Illumination systems for nucleic acid sequencing

Abstract

The present disclosure describes illumination methods and systems for illumination and sequencing applications, which can be used, for example, for microscopes and sequencing platforms. The methods and systems of the present disclosure may provide large area, uniform illumination, which may reduce errors and improve system throughput.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202480021405.5 (22) Application Date 2024.01.24 (30) Priority Data 63 / 481,583 2023.01.25 US 63 / 489,150 2023.03.08 US (85) PCT International Application Entering National Phase Date 2025.09.23 (86) PCT International Application Application Data PCT / US2024 / 012802 2024.01.24 (87) PCT International Application Publication Data WO2024 / 158927 EN 2024.08.02 (71) Applicant: Element Biosciences, Inc. Address: California, USA (72) Inventors: R. Hudima, S.X. Chen, A. Gulbani, M. Previt, Y. Jiang, C. Jin, D. Fuller, C. Niemann, G. Pirand, D. Rong, G. Jem, J. Naismith (74) Patent Agency: King & Wood Mallesons, Beijing 11256 Patent Attorneys: Chen Wenping, Wang Beinan (51) Int.Cl. G02B 6 / 00 (2006.01) H04N 23 / 55 (2006.01) (54) Invention Title: Illumination System for Nucleic Acid Sequencing (57) Abstract: This disclosure describes an illumination method and system for illumination and sequencing applications, which can be used, for example, in microscopes and sequencing platforms. The methods and systems of this disclosure provide large-area, uniform illumination, which can reduce errors and improve system throughput. Claims 6 pages, Description 54 pages, Drawings 35 pages, CN 120936922 A 2025.11.11 CN 1 20 93 69 22 A 1. An optical system comprising: a stage configured to hold a solid support; a light source configured to illuminate the solid support; and an optical assembly at least partially disposed in an optical path from the stage to the light source, wherein the optical assembly is configured to provide illumination over an area of ​​the solid support greater than about 20 square millimeters (mm2), wherein the peak-to-valley variation is at most about 5%. 2. The optical system of claim 1, wherein the optical assembly does not include an objective lens. 3. The optical system of claim 2, wherein the optical system does not include the objective lens. 4. The optical system of claim 1, wherein the optical assembly does not include a barrel lens. 5. The optical system of claim 4, wherein the optical system does not include the barrel lens. 6. The optical system of claim 1, wherein the stage is not adjusted on the optical axis of the system.7. The optical system of claim 1, wherein the irradiance of the illumination is at least about 40 milliwatts per square millimeter. 8. The optical system of claim 1, wherein the optical component is configured to receive emitted light from the solid support. 9. The optical system of claim 8, wherein the numerical aperture (NA) of the optical component is at least about 0.3. 10. The optical system of claim 8, wherein the wavelength of the emitted light is from about 500 nanometers to about 750 nanometers. 11. The optical system of claim 1, wherein the working distance of the optical component is at least about 1 mm to 25 mm. 12. The optical system of claim 1, further comprising a motion coil housed within the optical component, the motion coil being configured to move a focusing element within the optical path of the optical system. 13. The optical system of claim 1, wherein a motor located outside the optical system is configured to move the focusing element along the optical axis in one or both directions. 14. The optical system of claim 13, wherein the motor is directly coupled to a portion of the first, second, or third housing of the optical component, and the portion of the first, second, or third housing of the optical component is directly coupled to the focusing element. 15. The optical system of claim 1, wherein the light source is a pulsed light source. 16. The optical system of claim 1, wherein the composite root mean square error of the optical system is less than about 0.05. 17. The optical system of claim 1, wherein the illumination efficiency of the optical component is at least about 90%. 18. The optical system of claim 1, wherein the region is greater than 30 mm². 19. The optical system of claim 1, wherein the region is greater than 50 mm² or 60 mm². 20. The optical system of claim 1, further comprising the solid support within the stage. 21. The optical system of claim 20, wherein the solid support comprises two or more surfaces having one or more samples imaged by the optical system fixed thereon. 22. The optical system of claim 21, wherein the solid support comprises three or more surfaces having one or more samples imaged by the optical system fixed thereon. (Claims 1 / 6, Page 2, CN 120936922 A) 23. The optical system of claim 22, wherein the three or more surfaces are axially displaced from each other at least along the optical axis of the optical system. 24. The optical system of claim 20, wherein the solid support comprises a probe configured to bind nucleic acid molecules.25. The optical system of claim 24, wherein the probe is attached to the surface of the solid support. 26. The optical system of claim 1, wherein the light source is a laser light source. 27. The optical system of claim 1, wherein the optical component includes a dichroic filter configured to transmit the illumination. 28. The optical system of claim 1, wherein the optical component includes: a first segment including a first housing including a first plurality of lenses; a second segment including a second housing; and a third segment including a third housing including a second plurality of lenses. 29. The optical system of claim 28, wherein the first segment and the third segment are optically aligned. 30. The optical system of claim 28, wherein the first segment is positioned between the third segment and the stage. 31. The optical system of claim 28, wherein the third segment is positioned between the first segment and the image sensor of the optical system. 32. The optical system of claim 28, wherein the first plurality of lenses are movable along an optical path ranging from about 0 to about 2 mm. 33. The optical system of claim 28, wherein the first plurality of lenses includes asymmetric biconvex lenses. 34. The optical system of claim 28, wherein the second plurality of lenses comprises an asymmetric biconcave lens. 35. The optical system of claim 34, wherein the asymmetric biconcave lens is an aspherical asymmetric biconcave lens. 36. The optical system of claim 1, wherein the optical system is configured to acquire an image of the solid support without moving an optical compensator into the optical path between the solid support and the detector of the optical system. 37. The optical system of claim 1, wherein the optical system is configured to acquire an image of the solid support without removing an optical compensator from the optical path between the sample and the detector of the optical system. 