow cell may be positioned between a first objective lens and a second objective lens, with the first objective lens facing the first substrate and the second objective lens facing the second substrate.

[0048] The first optical axis and the second optical axis may be collinear optical axes.

[0049] In another example, a double-sided substrate includes a first planar surface and an array of analyte binding sites on the first planar surface, a second planar surface and an array of analyte binding sites on the second planar surface.

[0050] The first planar surface may be an outer surface of a first substrate and the second planar surface may be an outer surface of a second substrate, the first and second substrates being bonded together on their inner surfaces.

[0051] In another example, the imaging system includes a stage configured to hold a carrier having a specimen; a light source configured to illuminate the specimen with a light beam; a detector configured to detect light; a lens system configured to focus light from the specimen onto a detector after the light beam illuminates the specimen; the carrier is configured to be mounted to a stage while the specimen is illuminated by a light beam; The light beam is characterized by an optical path, A lens system is characterized by its optical axis, the light beam is configured to propagate through the lens system and illuminate the specimen; The optical path of the light beam incident on the carrier is not parallel to the optical axis of the lens system.

[0052] The imaging system may further include a controller configured to vary the distance between the carrier and the lens system to focus light from the specimen onto the detector.

[0053] The system may further include one or more processors configured to perform the following operations. calculating a first light intensity based on the light detected by the first pixel block of the detector; calculating a second light intensity based on the light detected by a second block of pixels of the detector; comparing the first light intensity to the second light intensity to generate a focus error signal; Based on the focus error signal, movement of the lens system relative to the carrier is controlled.

[0054] The carrier may be a flow cell.

[0055] The detector may be an array detector configured to focus a spot of light incident on the detector.

[0056] The array detector may be a one-dimensional array.

[0057] The carrier may include a first surface and a second surface; The first surface may be separated from the second surface by a width.

[0058] The depth of focus of the lens system may be less than the width separating the first surface from the second surface of the carrier.

[0059] The analytes may include a first analyte attached to a first surface, and a second analyte attached to a second surface.

[0060] The stage may be configured to move such that the lens system focuses light from a first specimen onto the detector and, but not simultaneously, focuses light from a second specimen onto the detector.

[0061] The light beam may be defined by a beam width measured at the carrier, which may be less than 2.0 mm and / or greater than 10 microns.

[0062] In another example, a method of imaging includes the steps of attaching a carrier to a stage, the carrier including a specimen; illuminating the specimen with a light source by transmitting a light beam of the light source through a lens system to the specimen; The light beam is characterized by an optical path, A lens system is characterized by its optical axis, a step in which the optical path of the light beam incident on the carrier is not parallel to the optical axis of the lens system; focusing light from the specimen onto a detector using a lens system; and detecting light from the specimen using a detector.

[0063] The imaging method may further include controlling movement of the lens system relative to the carrier in a direction parallel to an optical axis of the lens system to focus light from the specimen onto a detector.

[0064] The step of controlling the movement of the lens system relative to the carrier includes: calculating a first light intensity, the first light intensity being calculated based on light detected by a first pixel block of the detector; calculating a second light intensity, the second light intensity being calculated based on light detected by a second pixel block of the detector; comparing the first light intensity and the second light intensity to generate a focus error signal; and controlling movement of the lens system relative to the carrier based on the focus error signal.

[0065] In another embodiment, the imaging method comprises: defining a first block of pixels of the detector; defining a second block of pixels of the detector; illuminating the specimen with an off-axis light beam; detecting the light from the sample with a detector after the light from the sample passes through the lens system; calculating a first light intensity, the first light intensity being calculated based on light detected by a first pixel block; calculating a second light intensity, the second light intensity being calculated based on light detected by a second pixel block; comparing the first light intensity and the second light intensity to generate a focus error signal; and adjusting the distance between the lens system and the specimen based on the focus error signal. [Brief description of the drawings]

[0066] [Figure 1a] An example of a flow cell 100 is shown diagrammatically. [Figure 1b] 1b shows a schematic representation of the flow cell of FIG. 1a in a holder. [Figure 1c] 1 shows another example of a flow cell and holder. [Figure 1d] 1c showing the holder of FIG. 1c holding the flow cell of FIG. [Figure 1e] 1 shows another example of a flow cell in schematic form. [Diagram 2] 1 illustrates a schematic diagram of an example imaging system. [Diagram 3] 2 illustrates a schematic diagram of another example of an imaging system. [Figure 4a] 1 shows the flow cell oriented relative to the objective lens of the imaging system. [Figure 4b] Figure 4a shows the flow cell in an inverted orientation. [Diagram 5] 2 illustrates a schematic diagram of another example of an imaging system. [Figure 6] 6 shows a flow cell positioned between two objective lenses of the imaging system of FIG. 5. [Figure 7] 1 illustrates another example of an imaging system. [Figure 8] 1 illustrates another example of an imaging system. [Figure 9] 1 illustrates another example of an imaging system. [Figure 10] 1 illustrates another example of an imaging system. [Figure 11] 1 illustrates another example of an imaging system. [Figure 12] 1 illustrates another example of an imaging system. [Figure 13] 1 illustrates another example of an imaging system. [Figure 14] 1 illustrates another example of an imaging system. [Figure 15] 1 illustrates another example of an imaging system. [Figure 16] 1 illustrates another example of an imaging system. [Figure 17] 1 illustrates another example of an imaging system. [Figure 18] 1 illustrates another example of an imaging system. [Figure 19] 4 shows another example of a double-sided board. [Figure 20] 1 illustrates an embodiment of an autofocus system. [Figure 21] 1 illustrates one embodiment of reflections from an off-axis narrow illumination beam. [Figure 22] 13 shows an embodiment of spots from reflection based on different flow cell materials. [Figure 23]13 shows an embodiment of spots from reflection based on different flow cell materials. [Figure 24] 13 shows an embodiment of spots from reflection based on different flow cell materials. [Diagram 25] 13 is a composite image of an image from an embodiment focused on two reflective spots. [Figure 26] 1 is an embodiment of a pixel group for an autofocus algorithm. [Figure 27] 13 shows a graph of an embodiment of moving a spot across two pixel groups. [Figure 28] 13 shows a graph of an embodiment of sum and difference light from pixel groups as focus changes. [Figure 29] 1 illustrates a graph of one embodiment of an autofocus signal. [Diagram 30] 1 shows a heat map of the surface height of one embodiment of a flow cell. [Diagram 31] 1 shows a heat map of the thickness of one embodiment of a flow cell. [Diagram 32] 1 shows a chart of delay times for one embodiment of a detector. [Diagram 33] 13 shows a chart used to calculate autofocus error during scanning of an embodiment of a surface of a flow cell. [Diagram 34] 13 shows a chart used to calculate autofocus error during scanning of an embodiment of a surface of a flow cell. [Diagram 35] 1 shows a series of images of one embodiment of a surface during a focal sweep. [Diagram 36] 13 is a chart of one embodiment of focus scores during a focus sweep. [Figure 37] A series of images of a surface during a focus sweep are shown. [Figure 38] 1 is a flow chart of one embodiment of a process for imaging a flow cell. [Figure 39] 1 is a flow chart of an embodiment of a process for autofocus of a flow cell surface. [Diagram 40]FIG. 1 illustrates a block diagram of one embodiment of a computer system. [Diagram 41] 2 illustrates a schematic diagram of another example of an imaging system. [Diagram 42] 2 illustrates a schematic diagram of another example of an imaging system. [Diagram 43] 2 illustrates a schematic diagram of another example of an imaging system.

[0067] All of the drawings are not to scale and, where necessary, reference numbers are repeated among the drawings to indicate corresponding elements. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0068] (Flow cell) FIG. 1 a shows an example of a flow cell 100 . The flow cell 100 includes a first substrate 102 and a second substrate 104 positioned such that a first surface 106 of the first substrate 102 faces a second surface 108 of the second substrate 104. The surfaces 106, 108 are spaced apart and define a fluid passageway 100 between them.

[0069] The surfaces 106, 108 are configured to receive an analyte 112 for analysis. The analyte 112 may be a nucleic acid material, such as DNA or RNA, to be sequenced, or other biological or non-biological / synthetic material to be analyzed. In one particular example, the analyte 112 may be DNA nanoballs or other individual nucleic acid samples to be sequenced or analyzed. The analyte 112 may be partially or entirely disposed on the first and second surfaces 106, 108 in a spaced apart array of discrete units. Although not shown, the surfaces 106, 108 may include an array of separate attachment sites spaced apart from one another, such that each analyte unit 112 may be held apart from adjacent analyte units 112. For purposes of illustration, the figures show only a small number of discrete analyte 112 sites, but it should be understood that the array may include up to millions or billions of discrete analyte sites spaced apart at a pitch that may be on the order of tens or hundreds of nanometers.

[0070] The flow cell 100 is configured to allow reagents and other fluids to be flowed through a fluid passageway 110 to perform a sequencing or other reaction on the analyte 112. In one example, during a sequencing reaction, fluorescently tagged molecules can selectively bind to portions of the analyte 112. As described in more detail below, an optical imaging system can be used to stimulate and detect fluorescent emissions from the tagged analyte 112 to generate sequencing or other data related to the analyte 112.