38. The optical system of claim 1, wherein the solid support is a flow cell. 39. The optical system of claim 1, wherein the optical component is configured to generate one or more spatial contractions transverse to the optical path of light traveling through it. 40. The optical system of claim 1, wherein the optical component is configured to generate one or more field curvature corrections transverse to the optical path of light traveling through it. 41. The optical system of claim 1, wherein the optical component is configured to generate at least one field curvature correction in a first, second, or third segment transverse to the optical path through which light travels. 42. A method for analyzing biomolecules, the method comprising: (a) providing a solid support comprising the biomolecules containing a marker;(b) Illuminating the biomolecule containing the marker using an optical system including a light source to generate signal light or a variation thereof, wherein the illumination is provided over an area of ​​the solid support greater than about 20 square millimeters (mm2), wherein the peak-to-valley variation is at most about 5%; Claims 2 / 6, page 3, CN 120936922 A (c) Detecting the signal light or the said variation thereof using a detector of the optical system; and (d) Processing the signal light or the said variation thereof at least partially to analyze the biomolecule. 43. The method of claim 42, wherein the biomolecule is a nucleic acid molecule, a protein, or a polypeptide. 44. The method of claim 43, wherein the biomolecule is a nucleic acid. 45. The method of claim 42, further comprising, prior to (a), binding the biomolecule to a probe bound to the solid support and conjugating the marker to the biomolecule. 46. The method of claim 42, wherein the marker is conjugated to the biomolecule by hybridization. 47. The method of claim 42, wherein the optical system does not include an objective lens. 48. The method of claim 42, wherein the solid support is not moved along the optical axis of the optical system. 49. The method of claim 48, wherein multiple images of the solid support are acquired without moving the solid support along the optical axis. 50. The method of claim 42, wherein the irradiance of the illumination is at least about 40 milliwatts per square millimeter. 51. The method of claim 42, wherein the wavelength of the signal light is from about 500 nanometers to about 750 nanometers. 52. The method of claim 42, wherein the detection in (c) is performed using an optical element with a numerical aperture of at least about 0.3. 53. The method of claim 42, further comprising, in (b), using a motion coil within the optical system to move a focusing element within the optical path of the optical system, thereby changing the focus of the optical system on the solid support. 54. The method of claim 42, wherein the light source is a pulsed light source. 55. The method of claim 42, wherein the illumination is provided with an efficiency of at least about 90%. 56. The method of claim 42, further comprising repeating (b)-(d) on additional biomolecules coupled to another surface of the solid support. 57. The method of claim 42, further comprising, after (c), removing the marker from the biomolecule. 58. The method of claim 57, further comprising repeating (a)-(d) on additional markers bound to another portion of the biomolecule.59. The method of claim 42, wherein the optical component is configured to generate one or more spatial contractions transverse to the optical path of light traveling through it. 60. The method of claim 42, wherein the optical component is configured to generate one or more field curvature corrections transverse to the optical path of light traveling through it. 61. The method of claim 42, wherein the optical component is configured to generate at least one field curvature correction transverse to the optical path of light traveling through it in a first, second, or third segment. 62. The method of claim 42, wherein (d) includes at least partially processing the signal light or its said variations to generate one or more solid support images, and analyzing the one or more solid support images to generate a base interpretation of the sample. 63. The method of claim 62, wherein each solid support image in the solid support images includes a field of view (FOV) greater than 20 square millimeters (mm²). 64. The method of claim 42, wherein the solid support is a flow cell. Claims 3 / 6, Page 4, CN 120936922, A 65. An optical system comprising: a stage configured to hold a solid support; a light source configured to illuminate the solid support; and a speckle eliminator optically coupled to the light source and disposed within an optical path from the light source to the stage. 66. The optical system of claim 65, further comprising an additional light source optically coupled to the speckle eliminator. 67. The optical system of claim 66, wherein light from the additional light source is configured to illuminate the solid support with light of a different wavelength than that of the light source. 68. The optical system of claim 66, wherein at least about four light sources are coupled to the speckle eliminator. 69. The optical system of claim 65, wherein the speckle eliminator is a vibrating speckle eliminator. 70. The optical system of claim 65, wherein the speckle eliminator is a passive speckle eliminator. 71. The optical system of claim 70, wherein the passive speckle eliminator comprises a diffuser plate. 72. The optical system of claim 65, wherein the speckle eliminator is a tension speckle eliminator. 73. The optical system of claim 65, wherein the speckle eliminator is configured to reduce speckle noise to at most about 5%. 74. The optical system of claim 65, wherein the solid support is a flow cell. 75. A method for analyzing biomolecules, the method comprising: (a) providing a solid support comprising a biological sample containing a marker; (b) illuminating the biological sample containing the marker using an optical system including a light source, thereby generating…(a) a signal light or a variation thereof, wherein the illumination is provided by a speckle eliminator in the optical path of the optical system; (b) detecting the signal light or the variation thereof using a detector of the optical system; and (d) at least partially processing the signal light or the variation thereof to analyze the biomolecule. 76. The method of claim 75, further comprising repeating (b)-(d) with an additional biological sample coupled to an additional surface of the solid support. 77. The method of claim 75, further comprising removing the marker from the biological sample after (c). 78. The method of claim 77, further comprising repeating (a)-(d) with an additional marker bound to the biological sample. 79. The method of claim 75, wherein the speckle eliminator uses vibration to perform speckle elimination on the illumination. 80. The method of claim 75, further comprising illuminating the solid support with an additional light source. 81. The method of claim 80, wherein the additional light source provides light of a different wavelength to the solid support. 82. The method of claim 80, wherein the additional light source is optically coupled to the speckle eliminator. 83. The method of claim 75, wherein the biological sample comprises a nucleic acid molecule, protein, or polypeptide. 84. The method of claim 83, wherein the biological sample comprises nucleic acid. 85. The optical system of claim 1, wherein the optical component is at least partially disposed within the optical path from the stage to the detector of the optical system. Claims 4 / 6 pages 5 CN 120936922 A 86. The optical system of claim 1, wherein the illumination system of the optical component is disposed within the optical path from the stage to the detector of the optical system. 87. A sample stage for holding a DNA sample for DNA sequencing reaction and imaging, the sample stage comprising: a stage including a top surface, wherein the stage is rotatable about a z-axis relative to an optical system of a sequencing system; one or more top stages positioned on the top surface of the stage, wherein each of the one or more top stages is configured to receive and fix one or more flow cell devices thereon, and wherein each of the one or more top stages is movable relative to the stage; a first motor configured to actuate the stage to rotate at a first resolution. The sample stage according to any one of the preceding claims, wherein the top surface is circular. 88. The sample stage according to any one of the preceding claims, wherein the first resolution is an angular resolution and is less than 0.1 degrees, 0.2 degrees, 0.5 degrees, 1 degree, 2 degrees, 3 degrees, 4 degrees, 5 degrees, 10 degrees, 20 degrees, 30 degrees, or 50 degrees.89. The sample stage according to any one of the preceding claims, wherein each flow cell device in the flow cell apparatus includes one or more samples to be sequenced fixed thereon. 90. The sample stage according to any one of the preceding claims, wherein at least one flow cell device in the flow cell apparatus includes an in-situ sample fixed thereon. 91. The sample stage according to any one of the preceding claims, wherein the sample stage further includes one or more second motors configured to individually actuate the one or more top stages relative to the base station at a second resolution. 92. The sample stage according to any one of the preceding claims, wherein the sample stage further includes a second motor configured to simultaneously actuate the one or more top stages relative to the base station at a second resolution. 93. The sample stage according to any one of the preceding claims, wherein the second resolution is less than 0.01 mm, 0.015 mm, 0.02 mm, 0.03 mm, 0.04 mm, 0.05 mm, 0.08 mm, 0.1 mm, 0.2 mm, or 1 mm. 94. The sample stage according to any one of the preceding claims, wherein the sequencing system includes a fluid control device in fluid communication with the flow cell devices positioned on the sample stage. 95. The sample stage according to any one of the preceding claims, wherein each of the one or more top stages is movable relative to the base in a sample plane. 96. The sample stage according to any one of the preceding claims, wherein a first top stage of the one or more top stages is movable independently relative to a second top stage of the one or more top stages. 97. The sample stage according to any one of the preceding claims, wherein a first top stage of the one or more top stages is movable simultaneously relative to the base and a second top stage of the one or more top stages. 98. The sample stage according to any one of the preceding claims, wherein each of the one or more top stages is movable relative to the base along a radius of the top surface of the base. 99. The sample stage according to any one of the preceding claims, wherein each of the one or more top stages is movable relative to the base with a radius orthogonal to the top surface of the base. 100. A method for sequencing multiple DNA samples positioned on a rotating sample stage, the method comprising: obtaining a sample stage, the sample stage including a base and one or more top stages positioned on a top surface of the base, wherein the base is rotatable about a z-axis relative to an optical system of a sequencing system; positioning and fixing a first flow cell device relative to a first top stage of the one or more top stages; positioning and fixing a second flow cell device relative to a second top stage of the one or more top stages; Claims 5 / 6 pages 6 CN 120936922 AOne or more sequencing reagents are dispensed into the first flow cell device using a first fluid control device; a first sample region of the first flow cell device is imaged using the optical system of the sequencing system; the first top stage is moved relative to the optical system in the x-y plane while preventing the second flow cell device from moving relative to the optical system; a second sample region of the first flow cell device is imaged using the optical system of the sequencing system; the sample stage is rotated at a predetermined angular resolution to position the second flow cell device relative to the optical system at a predetermined position; and the first sample region of the second flow cell device is imaged using the optical system of the sequencing system. 101. The method according to any one of the preceding claims, wherein moving the first top stage relative to the optical system in the x-y plane while preventing the second flow cell device from moving relative to the optical system comprises: moving the first top stage independently relative to the optical system at a predetermined distance along the radius of the top surface of the stage while preventing the second flow cell device from moving relative to the optical system. 102. The method according to any one of the preceding claims, wherein moving the first top stage relative to the optical system in the x-y plane while preventing movement of the second flow cell device relative to the optical system comprises: independently moving the first top stage relative to the optical system at a predetermined distance along a direction orthogonal to the radius of the top surface of the base while preventing movement of the second flow cell device relative to the optical system. 103. The method according to any one of the preceding claims, wherein the method further comprises: moving the first fluid control device or the second fluid control device to position the second flow cell device relative to the first fluid control device or the second fluid control device at a predetermined position. 104. The method according to any one of the preceding claims, wherein the first sample area or the second sample area comprises a block. 105. The method according to any one of the preceding claims, wherein each of the one or more top stages comprises a range of motion greater than 15 mm and less than 80 mm along the radius of the top surface of the base or orthogonal to the radius of the top surface of the base. 106. The method according to any one of the preceding claims, wherein each of the one or more top stages includes a range of motion greater than 25 mm and less than 100 mm along the radius of the top surface of the base or orthogonal to the radius of the top surface of the base. Claims 6 / 6 Page 7 CN 120936922 A Illumination system for nucleic acid sequencing