[0071] The substrates 102, 104 of the flow cell 100 of FIG. 1a can be fabricated from a material or materials that are substantially transparent to the emission wavelengths used to stimulate emission from the tagged analytes 112 and are also substantially transparent to the emission wavelengths of emission from the tagged analyte 112 sites. The substrates 102, 104 can also be a material or materials that do not generate substantial emission in the wavelength range of stimulated emission from the tagged analyte 112 sites (e.g., by fluorescence of the substrate material itself or by inelastic photon scattering processes such as Raman scattering). As used in this paragraph, "substantially" and "substantial" refer to a level that interferes with the stimulation and / or detection of emission from the tagged analyte 112 sites. In one embodiment, the substrates 102, 104 are both glass or other suitable optically transparent material.

[0072] In this example, analytes 112 (e.g., DNA nanoballs or other discrete nucleic acid analytes) on the inner surface of the substrate 102, 104 bind to discrete sites arranged in an array on the inner surface of the substrate 102, 104. These binding sites can be created by well-known lithography tools, such as 248 nm KrF (krypton fluoride), 193 nm ArF (argon fluoride) lithography systems, or electron beam lithography systems. The arrays are typically spaced apart from one another at ultra-high density, high density, medium density, or low density. At ultra-high density, the separation is less than 250 nm. At high density, the separation is in the range of 300-350 nm. At medium density, the separation is in the range of 400 nm-500 nm. At low density, the separation is 500 nm or greater. In some implementations (e.g., some low density implementations), two-dimensional patterning with photoresist is sufficient to isolate the DNA nanoballs or other discrete nucleic acid samples. In some implementations (e.g., some medium-density, high-density, or ultra-high-density implementations), smaller samples may be needed to reduce the risk of discrete samples not remaining in a single location, and three-dimensional patterning may be required for more efficient capture of fluorescence from tagged DNA nanoballs or other tagged nucleic acid samples. In such implementations, three-dimensional patterned well nanostructures can be developed with non-binding materials as the well walls and binding materials for the well bottoms to isolate DNA nanoballs.

[0073] The flow cell 100 of Figure 1a is optically symmetric: both substrates 102, 104 are of substantially the same thickness, material, shape, and are otherwise identical or nearly identical.

[0074] 1b shows the flow cell 100 held by a holder 114. The holder 114 contacts the flow cell 100 around the edges / perimeter of the flow cell so that the flow cell 100 can be imaged through both substrates 102, 104 without interference from the holder 114.

[0075] Figures 1c and 1d show another example of a holder 150 for holding a flow cell 160. As shown in Figures 1c and 1d, the holder 150 holds the flow cell 160 by its edge, leaving the substrate of the flow cell uncovered so that imaging can be done through the substrate.

[0076] FIG. 1e shows another example of a flow cell 180. The flow cell 180 includes a first substrate 102 with a surface 106 configured to receive an analyte 112 for analysis, and a second substrate 182 with a surface 108 configured to receive an analyte 112 for analysis. Unlike the flow cell 100 of FIG. 1a, the flow cell 180 of FIG. 1e is not optically symmetric. In FIG. 1e, the substrate 102 is optically transparent, and the substrate 182 is made of a different, non-optically transparent material and is also thicker. In the particular example of FIG. 1e, the substrate 102 is SiO2 and the substrate 108 is Si.

[0077] (Flow cell - Example of manufacturing method) Typically, a substrate with an array of attachment sites can be diced from a full 8-inch wafer, for example by laser dicing or saw dicing. For glass substrates, such as substrates 102, 104 of flow cell 100 of FIG. 1a, the dicing tolerance from the whole wafer can be ±50 μm in both xy plane directions. For the symmetric flow cell 100 of FIG. 1a, the diced bottom glass substrate 104 can have four drill holes and two top and bottom trenches as fluid inlet and outlet ports, while the diced top glass substrate 102 has no drill holes and no trenches.

[0078] The two substrates of the flow cell 100 can be supported by adhesive materials, such as UV / Visible light curable adhesive mixed with polystyrene beads with specific sizes, or pressure sensitive adhesive (PSA) with specific thickness to define the spacing structure. The UV / Visible light curable adhesive can be applied as multiple adhesive dots, lines, or specific channel structures on one of the glass surfaces to bond the upper and lower glass substrates 102, 104. Another approach is to use a pre-cut PSA tape with a predetermined channel shape to bond the two glass substrates 102, 104.

[0079] During the manufacturing of the flow cell 100, the bottom glass substrate can be held by a vacuum pre-assembled chuck aligned by alignment pins, and the UV adhesive can be applied onto the surface 108 of the bottom glass substrate 104 using an automated adhesive application system. A designed application program can be executed to apply the adhesive with beads onto the surface in a desired pattern. The top glass substrate 102 can be lifted by a vacuum weight with ball-shaped pins and placed onto the adhesive pattern by aligning the pre-assembled chuck holes to ensure full contact with the adhesive on the bottom glass substrate 104. A uniform downward force from the weight can be applied to the glass substrate until the adhesive is fully cured by exposure to UV light. The curing time can vary depending on the curing requirements of the adhesive. The weight applied to the glass substrates 102, 104 can be a uniform load to ensure uniformity of the flow cell fluid passage gap, while the size of the spacer used can define a specific flow cell gap or spacing. In general, the gap height of a flow cell can be defined as the distance between the top and bottom glass surfaces measured perpendicular to the plane of the flow cell. The gap height is, in one example, about 50 μm, with a tolerance of ±5 μm.

[0080] In another example, the fabrication of the flow cell can use a pre-cut channel pressure sensitive adhesive (PSA) tape as the adhesive material. The thickness of the PSA tape can act as a mechanical spacer that defines the gap height of the flow cell. The pre-cut channel PSA tape can be applied to the surface 106 of the top glass substrate 102. The top glass substrate 102 with the pre-cut channel PSA tape attached can then be lifted by a vacuum weight and placed on the surface 108 of the bottom glass substrate 104. The weight can remain on the glass substrate for a desired time to ensure that the glass substrate is in full contact with the PSA tape.

[0081] In both of the above mentioned manufacturing techniques, there may be a placement tolerance of 100-200 μm between the top glass substrate 102 and the bottom substrate 104 in the flow cell 100 .

[0082] (Fig. 2 Imaging system) Figure 2 illustrates generally one example of an optical imaging system 200. In the example of Figure 2, radiation from a radiation source 204 is directed towards the flow cell 100 and stimulates emission from a tag attached to a portion of the specimen site 112. The emission is imaged by a detector 222. The optical imaging system 200 is controlled by a controller 226, which may be one or more computers or other devices configured to control the various components of the system 200 and process data collected by the various components of the system.

[0083] In the example of Figure 2, the radiation source 204 is a laser configured to emit laser light that stimulates fluorescent emission by the fluorescently tagged analytes 112 in the flow cell 100. The laser light from the laser 204 passes through conditioning optics 206 and is guided through optics 208 and objective lens 210 to the flow cell 100. The directing optics 208 may be a dichroic beam splitter or other optical components configured to reflect wavelengths of light from the radiation source 204 while allowing other wavelengths of light (including fluorescent emission from the analytes 112) to pass through the directing optics 208 and reach the detector 222 along optical path 228. Although Figure 2 shows only a single radiation source 204 for stimulating fluorescent emission by the analytes 112, additional radiation sources operating at different wavelengths may be included along with additional conditioning and directing optics for these additional radiation sources.

[0084] The XY stage 202 translates the flow cell 100 in the x and y directions (perpendicular to the optical axis 210 a of the objective lens 210 ) so that a laser beam from a radiation source 204 can be scanned across the flow cell 100 .

[0085] 2, detector 222 images fluorescent emissions from analyte 112. Detector 222 may be any suitable camera or other device configured to image induced emissions from a tag attached to analyte 112. Detector 222 may include a charge-coupled device image sensor (CCD), a complementary metal-oxide semiconductor image sensor (CMOS), or other suitable image sensor. Detector 222 may be a time-delay integration (TDI) detector.

[0086] In the example of Figure 2, laser light directed at the flow cell can simultaneously stimulate fluorescent emission from tagged analytes attached to both surfaces 106, 108 of the flow cell 100. The imaging system 200 includes an autofocus subsystem that facilitates imaging of fluorescent emission from analytes on one surface of the flow cell 100 without undue interference from fluorescent emission from analytes on the other surface of the flow cell 100. In the example of Figure 2, the autofocus subsystem includes a radiation source 214 (e.g., an infrared laser), directing optics 218, 220, and a detector 216 that receives light reflected from the radiation source 214 by the surface of the flow cell 100. A controller 226 receives data from the detector 216 and, based on the data, drives the z translation stage 212 to translate the objective lens 210 in the z direction along the optical axis of the objective lens 210a.

[0087] (Fig. 3 Imaging system) 3 illustrates another example of an imaging system 300. In this example, the system includes an actuator 302 configured to change the orientation of a flow cell 100 within the imaging system 300. For example, the actuator 302 can be configured to flip the flow cell between an orientation in which the first substrate 102 is above the second substrate 104 (i.e., as shown in FIG. 4a) and an orientation in which the second substrate 104 is above the first substrate 102 (i.e., as shown in FIG. 4b).