[0001] Cross-reference

[0002] This application claims U.S. Provisional Application No. 63 / 481,583, filed January 25, 2023 and March 8, 2023.Priority is claimed in U.S. Provisional Application No. 63 / 489,150, the entire contents of which are incorporated herein by reference. Summary of the Invention

[0003] This document describes an optical system for an imaging module for sequencing nucleic acids in DNA samples. The optical system and method described herein are capable of illuminating multiple surfaces of a flow cell that are axially displaced from each other with an illumination field of relatively uniform illumination power density. This illumination field is wider than that provided by some illumination systems, thus facilitating increased sequencing throughput over the set system runtime. Speckle eliminators, which are cost-effective and easy to implement, can be used to advantageously reduce speckle noise in the illumination system. Therefore, the illumination system and method of this document can increase the effectiveness and efficiency of sequencing analyses, including next-generation sequencing (NGS).

[0004] In some cases, the optical systems and optical components of this disclosure can provide a wide range of temporal color imaging. Such imaging can enhance sequencing or other imaging performance, where wide-area high-resolution imaging can increase throughput and reduce the time required for surface imaging. The optical systems and optical components of this disclosure can provide a reduced volume of optical systems and optical components, which can reduce the coverage area and enable new system architectures. The optical systems and assemblies disclosed herein can be used to reduce the number of optical components (e.g., lenses, etc.), thereby reducing the number of elements to be aligned and the number of system failure points, extending uptime, and reducing manufacturing burden. The optical systems and assemblies disclosed herein can eliminate stage movement in the system's z-direction (e.g., along the optical axis, relative to the optical assembly), which can simplify setup and enhance the reliability of the optical system.

[0005] In some embodiments, the aspects disclosed herein provide an optical system comprising: a stage configured to hold a solid support; a light source configured to illuminate the solid support; and an optical assembly at least partially disposed within an optical path from the stage to the light source, wherein the optical assembly is configured to provide illumination over an area of ​​the solid support greater than about 20 square millimeters (mm²), wherein the peak-to-valley variation is at most about 5%. In some embodiments, the optical assembly does not include an objective lens. In some embodiments, the optical system does not include the objective lens. In some embodiments, the optical assembly does not include a barrel lens. In some embodiments, the optical system does not include the barrel lens. In some embodiments, the stage is not adjusted along the optical axis of the system. In some embodiments, the irradiance of the illumination is at least about 40 milliwatts per square millimeter. In some embodiments, the optical component is configured to receive emitted light from the solid support. In some embodiments, the numerical aperture (NA) of the optical component is at least about 0.3. In some embodiments, the wavelength of the emitted light is from about 500 nanometers to about 750 nanometers. In some...In some embodiments, the working distance of the optical component is at least about 1 mm to 25 mm. In some embodiments, the optical system further includes a motion coil housed within the optical component, the motion coil being configured to move a focusing element within the optical path of the optical system. In some embodiments, a motor located outside the optical system is configured to move the focusing element along the optical axis in one or both directions. In some embodiments, the motor is directly coupled to a portion of a first, second, or third housing of the optical component, and a portion of the first, second, or third housing of the optical component is directly coupled to the focusing element. In some embodiments, the light source is a pulsed light source. In some embodiments, the composite root mean square error of the optical system is less than about 0.05. In some embodiments, the illumination efficiency of the optical component is at least about 90%. In some embodiments, the area is greater than 30 mm². In some embodiments, the area is greater than 50 mm² or 60 mm². In some embodiments, the optical system further includes the solid support within the stage. In some embodiments, the solid support includes two or more surfaces having one or more samples imaged by the optical system fixed thereon. In some embodiments, the solid support includes three or more surfaces having one or more samples imaged by the optical system fixed thereon. In some embodiments, the three or more surfaces are axially displaced relative to each other at least along the optical axis of the optical system. In some embodiments, the solid support includes a probe configured to bind nucleic acid molecules. In some embodiments, the probe binds to the surface of the solid support. In some embodiments, the light source is a laser source. In some embodiments, the optical assembly includes a dichroic filter configured to transmit the illumination. In some embodiments, the optical assembly includes: a first segment including a first housing including a first plurality of lenses; a second segment including a second housing; and a third segment including a third housing including a second plurality of lenses. In some embodiments, the first segment and the third segment are optically aligned. In some embodiments, the first segment is positioned between the third segment and the stage. In some embodiments, the third segment is positioned between the first segment and an image sensor of the optical system. In some embodiments, the first plurality of lenses are movable along an optical path ranging from about 0 to about 2 mm. In some embodiments, the first plurality of lenses include asymmetric biconvex lenses. In some embodiments, the second plurality of lenses include asymmetric biconcave lenses. In some embodiments, the asymmetric biconcave lens is an aspherical asymmetric biconcave lens. In some embodiments...In some embodiments, the optical system is configured to acquire an image of the solid support without moving the optical compensator into the optical path between the solid support and the detector of the optical system. In some embodiments, the optical system is configured to acquire an image of the solid support without removing the optical compensator from the optical path between the sample and the detector of the optical system. In some embodiments, the solid support is a flow cell. In some embodiments, the optical component is configured to generate one or more spatial contractions transverse to the optical path of light traveling through it. In some embodiments, the optical component is configured to generate one or more field curvature corrections transverse to the optical path of light traveling through it. In some embodiments, the optical component is configured to generate at least one field curvature correction transverse to the optical path of light traveling through it in a first, second, or third segment.

[0006] On the other hand, this disclosure provides a method for analyzing biomolecules, comprising: (a) providing a solid support comprising the biomolecule containing a label; (b) illuminating the biomolecule containing the label using an optical system including a light source to generate signal light or a variation thereof, wherein the illumination is provided over an area of ​​the solid support greater than about 20 square millimeters (mm2), wherein the peak-to-valley variation is at most about 5%; (c) detecting the signal light or the said variation thereof using a detector of the optical system; and (d) processing the signal light or the said variation thereof at least partially to analyze the biomolecule. In some embodiments, the biomolecule is a nucleic acid molecule, a protein, or a polypeptide. In some embodiments, the biomolecule is a nucleic acid. In some embodiments, the method further comprises, prior to (a), binding the biomolecule to a probe bound to the solid support and conjugating the label to the biomolecule. In some embodiments, the label is conjugated to the biomolecule by hybridization. In some embodiments, the optical system does not include an objective lens. In some embodiments, the solid support does not move along the optical axis of the optical system. In some embodiments, multiple images of the solid support are acquired without moving the solid support along the optical axis. In some embodiments, the irradiance of the illumination is at least about 40 milliwatts per square millimeter. In some embodiments, the wavelength of the signal light is from about 500 nanometers to about 750 nanometers. In some embodiments, the detection in (c) is performed using an optical element with a numerical aperture of at least about 0.3. In some embodiments, the method further includes, in (b), using a motion coil within the optical system to move a focusing element within the optical path of the optical system, thereby changing the focal point of the optical system on the solid support. In some embodiments, the light source is a pulsed light source. In some...In some embodiments, the illumination is provided with an efficiency of at least about 90%. In some embodiments, the method further includes repeating (b)-(d) on additional biomolecules coupled to another surface of the solid support. In some embodiments, the method further includes removing the marker from the biomolecule after (c). In some embodiments, the method further includes repeating (a)-(d) on additional markers bound to another portion of the biomolecule. In some embodiments, optical components are configured to generate one or more spatial contractions transverse to the optical path of light traveling through it. In some embodiments, optical components are configured to generate one or more field curvature corrections transverse to the optical path of light traveling through it. In some embodiments, optical components are configured to generate at least one field curvature correction transverse to the optical path of light traveling through it in a first, second, or third segment. In some embodiments, (d) includes at least partially processing the signal light or its said variations to generate one or more solid support images and analyzing the one or more solid support images to generate a base interpretation of the sample. In some embodiments, each solid support image in the solid support images includes a field of view (FOV) greater than 20 square millimeters (mm2). In some embodiments, the solid support is a flow cell.