[0088] When the flow cell is in the orientation shown in Figure 4a, the system 300 is configured to image radiation emitted from tagged analytes on the first surface 106 of the flow cell 100. In the orientation shown in Figure 4a, the objective lens 210 is focused on or near the first surface 106 of the flow cell 100 (or on the analytes 112 attached to the first surface 106), such that the detector 222 captures a focused image of stimulated emission by the tagged analytes on the first surface 106, but does not capture a fully focused image of stimulated emission by the tagged analytes on the second surface 108.

[0089] When the flow cell is in the orientation shown in Figure 4b, the system 300 is configured to image radiation emitted from tagged analytes on the second surface 108 of the flow cell 100. In the orientation shown in Figure 4b, the objective lens 210 is focused on or near the second surface 108 of the flow cell 100 such that the detector 222 captures an image of stimulated emission by tagged analytes on the second surface 108, but does not capture an image of stimulated emission by tagged analytes on the first surface 106.

[0090] Returning to FIG. 3, the actuator 302 is configured to reorient the flow cell between the orientation shown in FIG. 4a and the orientation shown in FIG. 4a that is flipped 180 degrees from the orientation shown in FIG. 4a. In the example shown in FIG. 3, the actuator 302 is a component of a flow cell transport device 304. The flow cell transport device 304 may be a robotic armature or other multi-degree-of-freedom device configured to reposition and reorient the flow cell 100. The flow cell transport device 304 includes a gripper 306 for gripping the flow cell 100 (or for gripping a flow cell holder such as the holder 114). The actuator 302 may be a rotary joint or other suitable mechanical link that allows the flow cell transport device 304 to reverse the orientation of the gripper 306.

[0091] In addition to being able to invert the flow cell 100, the flow cell transport device 304 is also configured to move the flow cell 100 between various stations. In Figure 3, the flow cell 100 is positioned at an imaging station, and the flow cell transport device 304 moves the flow cell 100 to other stations, such as stations 308, 310, where reagents are flowed through the flow cell 100 and other operations are performed to facilitate sequencing or other reactions with analytes, and at station 312, where the flow cell 100 can be temporarily held pending the availability of other stations.

[0092] (Fig. 3 Example of how the imaging system works) In one example of a method of operation of the imaging system 300 shown in Figure 3, the flow cell transport device 304 can position the flow cell 100 for imaging, with the flow cell 100 initially oriented as shown in Figure 4a, with the first surface 106 closer to the objective lens 210 than the second surface 108. Then, based on feedback from the autofocus subsystem, the system 300 focuses the objective lens 210 to image emissions from the fluorescently tagged analytes 112 on the first surface 106 of the flow cell 100, as shown in Figure 4a. Radiation from the radiation source 204 is then scanned across the analyte array 112 of the flow cell 100, while the detector 222 captures an image of stimulated emissions from the tagged analyte 112 sites on the first surface 106 of the flow cell 100.

[0093] The flow cell transport device 304 can then reorient the flow cell 100 to the orientation shown in Figure 4b, with the second surface 108 closer to the objective lens 210 than the first surface 106. Based on feedback from the autofocus subsystem, the system then focuses the objective lens 210 to image emissions from the fluorescently tagged analytes 112 on the second surface 108 of the flow cell 100, as shown in Figure 4b. Radiation from the radiation source 204 is then scanned across the analyte array 112 of the flow cell 100, while the detector 222 captures an image of the stimulated emissions from the tagged analytes on the second surface 108 of the flow cell 100.

[0094] The flow cell transport device 304 can then relocate the flow cell 100 to another station and position the new flow cell for imaging.

[0095] (Fig. 5 Imaging system) Figure 5 shows another example of an imaging system 500. In this example, the imaging system 500 includes two imagers for simultaneously imaging radiation emitted at both sides 106, 108 of the flow cell 100. The imaging system 500 of Figure 5 includes the same components as the imaging system 200 of Figure 2, and also includes an additional objective lens 510, a Z translation stage 512, a detector 522, detector optics 524, and autofocus components 514, 516, 518, 520.

[0096] In this example, the autofocus components 214, 216, 218, and 220 facilitate focusing the objective lens 210 onto the inner surface of the double-sided flow cell 100 closest to the objective lens 210, i.e., the first surface 106 of the first substrate 102. And, the focus components 514, 516, 518, and 520 facilitate focusing the objective lens 510 onto the inner surface of the double-sided flow cell 100 closest to the objective lens 510, i.e., the second surface 108 of the second substrate 104 (see FIG. 6). The system 500 can be configured such that the IR laser or other radiation generated by the radiation source 214 for the autofocus objective lens 210 does not interfere with the autofocusing of the other objective lens 510. For example, the geometry of each of the autofocus subsystems can be configured such that the IR laser or other radiation used for the autofocus objective lens 210 is not detected by or interferes with the detector 516 of the other autofocus objective lens, and vice versa. Alternatively, the autofocus subsystem used for objective lens 210 can be configured to operate at a different wavelength than the autofocus subsystem used for objective lens 510.

[0097] By focusing each objective lens on the surface of the flow cell closest to that objective lens, the imaging system 500 does not need to correct for variations in the thickness of the water gap in the double-sided flow cell 100 .

[0098] 5 and 6, the flow cell 100 is positioned between two objective lenses 210, 510, with the objective lens 210 facing the first substrate 102 of the flow cell 100 and the objective lens 510 facing the second substrate 104 of the flow cell 100. Also as shown in Figures 5 and 6, the optical axes 210a of the objective lenses 210, 510 are collinear.

[0099] 5, radiation from the radiation source 204 simultaneously stimulates emission from tagged analytes 112 on both surfaces 106, 108 of the flow cell 100. In the particular example shown, a single laser beam is scanned across the flow cell along the x and y axes using the x / y stage 202 to simultaneously stimulate emission from tagged analytes 112 on both surfaces 106, 108 at the location of the laser beam.

[0100] As the laser beam is scanned across flow cell 100, system 500 simultaneously images radiation emitted from tagged analytes on surface 106 and radiation emitted from tagged analytes on surface 108. System 500 uses objective lens 210, optics 224 and detector 222 to image radiation emitted from tagged analytes on surface 106. System 500 uses objective lens 510, optics 524 and detector 522 to image radiation emitted from tagged analytes on surface 108.

[0101] (Fig. 5 Example of how the imaging system works) In one example of a method of operation of the imaging system 500 shown in FIG. 5, the flow cell 100 can be positioned between the objective lenses 210, 510 for imaging. Then, based on feedback from the autofocus subsystem, the system 500 focuses the objective lens 210 to image emissions from the fluorescently tagged analytes on the first surface 106 of the flow cell 100, and focuses the objective lens 510 to image emissions from the fluorescently tagged analytes on the second surface 108, as shown in FIG. 6. In this example, the radiation source 214, detector 216, and directing optics 218, 220 are used in collecting data to position the objective lens 210 for focused imaging of emissions from the fluorescently tagged analytes on the first surface 106 of the flow cell. The radiation source 514, detector 516, and directing optics 518, 520 are then used in collecting data to position the objective lens 510 for focused imaging of emissions from the fluorescently tagged analytes on the second surface 108 of the flow cell. Radiation from the radiation source 204 is then scanned across the analyte array 112 of the flow cell 100 while the detector 222 captures an image of the stimulated emission from the tagged analytes on the first surface 106 of the flow cell 100 and the detector 522 simultaneously acquires an image of the stimulated emission from the tagged analytes on the second surface 108 of the flow cell. As the radiation source 204 is scanned, the autofocus subsystem can adjust the focus of the objective lenses 210, 510 to account for variations in the thickness of the water gap between the first surface 106 and the second surface 108.

[0102] (Fig. 7 Imaging system) FIG. 7 shows another example of an imaging system 700 including two imaging subsystems for simultaneously imaging radiation emitted on both sides of a flow cell. The imaging system 700 of FIG. 7 includes a single radiation source 704 for simultaneously stimulating radiation from tagged analytes on both sides of the flow cell (e.g., surfaces 106, 108 of the flow cell 100 shown in FIG. 1a). There are separate optical subsystems for imaging each side of the flow cell. The objective lens 710a, the detector 722a, and the autofocus subsystem 730a are configured to image one of the surfaces of the flow cell (e.g., the first surface 106 of the flow cell 100 in FIG. 1a). And the objective lens 710b, the detector 722b, and the autofocus subsystem 730b are configured to image the other surface (e.g., the second surface 108 of the flow cell 100 in FIG. 1a).

[0103] Figures 8-10 show additional views of the imaging system 700 of Figure 7. Figure 8 shows the imaging system 700 in plan view along with a transport 740 for positioning the flow cell 100 between two objective lenses 710a, 710b. Figures 9 and 10 show enlarged views of the flow cell 100 in the holder 150 positioned between the two objective lenses 710a, 710b, with the optical axes of the two objective lenses aligned collinearly.

[0104] 11 and 12 show additional views of the transport 740 of the imaging system 700. FIG. 11 shows an x-stage 742 of the transport 40, and FIG. 12 shows both the x-stage 742 and the y-stage 744 of the transport 740. The x-stage 742 is configured to translate the flow cell 100 along an x-axis 746. The y-stage 744 is configured to translate the flow cell 100 along a y-axis 748. The transport 740 can be configured for both gross motion (bringing the flow cell 100 to a location between the two objective lenses of the imaging system 700) and fine motion (translating the flow cell 100 along the x-axis 746 and y-axis 748 to scan radiation across the flow cell 100).

[0105] 13-18 show the use of a transport 740 to bring a flow cell 100 to a position between two objective lenses of an imaging system 700. In FIG. 13, the flow cell 100 is positioned in a holder 150 of the transport 740. In FIG. 14, the y-stage 744 is actuated to translate the flow cell 100 and holder 150 downward through a slot 750 extending through the x-stage 742 (see FIG. 11). In FIG. 15, the x-stage 742 is actuated to translate the flow cell 100 and holder 150 to the objective lenses of the imaging system 700, and in FIGS. 16 and 17, the y-stage 742 is actuated to translate the flow cell 100 and holder 150 back through the slot 750 in the x-stage 742 so that the flow cell 100 is positioned between the objective lenses of the imaging system 700.

[0106] (Fig.41 Imaging system) 41 shows another example of an imaging system 600. In this example, the objective lens 210 is an immersion objective lens, e.g., a water immersion objective lens. The imaging system 600 is configured such that radiation emitted at the first and second surfaces 106, 108 of the flow cell 180 can be imaged by the objective lens 210 through equal or substantially equal thicknesses of fluid, even though the two surfaces 106, 108 are separated by a fluid passage.

[0107] The distal end of the objective lens 210 is submerged in the fluid 602 (e.g., water) so that there is no air gap between the distal end of the objective lens 210 (or the distal lens of the objective lens 210) and the flow cell 180. The fluid can be held in a reservoir covering the top substrate of the flow cell 180. In the particular example shown, the walls of the holder 114 hold the fluid 602 in a reservoir on top of the first substrate 102. In one non-limiting example, the depth of the reservoir can be 200-500 micrometers deep, or about 350 micrometers deep. In other examples, the fluid can be held in a fixed space above the flow cell 108 in other ways or can be at a different depth. The imaging system 600 includes a fluid monitoring and delivery subsystem 606 configured to maintain the fluid 602 at a desired level.

[0108] In some implementations, the fluid 602 above the flow cell 180 may have the same or substantially the same optical properties (e.g., refractive index) as the fluid in the fluid passage between the first substrate 102 and the second substrate 182 of the flow cell 180. In some implementations, the fluid 602 above the flow cell 180 may be the same or substantially the same as the fluid in the fluid passage between the first substrate 102 and the second substrate 182 of the flow cell 180.

[0109] In this particular example, the distal end of the objective lens 210 becomes immersed in the fluid 602 during translation by the XY stage 202, which can cause undesirable turbulence in the fluid 602 and affect image quality. In some implementations, the objective lens 210 and other components of the system can be configured to reduce any turbulence in the fluid caused by the motion. For example, in some configurations, the objective lens 210 may include a flat distal surface to promote a more laminar flow of the fluid relative to the objective lens and reduce turbulence. In these or other configurations, the objective lens 210 (or at least the portion of the objective lens 210 that is immersed in the fluid 602) can be non-tapered (e.g., cylindrical) to promote a more laminar flow of the fluid relative to the objective lens and reduce turbulence.

[0110] In the imaging system 600 of FIG. 41, the z-translation stage 212 is configured to translate the objective lens 210 along the optical axis 210a to move the objective lens 210 from a location where the objective lens 210 is positioned to image radiation emitted from tagged analytes on the first surface of the flow cell 180 to a location where the objective lens 210 is positioned to image radiation emitted from tagged analytes on the second surface 108 of the flow cell 180. FIG. 42 shows the objective lens 210 positioned to image radiation emitted from tagged analytes on the first surface 106 of the flow cell 180. FIG. 43 shows the objective lens 210 positioned to image radiation emitted from tagged analytes on the second surface 108 of the flow cell 180.

[0111] The imaging system 600 is configured such that a vertical distance 604 ( FIG. 42 ) between a distal surface of the objective lens 210 and the first surface 106 when the objective lens 210 is positioned to image radiation emitted from tagged analytes on the first surface 106 is the same or substantially the same as a vertical distance 608 ( FIG. 43 ) between a distal surface of the objective lens 210 and the second surface 108 when the objective lens 210 is positioned to image radiation emitted from tagged analytes on the second surface 108. Furthermore, the vertical length of the optical path extending through the fluid (including the fluid 602 in the region above the flow cell 180 and the fluid in the fluid passages of the flow cell 180) and the vertical length of the optical path extending through the flow cell substrate 102 are the same for the two objective lens positions shown in FIG. 42 and FIG. 43. In FIG. 42 , the vertical segment of the optical path passing through the fluid is labeled d1. In FIG. 43, vertical segments of the optical path that travel through the fluid (including the fluid 602 above the flow cell 180 and the fluid within the fluid passages of the flow cell 180) are labeled d2 and d3, respectively.

[0112] The imaging system 600 is configured such that d1 (when the objective lens 210 is positioned to image radiation emitted at the first surface 106) is equal to or approximately equal to d2+d3 (when the objective lens 210 is positioned to image radiation emitted at the second surface 108). When the objective lens 210 is repositioned to image radiation emitted at the second surface 108, the objective lens 210 is translated by the z translation stage 212 deeper into the fluid 602 a distance equal to or approximately equal to d3 (the height of the fluid passage in the flow cell 180).

[0113] (Equivalent Optical Path) The imaging system 600 of Figures 41-43 is configured such that the optical path along the optical axis of the objective lens 210a between the distal end of the objective lens 210 and the surface of the flow cell 180 being imaged is equal or substantially equal regardless of whether radiation emitted at the first surface 106 or the second surface 108 is being imaged. The optical path extends vertically through the same thickness of fluid and substrate regardless of whether the objective lens 210 is positioned to image radiation emitted at the first surface 106 (Figure 42) or the second surface 108 (Figure 43). Thus, an optical system including a high numerical aperture (NA) objective lens, or other optical system designed specifically for the particular imaging condition, can be used to image radiation emitted at two different surfaces of the flow cell 180 without introducing undesirable aberrations or otherwise adversely affecting imaging quality. The systems 300, 500 of Figures 3 and 5 also have equal optical paths between the two surfaces being imaged. In these examples, the flow cell 100 is optically symmetric (both substrates 102, 104 are the same or substantially the same material and thickness) and the optical path between the distal end of the objective lens and the surface being imaged is otherwise equivalent.

[0114] (Figure 41 Example of how the imaging system works) In one example of a method of operation of the imaging system 600, shown in Figure 41, the flow cell 180 can be positioned for imaging and the fluid monitoring / delivery subsystem 606 ensures that the fluid 602 sufficiently fills the reservoir above the flow cell 180. Then, based on feedback from the autofocus subsystem, the system 600 focuses the objective lens 210 to image emissions from the fluorescently tagged analytes 112 on the first surface 106 of the flow cell 180, as shown in Figure 42. Radiation from the radiation source 204 is then scanned across the analyte 112 array of the flow cell 180 while the detector 222 captures an image of the stimulated emissions from the tagged analyte 112 sites on the first surface 106 of the flow cell 180.

[0115] The z-translation stage 212 can then vertically translate the objective lens 210 downward a distance equal to or substantially equal to the height of the fluid passage of the flow cell 180 (50 microns in one non-limiting example). Then, based on feedback from the autofocus subsystem, the system focuses the objective lens 210 to image emissions from the fluorescently tagged analytes 112 on the second surface 108 of the flow cell 180, as shown in Figure 43. Radiation from the radiation source 204 is then scanned across the analyte 112 array of the flow cell 180 while the detector 222 captures an image of the stimulated emissions from the tagged analytes on the second surface 108 of the flow cell 180.

[0116] (Other double-sided boards) 1a, previously described, illustrates an example of a flow cell 100 including a first substrate 102 and a second substrate 104 positioned such that a first inner surface 106 of the first substrate 102 faces and is spaced apart from a second inner surface 108 of the second substrate 104 to define a fluid passageway 100 between the surfaces. The inner surfaces 106, 108 are configured to receive a sample 112 for analysis.

[0117] 19 shows another example of a double-sided substrate 900 in which the two surfaces configured to receive analytes 112 for analysis are exterior surfaces 906, 908 rather than interior surfaces. The double-sided substrate can be formed by first forming binding sites for the analytes 112 on the surfaces 906, 908 of two separate substrates 902, 904, respectively, and then bonding the two substrates 902, 904 together such that the surfaces 906, 908 with the binding sites formed for the analytes 112 are on the exterior of the double-sided substrate.

[0118] These binding sites for the analytes 112 can be created by well-known lithography tools, such as 248 nm KrF (krypton fluoride) lithography systems, 193 nm ArF (argon fluoride) lithography systems, or electron beam lithography systems. The arrays are typically spaced apart from one another at ultra-high density, high density, medium density, or low density. At ultra-high density, the separation is less than 250 nm. At high density, the separation is in the range of 300-350 nm. At medium density, the separation is in the range of 400 nm-500 nm. At low density, the separation is 500 nm or greater. In some implementations (e.g., some low density implementations), two-dimensional patterning with photoresist is sufficient to isolate DNA nanoballs or other discrete nucleic acid samples. In some implementations (e.g., some medium-density, high-density, or ultra-high-density implementations), smaller samples may be required to mitigate the risk of discrete samples not remaining in a single location, and three-dimensional patterning may be required for more efficient capture of fluorescence from tagged DNA nanoballs or other tagged nucleic acid samples. In such implementations, three-dimensional patterned well nanostructures can be developed with non-binding material as the well walls and binding material for the well bottom to isolate the DNA nanoballs.