[0007] In some embodiments, the aspects disclosed herein provide an optical system comprising: a stage configured to hold a solid support; a light source configured to illuminate the solid support; and a speckle eliminator optically coupled to the light source and disposed within an optical path from the light source to the stage. In some embodiments, the speckle eliminator is configured to reduce speckle noise introduced between the light source and the stage. In some embodiments, the optical system further includes an additional light source optically coupled to the speckle eliminator. In some embodiments, light from the additional light source is configured to illuminate the solid support with light of a different wavelength than that of the light source. In some embodiments, at least about four light sources are coupled to the speckle eliminator. In some embodiments, the speckle eliminator is a vibratory speckle eliminator. In some embodiments, the speckle eliminator is a passive speckle eliminator. In some embodiments, the passive speckle eliminator includes a diffuser plate. In some embodiments, the speckle eliminator is a tension speckle eliminator. In some embodiments, the speckle eliminator is configured to reduce speckle noise to at most about 5%. In some embodiments, the solid support is a flow cell.

[0008] In some embodiments, aspects disclosed herein provide a method for analyzing biomolecules, the method comprising: (a) providing a solid support comprising a biological sample containing a marker; (b) using a light source...An optical system illuminates the biological sample containing the marker, thereby generating signal light or a variation thereof, wherein the illumination is provided by a speckle eliminator in the optical path of the optical system; (c) the signal light or the variation thereof is detected using a detector of the optical system; and (d) the signal light or the variation thereof is processed at least partially to analyze the biomolecule.

[0009] In some embodiments, the method further includes repeating steps (b)–(d) on another biological sample coupled to another surface of the solid support. In some embodiments, the method further includes removing the marker from the biological sample after (c). In some embodiments, the method further includes repeating (a)–(d) on another marker bound to another portion of the biological sample. In some embodiments, the speckle eliminator uses vibration to perform speckle elimination on the illumination. In some embodiments, the method further includes illuminating the solid support using an additional light source. In some embodiments, the additional light source provides light of a different wavelength to the solid support. In some embodiments, the additional light source is optically coupled to the speckle eliminator. In some embodiments, the biological sample includes nucleic acid molecules, proteins, or peptides. In some embodiments, the biological sample includes nucleic acids. In some embodiments, the optical components are at least partially disposed within the optical path from the stage to the detector of the optical system (see page 3 / 54 of CN 120936922 A). In some embodiments, the illumination system of the optical components is disposed within the optical path from the stage to the detector of the optical system. In another aspect, the present invention provides a sample stage for holding a DNA sample for use in a DNA sequencing reaction and imaging, the sample stage comprising: a stage including a top surface, wherein the stage is rotatable about a z-axis relative to the optical system of the sequencing system; one or more top stages positioned on the top surface of the stage, wherein each of the one or more top stages is configured to receive and fix one or more flow cell devices thereon, and wherein each of the one or more top stages is movable relative to the stage;

[0010] a first motor configured to actuate the stage to rotate at a first resolution. In some embodiments, the top surface is circular. In some embodiments, the first resolution is an angular resolution and is less than 0.1 degrees, 0.2 degrees, 0.5 degrees, 1 degree, 2 degrees, 3 degrees, 4 degrees, 5 degrees, 10 degrees, 20 degrees, 30 degrees, or 50 degrees. In some embodiments, each flow cell device includes one or more samples to be sequenced fixed thereon. In some embodiments, at least one flow cell device includes an in-situ sample fixed thereon. In some embodiments, the sample stage further includes one or more second motors, wherein the first motor...Two motors are configured to individually actuate one or more top stages relative to the substrate at a second resolution. In some embodiments, the sample stage further includes a second motor configured to simultaneously actuate one or more top stages relative to the substrate at a second resolution. In some embodiments, the second resolution is less than 0.01 mm, 0.015 mm, 0.02 mm, 0.03 mm, 0.04 mm, 0.05 mm, 0.08 mm, 0.1 mm, 0.2 mm, or 1 mm. In some embodiments, the sequencing system includes a fluid control device in fluid communication with a flow cell device positioned on the sample stage. In some embodiments, each of the one or more top stages is movable relative to the substrate in a sample plane. In some embodiments, a first top stage of the one or more top stages is movable independently relative to a second top stage of the one or more top stages. In some embodiments, a first top stage of the one or more top stages is movable simultaneously relative to the substrate and a second top stage of the one or more top stages. In some embodiments, each of the one or more top stages is movable relative to the substrate along a radius of the top surface of the substrate. In some embodiments, each of the one or more top stages is movable relative to the base with a radius orthogonal to the top surface of the base.