[0119] Unlike the flow cell 100 of FIG. 1a, the double-sided substrate 900 does not have a fluid passage between the two substrates, and the analyte 112 binding sites are on the outwardly facing surfaces 906, 908 of the substrates 902, 904. Sequencing or other reactions can be performed on the analytes 112 on the double-sided substrate 900 by successively dipping or immersing the double-sided substrate 900 in reagents and other fluids. In one example, during a sequencing reaction, fluorescently tagged molecules can selectively bind to portions of the analytes 112 on the surfaces 906, 908 of the double-sided substrate 900. Sequencing reactions can be performed on the double-sided substrate 900, the same as or similar to the immersion reaction protocols described in U.S. Patent Publication No. 2020 / 00318177 (published October 8, 2020, Yang et al.).

[0120] The double-sided substrate 900 can be imaged by the imaging system described above. For example, in the imaging system 300 shown in FIG. 3, the flow cell transport device 304 can position the double-sided substrate 900 for imaging, where the double-sided substrate 900 is initially oriented such that the first surface 906 is closer to the objective lens 210 than the second surface 908. Then, based on feedback from the autofocus subsystem, the system 300 focuses the objective lens 210 to image the emission from the fluorescently tagged analytes 112 on the first surface 906 of the double-sided substrate 900. Then, radiation from the radiation source 204 is scanned across the array of analytes 112 on the first surface 906, while the detector 222 captures an image of the stimulated emission from the tagged analyte 112 sites. Then, the flow cell transport device 304 can reorient the double-sided substrate 900 such that the second surface 908 is closer to the objective lens 210 than the first surface 906. Based on feedback from the autofocus subsystem, the system then focuses the objective lens 210 to image the emission from the fluorescently tagged analytes 112 on the second surface 908. Radiation from the radiation source 204 is then scanned across the array of analytes 112 on the second surface 908 while the detector 222 captures an image of the stimulated emission from the tagged analytes on the second surface 908. The transport device 304 can then reposition the double-sided substrate 900 to another station and position a new double-sided substrate 900 for imaging.

[0121] As another example, in the imaging system 500 of FIG. 5, the double-sided substrate 900 can be positioned between the objective lenses 210, 510 for imaging. Then, based on feedback from the autofocus subsystem, the system 500 focuses the objective lens 210 to image the emission from the fluorescently tagged analytes on the first surface 906 of the double-sided substrate 900, and focuses the objective lens 510 to image the emission from the fluorescently tagged analytes on the second surface 908. In this example, the radiation source 214, the detector 216, and the directing optics 218, 220 are used in collecting data to position the objective lens 210 for focused imaging of the emission from the fluorescently tagged analytes on the first surface 906. And, the radiation source 514, the detector 516, and the directing optics 518, 520 are used in collecting data to position the objective lens 510 for focused imaging of the emission from the fluorescently tagged analytes on the second surface 908. Radiation from the radiation source 204 or multiple radiation sources (e.g., one radiation source configured to stimulate emission of tagged analytes 112 on the first surface 906 and a second radiation source configured to stimulate emission of tagged analytes 112 on the second surface 908) is then scanned across the analyte 112 array while the detector 222 captures an image of stimulated emission from the tagged analytes on the first surface 906 of the flow cell and the detector 522 simultaneously captures an image of stimulated emission from the tagged analytes on the second surface 908 of the flow cell. As the radiation source 204 is scanned, the autofocus subsystem can adjust the focus of the objective lenses 210, 510 to account for variations in the first and second surfaces 906, 908.

[0122] (Autofocus System) The objective lens used to focus light from the flow cell onto the detector (e.g., objective lens 210 in FIG. 2) often has a narrow depth of focus (e.g., on the order of ¼ μm). The thickness of the water gap in the flow cell may vary from flow cell to flow cell across the area of ​​each flow cell, and the variation is greater than the depth of focus of the objective lens. In some embodiments, it is desirable to have a system that can focus on one or both sides of a double-sided flow cell without being affected by reflections from other surfaces in order to obtain acceptable image quality of analytes on one or both sides of a double-sided flow cell.

[0123] In some configurations, the sample (e.g., analyte) is illuminated with an off-axis beam with a small diameter. Illuminating the sample with a small off-axis beam is an arrangement that can provide good sensitivity and / or allow isolation of reflections from the surface of the flow cell. An array detector can be used to track spots (e.g., spots of analyte and / or reflections). By appropriately defining pixel blocks, the desired spots can be isolated and used to control where the system thinks the "focus" is located. The use of summation and / or difference algorithms allows for fast and / or efficient generation of a focus error signal (e.g., this approach is numerically faster than measuring spot centroids).

[0124] (Fig.20 Autofocus system) 20, an embodiment of a system 2000 for auto-focusing a flow cell is shown. System 2000 includes a stage 2004 configured to hold a carrier 2008 (e.g., a flow cell), a light source 2012 configured to emit a light beam 2016 that illuminates a specimen on the carrier 2008 (light beam 2016 characterized by an optical path 2020), a detector 2024, and a lens system 2028 characterized by an optical axis 2030.

[0125] The carrier 2008 is configured to be mounted on a stage while the specimen is illuminated by the light beam 2016. The stage 2004 is configured to move the carrier 2008 in the X / Y plane. The stage 2004 is also configured to move the carrier 2008 along a Z-axis (also referred to as a Z-stage), in this embodiment, which is defined parallel to the optical axis 2030 of the lens system 2028. Controlling the movement along the Z-axis changes the distance between the lens system 2028 and the carrier 2008. In some embodiments, the stage 2004 moves the carrier 2008 in the X / Y plane (e.g., the XY stage 202 in FIG. 2) and uses another stage (e.g., the Z translation stage 212 in FIG. 2) to control the distance between the lens system 2028 and the carrier 2008.

[0126] The light source 2012 is a laser (e.g., an infrared laser). The light source 2012 is configured to illuminate the specimen on the carrier 2008 by generating a light beam 2016. The light beam 2016 is configured to propagate through a lens system 2028 and illuminate the specimen on the carrier 2008.

[0127] The detector 2024 (e.g., an array detector) is configured to detect light. In some embodiments, the detector 2024 comprises a TDI (time delay integration) sensor. The lens system 2028 is configured to focus light from the specimen onto the detector 2024 after the light beam 2020 illuminates the specimen. A first pixel block and a second pixel block of the detector 2024 are defined. A first light intensity is calculated based on the light detected by the first pixel block of the detector 2024. A second light intensity is calculated based on the light detected by the second pixel block of the detector 2024. The first light intensity is compared to the second light intensity to generate a focus error signal. An analog output 2038 is generated based on the focus error signal and provided to the controller 2040. The controller 2040 is configured to change the distance between the carrier 2008 and the lens system 2028 by sending a control signal to the stage 2004 to move the carrier 2008 in the Z direction. The focus of the light on the detector 2024 is adjusted by moving the carrier 2008 in the Z direction or by moving the lens system 2028 in the Z direction.

[0128] (Figure 21 Narrow beam reflection from a flow cell) In some imaging systems, light from a light source fills half the pupil of a lens system. In these systems, reflections from various surfaces are mixed at a detector. Figure 21 shows one embodiment of reflections from an illumination beam 2104, where reflections from surfaces can be distinguished. Instead of filling half the pupil, the illumination beam 2104 in Figure 21 is narrow and provides off-axis illumination.

[0129] FIG. 21 shows a coverslip 2108 and a flow layer 2112. The coverslip 2108 includes a top surface 2120 and a bottom surface 2124. The flow layer 2112 is between the bottom surface 2124 of the coverslip 2108 and a substrate 2128 (e.g., a silicon or glass substrate). FIG. 21 also shows an optical axis 2030 of the lens system 2028 of FIG. 20. The incident beam 2104 propagates along an optical path 2132. The optical path 2132 is incident on the top surface 2120 of the coverslip 2108 at an angle theta (θ) relative to the optical axis 2030.

[0130] The incident beam 2104 has a beam width w. The beam width w is narrow compared to the pupil of a lens system (e.g., lens system 2028 of FIG. 20). In some embodiments, the beam width w (e.g., as measured at a first entrance surface, such as top surface 2120 of FIG. 21) is no more than ⅓, ¼, or ⅙ of the diameter of the entrance pupil.

[0131] The incident beam 2104 produces a first reflection 2131 from the top surface 2120, a second reflection 2132 from the bottom surface 2124, and a third reflection 2133 from the substrate 2128. Because the incident beam 2104 has a narrow width w, the spots formed by the first reflection 2131, the second reflection 2132, and the third reflection 2133 do not overlap and appear as separate and distinct spots on a detector (e.g., on detector 2024 in FIG. 20).

[0132] The incident beam 2104 is incident on one side of an objective lens (e.g., lens system 2028 in FIG. 20). The reflected light, after passing through the lens system, is incident on a detector. The location and / or movement of the spot on the detector provides information about the focal position (e.g., in the z direction) of various surfaces. The algorithm is used by one or more processors (e.g., processor 2034 in FIG. 20) to generate an analog output signal, which is used as feedback for the Z-stage motion control electronics.

[0133] (Figures 22-24 Light-focusing spots using various flow cell materials) 22-24 show embodiments of spots from reflections based on various flow cell materials. FIGS. 22-24 show simulations (ZEMAX) on the left and images on the right. The first spot 2204-1 (e.g., from the first reflection 2131 in FIG. 21) is from reflection on a first surface (e.g., top surface 2120 in FIG. 21). The second spot 2204-2 (e.g., from the second reflection 2132 in FIG. 21) is from reflection on a second surface (e.g., bottom surface 2124 in FIG. 21). The third spot 2204-3 (e.g., from the third reflection 2133 in FIG. 21) is from reflection on a third surface (e.g., substrate 2128 in FIG. 21).

[0134] In Fig. 22, there is an air gap between the second and third surfaces, and the second and third surfaces are glass. The three spots 2204 in Fig. 22 are of approximately equal brightness. The second spot 2204-2 and the third spot 2204-3 are closer to each other than the second spot 2204-2 to the first spot 2204-1 (e.g., because in Fig. 21 the thickness of the coverslip 2108 is greater than the thickness of the flow layer 2112).

[0135] 23, there is water between the second and third surfaces, which are glass. The second spot 2204-2 and the third spot 2204-3 are of lower intensity than the first spot 2204-1 due to the smaller difference in refractive index at these surfaces.

[0136] In Fig. 24, water is present between the second and third surfaces, the second surface being glass, and the third surface being silicon. The reflection from the silicon surface is much brighter than the reflection from the surface of the coverslip. The spacing between the first spot 2204-1 and the second spot 2204-2 is larger in Fig. 24 than in Fig. 23. This is because a thicker coverslip (250 μm thick) was used in Fig. 24 than in Fig. 23 (where a 170 μm coverslip was used).

[0137] The autofocus system can be configured to accommodate a variety of flow cell types, including those listed in Figures 22-24.

[0138] (Figures 25-29 Focusing using pixel groups) Figure 25 is a composite image (montage) of images from an embodiment focused on two reflective spots 2504. Figure 25 shows a first spot 2504-1 and a second spot 2504-2 moving from right to left past a focal position 2508 as the Z stage moves the carrier in the Z direction. The composite image includes eight slides 2512 numbered 1 through 8. The slides 2512 are from a focal sweep on a glass-glass flow cell with a Z step of 10 μm.

[0139] The first spot 2504-1 is from the substrate, the second spot 2504-2 is from the bottom surface of the coverslip, and the third spot 2504-3 is from the top surface of the coverslip, as seen on slides 2512-6, 2512-7, and 2512-8.

[0140] As the Z-stage moves up and down, the spot 2504 shifts on the detector (e.g., because the illumination beam is off-axis). The focus error can be measured based on the shift of the spot 2504 on the detector. Because the spots 2504 are independent, they can be focused on either surface of the flow cell without interference from the other. For example, the first spot 2504-1 on the substrate is focused on the third slide 2512-3. And the second spot 2504-2 on the bottom surface of the cover glass is focused on the sixth slide 2512-6.

[0141] FIG. 26 is an embodiment of a pixel group used in an autofocus algorithm. FIG. 26 shows a first spot 2604-1 and a second spot 2604-2 from a detector array. The first spot 2604-1 is due to reflection from the silicon substrate. The second spot 2604-2 is from the bottom surface of the cover slip. A first cell 2608-1 of a first pixel group is next to a second cell 2608-2 of a second pixel group. A first signal is generated by the sum of the light signals detected by the pixels in the first cell 2608-1. A second signal is generated by the sum of the light signals detected by the pixels in the second cell 2608-2. The first signal is compared to the second signal to generate an error signal.

[0142] The sum of the first and second signals is referred to as SUM. SUM provides information about whether there is enough light on the detector to measure focus (e.g., if SUM is equal to or greater than a predefined threshold, there is enough total light to run the focus algorithm). The subtraction of the second signal from the first signal is referred to as DIFF. DIFF provides a measure of the position of spot 2604 within cell 2608. Applicant has found that normalizing DIFF by SUM, such as AF signal=DIFF / SUM, helps generate an autofocus signal.

[0143] When the AF signal approaches zero, the spot 2604 approaches focus (i.e., the spot 2604 is on the boundary between the first cell 2608-1 and the second cell 2608-2). If the focus of the sample needs to be shifted, the cell 2608 can simply be shifted (e.g., left or right) as defined on the sensor. The width of the cell 2608 is designed to be wide enough to find the spot 2604 if it is slightly out of focus, but not so wide that it picks up light from the spot 2604 on other surfaces. In some embodiments, the width of the cell 2608 is less than or equal to half the distance between the centers of the spots 2604. The sensor shown is a two-dimensional sensor, but in some embodiments, a one-dimensional sensor is used (e.g., to provide a faster response) because the horizontal distribution of light is what changes the DIFF.

[0144] Figure 27 shows a graph of the signals from the cells as the spot moves across two pixel groups (e.g., cell 2608 in Figure 26). Figure 27 shows a first signal 2704-1 from the first cell (e.g., from light detected by the first cell 2608-1 in Figure 26 as the second spot 2604-2 moves from left to right) and a second signal 2704-2 from the second cell (e.g., from light detected by the second cell 2608-2 in Figure 26) as the Z stage moves up and down through the focus and the spot moves from one cell to the other.

[0145] Figure 28 shows an embodiment graph of the sum and difference of light from a group of pixels as focus changes. In Figure 28, a plot of the SUM and DIFF for a cell is shown. Note that the DIFF signal can have both positive and negative values.

[0146] 29 shows a graph of one embodiment of an auto focus (AF) signal 2904. The AF signal 2904 is calculated by dividing DIFF by SUM. The AF signal 2904 is a unitless number between -1 and +1. The AF signal 2904 provides the magnitude and direction of the focus error. When the AF signal 2904 is equal to zero (e.g., the spot is evenly between both cells 2608 in FIG. 26), the spot is in focus.

[0147] The AF signal and / or SUM are output to a Z-stage controller, for example a BrainBox (e.g., model ED-560) computer controlled output, or a custom electronic circuit that may include analog signal drivers.

[0148] (Fig. 30 to 31 Surface heat maps) Using the autofocus system, the water gap in the flow cell can be measured. The heat maps in Figures 30 and 31 cover an area of ​​60 x 60 mm on a T7 flow cell.

[0149] Figure 30 shows a heat map of the surface height of one embodiment of a flow cell. Figure 30 shows the height of the silicon surface of the flow cell. The height varies from +7 to -5 μm from the average.

[0150] FIG. 31 shows a heat map of thickness for one embodiment of a flow cell. A similar scan is performed on the surface of the cover slip of the flow cell. The Z position of the silicon surface is subtracted from the Z position of the cover slip to measure the water gap. FIG. 31 shows that the difference ΔZ is approximately equal to 29±4 μm. Correcting for refraction, the actual gap measurement ΔZ is ∼50±6 μm. This result is consistent with the quality control data for the flow cell. The gap variation is large compared to the depth of focus of the objective lens (e.g., lens system 2028 in FIG. 20).

[0151] (Fig. 32 to Fig. 34 Sensing lag) FIG. 32 shows a chart of the delay time of one embodiment of the detector as the Z stage moves. It was expected that there would be a delay in the implementation using a TDI camera and frame grabber. To measure the delay, the Z stage was driven up and down (changing the Z position) and the autofocus (AF) signal was recorded. The acquired data shows that the AF signal lags the actual Z stage movement by about 11 ms. With some algorithmic improvements, the delay was reduced to about 2 ms. To further reduce the delay, a one-dimensional sensor array and / or custom electronics can be used.

[0152] The ability of an AF system to remain in focus while scanning depends on (1) the flatness of the surface, and (2) the detector delay time. One way to measure tracking error is to scan back and forth along the surface (say, in the Y direction) and look at the Z delta.

[0153] Figures 33 and 34 show charts used to calculate the autofocus error during scanning of an embodiment of a surface of a flow cell. Figure 33 shows Y position vs. time, and Z position vs. time, as the autofocus tracks the surface. Figure 34 shows Z position vs. Y position at various scan speeds. The AF tracking error is half the vertical difference between two curves of the same color. For example, Blue (6 mm / s): error < 0.1 μm. Red (60 mm / s): error < 1 μm (too large in some embodiments).

[0154] (Fig. 35 to Fig. 37 Focal sweep) Figure 35 shows a series of images or slides 3504 of the surface of an embodiment of a coverslip during a focal sweep. The focal sweep is a coarse focus sweep to find the best focus setting. There are seven slides 3504 numbered 1 through 7. The images also show DNA nanoballs (DNBs).

[0155] Figure 36 is a chart of one embodiment of a focus score plot during a focus sweep. The horizontal axis corresponds to the slides from Figure 35. The first slide 3504-1 of Figure 35 corresponds to "-3" on the chart, and the seventh slide 3504-7 of Figure 35 corresponds to "3" on the chart. The location of the value "1" corresponds to the fifth slide 3504-5 of Figure 35, which is the sharpest focus state image.

[0156] Figure 37 shows a series of scanned images scanned at 30 mm / sec on a patterned coverslip surface, with the coverslip having DNB attached to it, the image in the middle is darker due to bleaching from the previous scan.