[0011] In some embodiments, aspects disclosed herein provide a method for sequencing a plurality of DNA samples positioned on a rotating sample stage, the method comprising: obtaining a sample stage including a base and one or more top stages positioned on the top surface of the base, wherein the base is rotatable about a z-axis relative to an optical system of a sequencing system; positioning and fixing a first flow cell device relative to a first top stage of the one or more top stages; positioning and fixing a second flow cell device relative to a second top stage of the one or more top stages; dispensing one or more sequencing reagents into the first flow cell device using a first fluid control device; imaging a first sample region of the first flow cell device using an optical system of the sequencing system; moving the first top stage relative to the optical system in the x-y plane while preventing movement of the second flow cell device relative to the optical system; imaging a second sample region of the first flow cell device using an optical system of the sequencing system; rotating the sample stage at a predetermined angular resolution to position the second flow cell device relative to the optical system at a predetermined position; and imaging the first sample region of the second flow cell device using an optical system of the sequencing system. In some embodiments, preventing the second flow cell device from moving relative to the optical system while simultaneously moving the first top stage relative to the optical system in the x-y plane includes: independently moving the first top stage relative to the optical system by a predetermined distance along the radius of the top surface of the base while simultaneously preventing the second flow cell device from moving relative to the optical system. In some embodiments, preventing the second flow cell device from moving relative to the optical system...Moving the first top stage relative to the optical system in the x-y plane while moving the system includes: independently moving the first top stage relative to the optical system at a predetermined distance along a direction orthogonal to the top surface of the base while preventing the second flow cell device from moving relative to the optical system. In some embodiments, the method further includes: moving a first fluid control device or a second fluid control device to position the second flow cell device relative to the first fluid control device or the second fluid control device at a predetermined position. In some embodiments, the first sample area or the second sample area includes a block. In some embodiments, the range of movement of each of the one or more top stages along a radius or orthogonal to the top surface of the base is greater than 15 mm and less than 80 mm. In some embodiments, the range of movement of each of the one or more top stages along a radius or orthogonal to the top surface of the base is greater than 25 mm and less than 100 mm. Brief Description of the Drawings

[0012] The novel features of the inventive concept are set forth in detail in the appended claims. The features and advantages of the inventive concept will be better understood by referring to the following detailed description, which elaborates on illustrative embodiments employing the principles of the inventive concept, and the accompanying drawings:

[0013] FIG1 shows a non-limiting example of an illumination system for the optical components of this invention, which includes an illumination subsystem and a beam transmission subsystem.

[0014] FIG2 shows a non-limiting example of an illumination subsystem of this invention.

[0015] FIGS. 3A to 3C show the uniformity of the illumination power density of the illumination system shown in FIG1 of this invention. FIG3A shows an example image of the illumination field and the associated illumination intensity. FIG3B shows a linear trajectory of the illumination intensity along the major axis in FIG3A. FIG3C shows a linear trajectory of the illumination intensity along the minor axis in FIG3A.

[0016] FIG4 shows a non-limiting example of an illumination subsystem and a beam transmission system for the optical components.

[0017] FIG5 shows a non-limiting example of an illumination subsystem for the optical components.

[0018] FIG6 shows a speckle canceller and its relative position to the collimator of the beam transmission subsystem.

[0019] FIG7 shows a non-limiting example of an optical fiber and a beam transmission subsystem.

[0020] Figure 8 shows a non-limiting example of a liquid-core optical guide and a beam transmission subsystem.

[0021] Figures 9A to 9D show non-limiting examples of speckle cancellers, in which the speckle canceller is a mechanical vibration source loosely or fixedly attached to at least a portion of an optical fiber. Figure 9A shows a wound portion of the optical fiber according to some embodiments. Figure 9B shows a portion of the optical fiber around the vibration source according to some embodiments. Figure 9C shows a portion of the optical fiber around a fan vibration source according to some embodiments. Figure 9D shows a portion of the optical fiber around a fan vibration source according to some embodiments.A portion of the optical fiber.

[0022] Figure 10 shows a table of the corresponding speckle noise levels of different speckle eliminator configurations associated with the optical fiber.

[0023] Figure 11 shows a block diagram of a sequencing system for imaging DNA samples during DNA sequencing reactions according to some embodiments.

[0024] Figure 12 is a schematic diagram of various examples of multivalent molecular configurations. Left (Class I): Schematic diagram of a multivalent molecule having a “starburst” or “helix-skelter” configuration. Middle (Class II): Schematic diagram of a multivalent molecule having a dendritic macromolecular configuration. Right (Class III): Schematic diagram of multiple multivalent molecules formed by reacting streptavidin with a 4-arm or 8-arm PEG-NHS having biotin and dNTPs. Nucleotide units are designated as 'N', biotin is designated as 'B', and streptavidin is designated as 'SA'.

[0025] Figure 13 is a schematic diagram of an example of a multivalent molecule comprising a universal core attached to multiple nucleotide arms.

[0026] Figure 14 is a schematic diagram of an example of a multivalent molecule comprising a dendritic macromolecular core attached to multiple nucleotide arms.

[0027] Figure 15 shows a schematic diagram of an example of a multivalent molecule comprising a core attached to multiple nucleotide arms, wherein the nucleotide arms comprise biotin, spacers, linkers, and nucleotide units.

[0028] Figure 16 is a schematic diagram of an example of a nucleotide arm comprising a core attachment portion, spacers, linkers, and nucleotide units, as described in page 5 / 54 of CN 120936922 A.

[0029] Figure 17 shows the chemical structure of an example of a spacer (top) and the chemical structures of various linker examples, including 11-atom linkers, 16-atom linkers, 23-atom linkers, and N3 linkers (bottom).

[0030] Figure 18 shows the chemical structures of various linker examples, including linkers 1 to 9.

[0031] Figure 19 shows the chemical structures of various linker examples connected / attached to nucleotide units.

[0032] Figure 20 shows the chemical structures of various examples of linkers attached to / attached to nucleotide units.

[0033] Figure 21 shows the chemical structures of various examples of linkers attached to / attached to nucleotide units.

[0034] Figure 22 shows the chemical structures of various examples of linkers attached to / attached to nucleotide units.

[0035] Figure 23 shows the chemical structure of an example of a biotinylated nucleotide arm. In this example, the nucleotide unit is linked to the linker via a propargylamine attacher located at the 5-position of a pyrimidine base or the 7-position of a purine base.

[0036] Figure 24 shows a flowchart of a method for analyzing biomolecules according to some embodiments.

[0037] Figure 25 shows a flowchart of a method for analyzing biological samples according to some embodiments.

[0038] Figure 26 shows a perspective view of a non-limiting example of an imaging module or optical assembly.

[0039] Figure 27 shows a cross-sectional view of a non-limiting example of an imaging module or optical assembly.

[0040] Figure 28 shows a cross-sectional view of a non-limiting example of a single-channel timing color imaging module of Figures 26 and 27.

[0041] Figures 29A and 29B show examples of external actuator coupling according to some embodiments. Figure 29A shows a detailed view of the external actuator and optical assembly according to some embodiments. Figure 29B shows a distant view of the external actuator and optical assembly according to some embodiments.

[0042] Figure 30 shows an example of optical elements and associated focusing paths of an optical assembly according to some embodiments.

[0043] Figure 31 shows an example of optical elements and associated focusing paths of an optical assembly according to some embodiments.

[0044] Figures 32A and 32B provide the diffraction modulation transfer function (MTF) of an optical system according to some embodiments. Figure 32A shows an example MTF of an objective-based optical system. Figure 32B shows an example MTF of the optical system excluding the objective lens.

[0045] Figures 33A and 33B show wavefront analysis calculations of the optical system of this disclosure according to some embodiments. Figure 33A shows an example wavefront analysis calculation at position 1. Figure 33B shows an example wavefront analysis calculation at position 2.

[0046] Figure 34 shows a top surface optical performance curve according to some embodiments.

[0047] Figure 35 shows a bottom surface optical performance curve according to some embodiments.

[0048] Figure 36 shows a graph of the MTF of the optical system according to some embodiments.

[0049] Figure 37 shows a cumulative probability plot of achieving a given wavefront error according to some embodiments.

[0050] Figure 38 is a schematic diagram of a rotary stage for moving a sample relative to the objective lens of the optical system for imaging a sequencing reaction. Detailed Description

[0051] Next-generation sequencing (NGS) analysis systems require higher throughput and flexibility. This document discloses an optical system, its design, and its usage, which can provide one or more of the following advantages: wide field of view, uniform illumination power; reduced speckle noise using a cost-effective and easy-to-implement method; higher system throughput for fluorescence imaging-based genomics applications; compatibility with conventional flow cell devices and / or optical systems; flexibility in sample analysis or comparison (e.g., larger sample volume and / or increased sample types); reduced system size; reduced complexity and other requirements of optical components (e.g., simpler optical setup); larger field of view; and improved illumination power uniformity. Specification 6 / 54 pages 13 CN 120936922 A

[0052] This document discloses an imaging module configured for multi-channel fluorescence imaging. Each optical system may include multiple componentsImaging modules, or equivalently, multiple optical components, such as one imaging module per color channel, and one or more imaging modules may include the illumination system and image acquisition system disclosed herein, the image acquisition system being configured to acquire flow cell images of one or more samples fixed on a sample stage and positioned at a sample plane. The illumination system and / or image acquisition system may operate individually for each respective imaging module or may be shared among multiple imaging modules. As disclosed herein, according to some embodiments, imaging modules may be used interchangeably as optical components.

[0053] Image Acquisition System and Method

[0054] In some embodiments, the image acquisition system includes one or more image sensors and one or more objectives. In some embodiments, an optical system for imaging a next-generation sequencing (NGS) reaction (e.g., imager 116 in FIG. 11) may include one or more multi-channel fluorescence imaging modules, each corresponding to a different color channel. Each imaging module may include an image acquisition system having an image sensor and objective corresponding to that color channel. Objectives may be shared among multiple imaging modules. In some embodiments, each imaging module may include its own sensor and may not include any objectives. In some embodiments, each imaging module is configured to generate a flow cell image without using any objective lens.

[0055] In some embodiments, the imaging module includes three distinct segments. These segments can be optically aligned independently of each other and can be coupled together to form the imaging module. Having multiple segments can advantageously allow each segment to be manufactured and optically aligned independently. Each segment may include its own separate housing. Alternatively, the imaging module may have a housing that houses all three distinct segments. In some embodiments, a first segment houses a first set of lens elements therein. Some of these lens elements may be movable relative to the housing. For example, 1 to 3 and 5 to 8 in FIG. 28 are lens elements housed in the first segment. In some embodiments, a third segment partially houses a second set of lens elements, G2. For example, 9 to 13 in FIG. 28 are in the third segment. Various methods can be used to control the centering and angular alignment of optical elements between multiple segments or within a single segment. For example, alignment flipping techniques can be used for control, and angular alignment can be based on sub-units, utilizing alignment flipping techniques to control centering and angular alignment. The second segment may house an excitation dichroic beam splitter, such as 2770 shown in FIG. 26 and 27. In some embodiments, the two orthogonal lens groups can be actively aligned at a nominal 45° angle, which controls the pointing difference between the first lens element G1 and the second lens element group G2. In some embodiments, the active alignment of the two orthogonal lens groups can reduce alignment errors to a satisfactory range. This satisfactory range can be customized according to different applications. For example, alignment errors...This may include one or more of the following: eccentricity, tilt, lens spacing error, and the need for defocusing to meet the error budget.

[0056] As disclosed herein, the focusing of the imaging module is advantageously internalized. The imaging module enables a single lens element or an element therein to be moved relative to the housing of the imaging module to achieve focusing at least along the z-axis, rather than moving multiple lens elements, such as all objectives (one or more optical compensators), relative to the sample for focusing. In some embodiments, a single lens element may be moved relative to the housing to achieve focusing along the z-axis. In some embodiments, two lens elements may be moved together or separately relative to the housing to achieve focusing along the z-axis. As shown in Figures 29A and 29B, the lens elements may be mounted on linear bearings driven by external actuators so that the lens elements can be moved automatically a predetermined distance in a controlled manner. The lens elements may be moved along the optical axis of the optical assembly. The optical axis of the optical assembly between the detector and the stage (e.g., 2790 in Figure 26A) may be along the z-axis of the segment closest to the sample stage (e.g., the first segment). The optical axis of the optical component can be along an axis orthogonal to the z-axis of the segment closest to the image sensor (e.g., the third segment). The optical axis of the optical component between the light source and the stage (e.g., 2791 in FIG. 26A) can be along the z-axis of the segment closest to the sample stage (e.g., the first segment). The optical axis of the optical component can be along an axis orthogonal to the z-axis of the segment closest to the image sensor (e.g., the third segment). Specification 7 / 54 pages 14 CN 120936922 A