[0157] FIG. 38 Process for imaging the flow cell Figure 38 shows a flow chart of one embodiment of a process 3800 for imaging a flow cell. Process 3800 begins with step 3804 of illuminating a specimen with a light source. The specimen is on a carrier, and the carrier is mounted on a movable stage. The specimen is illuminated by transmitting a light beam of the light source through a lens system to the specimen. The light beam is characterized by an optical path. The lens system is characterized by an optical axis. The optical path of the light beam incident on the carrier is not parallel to the optical axis of the lens system (e.g., as shown in Figure 21).

[0158] In step 3808, the light from the specimen is focused onto the detector using a lens system. In step 3812, the light from the specimen is detected by the detector.

[0159] (Fig.39 Autofocus process) 39 shows a flow chart of one embodiment of a process 3900 for autofocus of a flow cell. The process 3900 begins with step 3904 of defining a first pixel block and a second pixel block on a detector. For example, the first cell 2608-1 and the second cell 2608-2 of FIG. 26 are defined on the sensor of the detector 2024 of FIG. 20.

[0160] In step 3908, the sample is illuminated with an off-axis light beam. Light from the sample is then detected by a detector after the light from the sample passes through a lens system (step 3912).

[0161] In step 3916, a first light intensity is calculated, a second light intensity is calculated, the first light intensity being calculated based on light detected by a first pixel block of the detector, and the second light intensity is calculated based on light detected by a second pixel block of the detector. The first light intensity is then compared to the second light intensity (e.g., calculating SUM and DIFF as described in Figures 26-29) to generate a focus error signal (e.g., to generate the correction signal of Figure 20, which is used to generate the control signal of Figure 20, and in some embodiments, the analog output 2038 of Figure 20 includes the AF signal from Figure 32) (step 3920).

[0162] In step 3924, the distance between the lens system and the specimen is adjusted based on the focus error signal. For example, the Z stage of FIG. 20 is moved closer and / or farther in the Z direction relative to the lens system 2028.

[0163] In some embodiments, the analyte is a first analyte and the method further comprises focusing on a second analyte on a second surface of the carrier, e.g., the first analyte is on the bottom surface of the coverslip and the second analyte is on a substrate (e.g., a glass or silicon substrate) of a flow cell or on the surface of a second coverslip.

[0164] (Fig.40 Computer Device) 40 is a simplified block diagram of a computing device 4000. The computing device 4000 can implement some or all of the functions, operations, and / or capabilities described above using electronic storage or processing, as well as other functions, operations, or capabilities not explicitly described. The computing device 4000 includes a processing subsystem 4002, a storage subsystem 4004, a user interface 4006, and / or a communication interface 4008. The computing device 4000 can also include other components (not explicitly shown), such as a battery, a power controller, and other components operable to provide various enhanced capabilities, etc. In various embodiments, the computing device 4000 may be implemented as a desktop or laptop computer, a mobile device (e.g., a tablet computer, a smartphone, a mobile phone), a wearable device, a media device, an application specific integrated circuit (ASIC), a digital signal processor (DSP), a digital signal processing device (DSPD), a programmable logic device (PLD), a field programmable gate array (FPGA), a processor, a controller, a microcontroller, a microprocessor, or any other electronic unit designed to perform any of the functions or combinations of functions described above.

[0165] The storage subsystem 4004 can be implemented using local storage and / or removable storage media, for example, disks, flash memory (e.g., secure digital cards, universal serial bus flash drives), or any other non-transitory storage media, or a combination thereof, and can include volatile and / or non-volatile storage media. The local storage can include random access memory (RAM), including dynamic RAM (DRAM), static RAM (SRAM), or battery-backed RAM. In some embodiments, the storage subsystem 4004 can store one or more applications and / or operating system programs executed by the processing subsystem 4002, including programs for implementing some or all of the above-described computer-implemented operations. For example, the storage subsystem 4004 can store one or more code modules 4010 for implementing one or more of the method steps described above.

[0166] A firmware and / or software implementation may be implemented using modules (e.g., procedures, functions, etc.). Any machine-readable medium tangibly embodied with instructions may be used in implementing the methodologies described herein. The code modules 4010 (e.g., instructions stored in a memory) may be implemented within a processor or external to the processor. The term "memory" as used herein may refer to long-term, short-term, volatile, non-volatile, or other types of storage media and is not limited to a particular type of memory, number of memories, or type of media on which memory is stored.

[0167] Additionally, the term "storage medium" or "storage device" may refer to one or more memories, including read-only memory (ROM), RAM, magnetic RAM, core memory, magnetic disk storage media, optical storage media, flash memory devices, and / or other machine-readable media for storing information. The term "machine-readable medium" includes, but is not limited to, portable or fixed storage devices, optical storage devices, wireless channels, and / or various other storage media capable of storing instructions and / or data.

[0168] Further, the embodiments can be implemented by hardware, software, scripting languages, firmware, middleware, microcode, hardware description languages, and / or any combination thereof. When implemented by software, firmware, middleware, scripting languages, and / or microcode, the program code or code segments for performing the tasks can be stored in a machine-readable medium, such as a storage medium. A code segment (e.g., code module 4010) or machine-executable instructions can represent a procedure, a function, a subprogram, a program, a routine, a subroutine, a module, a software package, a script, a class, or a combination of instructions, data structures, program statements. A code segment can be coupled to another code segment or a hardware circuit by passing and / or receiving information, data, arguments, parameters, and / or memory contents. Information, arguments, parameters, data, etc. can be passed, forwarded, or transmitted by any suitable means, such as memory sharing, message passing, token passing, network transmission, etc.

[0169] The implementation of the techniques, blocks, steps, and means described above can be done in various ways. For example, these techniques, blocks, steps, and means can be implemented in hardware, software, or a combination thereof. In the case of a hardware implementation, the processing unit can be implemented within one or more ASICs, DSPs, DSPDs, PLDs, FPGAs, processors, controllers, microcontrollers, microprocessors, other electronic units designed to perform the functions described above, and / or combinations thereof.

[0170] Each code module 4010 may comprise a set of instructions (code) embodied on a computer-readable medium that directs a processor of the computing device 4000 to perform a corresponding operation. The instructions can be configured to execute sequentially, in parallel (e.g., under different processing threads), or a combination thereof. After loading the code modules 4010 into a general-purpose computer system, the general-purpose computer is transformed into a special-purpose computer system.

[0171] A computer program incorporating various features described herein (e.g., one or more code modules 4010) can be encoded and stored on various computer-readable storage media. A computer-readable medium encoded with program code can be packaged with a compatible electronic device, or the program code can be provided separately from the electronic device (e.g., via Internet download or as a separately packaged computer-readable storage medium). The storage subsystem 4004 can store information useful for establishing a network connection using the communications interface 4008.

[0172] The user interface 4006 may include input devices (e.g., touch pad, touch screen, scroll wheel, click wheel, dials, buttons, switches, keypad, microphone, etc.) and output devices (e.g., video screen, indicator lights, speakers, headphone jack, virtual reality or augmented reality display, etc.) and supporting electronics (digital to analog or analog to digital converters, signal processors, etc.). A user can manipulate the input devices of the user interface 4006 to invoke functions of the computing device 4000, and can see and hear output from the computing device 4000 via the output devices of the user interface 4006. In some embodiments, the user interface 4006 may not be present (e.g., in the case of a process that uses an ASIC).

[0173] The processing subsystem 4002 can be implemented as one or more processors (e.g., an integrated circuit, one or more single-core or multi-core microprocessors, a microcontroller, a central processing unit, a graphics processing unit, etc.). In operation, the processing subsystem 4002 can control the operation of the computing device 4000. In some embodiments, the processing subsystem 4002 can execute various programs in response to program code and can maintain multiple simultaneously executing programs or processes. At a given time, some or all of the program code being executed can reside within the processing subsystem 4002 and / or within a storage medium, such as the storage subsystem 4004. Through programming, the processing subsystem 4002 can provide various functions to the computing device 4000. The processing subsystem 4002 can also execute other programs that control other functions of the computing device 4000, including programs that can be stored in the storage subsystem 4004.

[0174] The communication interface 4008 can provide voice and / or data communication capabilities for the computing device 4000. In some embodiments, the communication interface 4008 can include a radio frequency (RF) transceiver component for accessing wireless data networks (e.g., Wi-Fi networks, 3G, 4G / LTE, etc.), mobile communication technologies, short-range wireless communications (e.g., Bluetooth communication standard, NFC, etc.), other components, or a combination of technologies. In some embodiments, the communication interface 4008 can provide a wired connection (e.g., Universal Serial Bus, Ethernet, Universal Asynchronous Receiver / Transmitter, etc.) in addition to or instead of a wireless interface. The communication interface 4008 can be implemented using a combination of hardware (e.g., driver circuits, antennas, modulators / demodulators, encoders / decoders, and other analog and / or digital signal processing circuits) and software components. In some embodiments, the communication interface 4008 can support multiple communication channels simultaneously. In some embodiments, the communication interface 4008 is not used.

[0175] It will be understood that computing device 4000 is exemplary and that variations and modifications are possible, and that the computing device may have various functions not specifically described (e.g., voice communication over a cellular network) and may include appropriate components for such functions.

[0176] Additionally, while computing device 4000 is described with reference to particular blocks, it should be understood that these blocks are defined for convenience of description and are not intended to imply a particular physical arrangement of components. For example, processing subsystem 4002, storage subsystem 4004, user interface 4006, and / or communication interface 4008 can reside within one device or can be distributed across multiple devices.

[0177] Furthermore, the blocks need not correspond to physically separate components. The blocks can be configured to perform various operations, for example by programming a processor or providing appropriate control circuitry, and the various blocks may or may not be reconfigurable depending on how the initial configuration is obtained. Embodiments of the invention can be realized in a variety of apparatuses, including electronic devices implemented using a combination of circuitry and software. The electronic devices described herein can be implemented using a computing device 4000.

[0178] Various features described herein, e.g., methods, apparatus, computer-readable media, etc., can be implemented using a combination of dedicated components, programmable processors, and / or other programmable devices. The processes described herein can be implemented on the same processor or different processors. Where components are described as being configured to perform certain operations, such configuration can be achieved, for example, by designing electronic circuitry to perform the operations, by programming a programmable electronic circuit (such as a microprocessor) to perform the operations, or by a combination thereof. Additionally, while the above-described embodiments refer to specific hardware and software components, those skilled in the art will appreciate that different combinations of hardware and / or software components can also be used, and that certain operations described as being implemented in hardware can be implemented in software, and vice versa.

[0179] Specific details are given in the above description to provide an understanding of the embodiments. However, it will be understood that the embodiments can be practiced without these specific details. In some cases, well-known circuits, processes, algorithms, structures, and techniques may be shown without unnecessary detail to avoid obscuring the embodiments.

[0180] While the principles of the present disclosure have been described above in conjunction with specific devices and methods, it will be understood that this description is made merely as an example and is not intended to limit the scope of the present disclosure. The embodiments have been chosen and described in order to explain the principles and practical applications of the invention, so that others skilled in the art will be able to utilize the invention in various embodiments and with various modifications as may be suitable for the particular applications envisioned. It will be understood that this description is intended to cover modifications and equivalents.

[0181] It is also noted that the embodiments may be described as a process, which is depicted as a flowchart, a flow diagram, a data flow diagram, a structure diagram, or a block diagram. Although a flowchart may describe operations as a sequential process, many of the operations may be performed in parallel or simultaneously. In addition, the order of operations may be rearranged. A process is terminated when its operations are completed, but may include additional steps not included in the diagram. A process may correspond to a method, a function, a procedure, a subroutine, a subprogram, etc.

[0182] The terms "a," "an," or "the" are intended to mean "one or more," unless specifically stated to the contrary. All patents, patent applications, publications, and descriptions referred to herein are incorporated by reference in their entirety for all purposes. None are admitted to be prior art.

Claims

1. a stage configured to hold a carrier configured to receive the specimen; a light source configured to illuminate the specimen with a light beam, the light beam characterized by an optical path; a detector configured to detect light; a lens system configured to focus light from the specimen onto a detector after the light beam illuminates the specimen; A lens system is characterized by an optical axis, the light beam is configured to propagate through the lens system and illuminate the specimen; An imaging system in which the optical path of the light beam incident on the carrier is not parallel to the optical axis of the lens system.

2. The imaging system of claim 1 , further comprising a controller configured to vary the distance between the carrier and the lens system to focus light from the specimen onto the detector.

3. calculating a first light intensity based on light detected by a first pixel block of the detector; calculating a second light intensity based on the light detected by a second pixel block of the detector; comparing the first light intensity to the second light intensity to generate a focus error signal; controlling movement of the lens system relative to the carrier based on the focus error signal; The imaging system of claim 2 , further comprising one or more processors configured to:

4. 10. The imaging system of claim 1, wherein the detector is an array detector configured to focus a spot of light incident on the detector.

5. The imaging system of claim 4 , wherein the array detector is a one-dimensional array.

6. the carrier comprises a flow cell; 10. The imaging system of claim 1, wherein the flow cell comprises a first sample receiving surface and a second sample receiving surface separated by a width.

7. The imaging system of claim 6 , wherein the depth of focus of the lens system is less than the width.

8. 7. The imaging system of claim 6, wherein the stage is configured to move such that the lens system focuses light from the first sample receiving surface onto the detector and, but not simultaneously, focuses light from the second sample receiving surface onto the detector.

9. A light beam is defined by its beam width, the beamwidth is measured at the carrier; The imaging system of claim 1 , wherein the beam width is less than or equal to 2 mm and / or greater than or equal to 10 microns.

10. The imaging system of claim 6 , wherein the lens system comprises an immersion objective lens with a distal lens surface configured to be immersed in a fluid.

11. (a) in a first configuration, the system is configured to image radiation emitted from a specimen attached to the first specimen receiving surface; the distal lens surface is spaced a first perpendicular distance from the first sample receiving surface; the first vertical distance includes the fluid segment and the substrate segment; (b) in a second configuration, the system is configured to image radiation emitted from a specimen attached to the second specimen receiving surface; the distal lens surface is spaced a second perpendicular distance from the second sample receiving surface; the second vertical distance includes the fluid segment and the substrate segment; (c) the first perpendicular distance is substantially the same as the second perpendicular distance; The imaging system of claim 10 , wherein the fluid segment at the first perpendicular distance is substantially the same as the fluid segment at the second perpendicular distance.

12. 12. The imaging system of claim 11, wherein the immersion objective lens is at least partially immersed in a reservoir above the first sample receiving surface such that there is no air gap between the distal lens surface and the first sample receiving surface.

13. 13. The imaging system of claim 12, further comprising an x-y translation stage configured to translate one of the immersion objective or the flow cell relative to the other of the immersion objective or the flow cell while the immersion objective is at least partially immersed in the reservoir during imaging.

14. 14. The imaging system of claim 13, wherein the fluid in the reservoir has substantially the same refractive index as the fluid in the fluid passage of the flow cell between the first and second analyte receiving surfaces.

15. a stage configured to hold a carrier having a specimen; a light source configured to illuminate the specimen with a light beam; a detector configured to detect light; a lens system configured to focus light from the specimen onto a detector after the light beam illuminates the specimen; the carrier is configured to be mounted on a stage while the specimen is illuminated by a light beam; A light beam is characterized by an optical path, A lens system is characterized by an optical axis, the light beam is configured to propagate through the lens system and illuminate the specimen; An imaging system in which the optical path of the light beam incident on the carrier is not parallel to the optical axis of the lens system.

16. 16. The imaging system of claim 15, further comprising a controller configured to vary the distance between the carrier and the lens system to focus light from the specimen onto the detector.

17. calculating a first light intensity based on light detected by a first pixel block of the detector; calculating a second light intensity based on the light detected by a second pixel block of the detector; comparing the first light intensity to the second light intensity to generate a focus error signal; controlling movement of the lens system relative to the carrier based on the focus error signal; 17. The imaging system of claim 16, further comprising one or more processors configured to:

18. The imaging system of claim 15 , wherein the carrier is a flow cell.

19. 16. The imaging system of claim 15, wherein the detector is an array detector configured to focus a spot of light incident on the detector.

20. 20. The imaging system of claim 19, wherein the array detector is a one-dimensional array.

21. the carrier has a first surface and a second surface; 16. The imaging system of claim 15, wherein the first surface is separated from the second surface by a width.

22. 22. The imaging system of claim 21, wherein the depth of focus of the lens system is less than the width.

23. The specimen is a first specimen, a first analyte is attached to a first surface; 22. The imaging system of claim 21, wherein the second analyte is attached to a second surface.

24. 24. The imaging system of claim 23, wherein the stage is configured to move such that the lens system focuses light from a first specimen onto the detector and, but not simultaneously, focuses light from a second specimen onto the detector.

25. A light beam is defined by its beam width, The beam width is measured at the carrier, 16. The imaging system of claim 15, wherein the beam width is less than or equal to 2 mm and / or greater than or equal to 10 microns.

26. attaching a carrier to the stage, the carrier containing the specimen; illuminating the specimen with a light source by transmitting a light beam of the light source through a lens system to the specimen; A light beam is characterized by an optical path, A lens system is characterized by an optical axis, a step in which the optical path of the light beam incident on the carrier is not parallel to the optical axis of the lens system; using a lens system to focus light from the specimen onto a detector; and detecting light from the specimen with a detector.

27. 27. The imaging method of claim 26, further comprising controlling movement of the lens system relative to the carrier in a direction parallel to an optical axis of the lens system to focus light from the specimen onto a detector.

28. The step of controlling the movement of the lens system relative to the carrier includes: calculating a first light intensity, the first light intensity being calculated based on light detected by a first pixel block of the detector; calculating a second light intensity, the second light intensity being calculated based on light detected by a second pixel block of the detector; comparing the first light intensity and the second light intensity to generate a focus error signal; and controlling movement of the lens system relative to the carrier based on the focus error signal.

29. defining a first block of pixels of the detector; defining a second block of pixels of the detector; illuminating the specimen with an off-axis light beam; detecting light from the specimen using a detector after the light from the specimen passes through a lens system; calculating a first light intensity, the first light intensity being calculated based on light detected by a first pixel block; calculating a second light intensity, the second light intensity being calculated based on light detected by a second pixel block; comparing the first light intensity and the second light intensity to generate a focus error signal; and adjusting the distance between the lens system and the specimen based on the focus error signal.

Images