[0057] In some embodiments, the range of motion of the lens element along the z-axis for focusing can be customized based on the size and dimensions of the flow cell. For example, when the lens element moves toward or away from the imaging sensor, the z-motion range for imaging from the top surface of the flow cell to the bottom surface of the flow cell can be about 810 μm. In some embodiments, the housing may include a hard travel limiter for limiting the range of travel of the lens element during focusing. For example, 8 in FIG. 28 is an element that can be moved to focus the imaging module. As another example, 5 in Figure 28 can be a movable element for focusing an imaging module. The travel range is sufficient to allow focusing on multiple surfaces without interfering with, or contacting, other lens elements. For example, the lens element for focusing can move approximately 0.1 to 5 mm toward the sensor and approximately 0.1 to 4.0 mm away from the sensor. In some embodiments, a travel range may be included to assist in handling decoupling or placement errors of the flow cell surface (e.g., the top surface) relative to the apex of the lens element. In some embodiments, the lens element actively aligns E7 with E1 to E6. Sample Stage and Method of Use

[0058] In some embodiments, the optical system described herein (e.g., for imaging a sample or for a sequencing reaction)Those may include a sample stage configured to hold samples and / or their corresponding supports (e.g., flow cell devices with solid supports) in a predetermined position relative to an optical system. In some embodiments, the sample stage may include a base and one or more top stages positioned thereon. FIG38 illustrates an exemplary sample stage 3800 with a base 3810 and a top stage 3820.

[0059] The base (e.g., 3810 in FIG38) may include a thickness along the z-axis and a top surface. The thickness of the base may be customized to various values, for example, in the range of 1 mm to 5 cm. The top stage (e.g., 3820) may be positioned on the top surface 3811 of the base. The top surface may be planar. The top surface may be of various geometries. In some embodiments, the top surface of the base may be, but is not limited to, circular, annular, elliptical, square, rectangular, or rhomboid. The dimensions of the top surface of the base are sufficient to position one or more top stages thereon for sequencing purposes. For example, the top surface is sufficient to position 5, 10, 20, 30 or more top stages thereon.

[0060] The stage can be configured to move relative to the optical system (116), for example, relative to the focal plane of the objective lens or the focal plane of the optical system herein, to allow the sample positioned on the stage to be focused for imaging. The stage can be configured to move in one or more directions in 3D space. For example, the stage can be configured to move along the x, y and / or z axes relative to the focal plane of the optical system. As another example, the stage can be configured to rotate about an axis (e.g., the z-axis) to focus different areas of the stage top surface, thereby focusing the sample positioned thereon relative to the focal plane of the optical component.

[0061] In some embodiments, the sample stage can be of various geometries. In some embodiments, the stage can be movable relative to the optical axis of the optical system. In some embodiments, the stage can be rotated about the optical axis or z-axis of the optical system.

[0062] One or more top stages can be of various geometries. For example, as shown in FIG38, the 5 top stages are rectangular. In some embodiments, the top platform may be, but is not limited to, circular, annular, elliptical, square, rectangular, or rhomboid. In some embodiments, each top platform may have a shape and size sufficient to hold one or more flow cell devices located thereon.

[0063] In some embodiments, one or more top platforms may be movable relative to the base along the radius of the top surface of the base, for example, along the y-axis, as shown in FIG38. In some embodiments, one or more top platforms may be movable relative to the base orthogonal to the top surface of the base, for example, along the x-axis, as shown in FIG38. In some embodiments, one or more top platforms may be movable in various directions in the x-y plane.

[0064] In some embodiments, the first top platform of one or more top platforms may be movable relative to the next top platform of one or more top platforms.At least one second top stage moves independently. In some embodiments, a first top stage of one or more top stages may move simultaneously relative to the base stage and at least a second top stage of one or more top stages.

[0065] In some embodiments, each top stage may have one or more flow cell devices fixed thereon (not shown on page 8 / 54 of the specification, 15 CN 120936922 A). In some embodiments, the flow cell devices are detachably fixed to the respective top stage. In some embodiments, movement of the top stage may cause the one or more flow cell devices fixed thereon to move in the same way. The flow cell devices may be fixed relative to the top stage such that there is no relative movement between the flow cell devices and the respective top stage when the top stage moves. The flow cell devices may be fixed by a variety of fixing or fastening methods, including but not limited to mechanically clamping the flow cell devices, fixing with magnetic or electromagnetic forces, positioning the flow cell devices in a suitable housing fastened to the top stage, coupling a pin or post of the top stage to a hole or groove of the flow device, and vice versa.

[0066] In some embodiments, each flow cell device has a sample fixed thereon. The sample may be a 2D DNA sample. The sample may be a 3D volumetric sample of cells and / or tissue in situ. In some embodiments, the sample may be a multiplexed sample. In some embodiments, the nucleotide diversity of the sample may be balanced or unbalanced.

[0067] FIG38 illustrates a non-limiting example of a sample stage 3800 for holding a sample imaged by the optical system 116 of the sequencing system 110 disclosed herein. In this particular embodiment, the stage 3810 of the sample stage has a circular top surface 3811. The top surface may include one or more top stages 3820 coupled thereon. In the embodiment shown in FIG38, five top stages are spaced apart on the top surface of the stage. In some embodiments, there are 1 to 30 top stages positioned on the sample stage. In some embodiments, the sample stage (e.g., the stage and the top stages) may be rotatable about an optical axis (e.g., the z-axis). When the stage rotates, the top stages fixed thereon may also rotate with the stage in the same rotational motion.

[0068] In some embodiments, the top stages may be movable relative to the stage. This movement may be separate from or simultaneous with the rotational motion of the stage and the top stages. For example, the rotation of the stage relative to the optical system and the linear movement of the top stage relative to the stage can occur simultaneously to position a predetermined sample region of the flow cell apparatus relative to the optical system for efficient imaging. Alternatively, the rotation of the stage relative to the optical system and the linear movement of the top stage relative to the stage can occur sequentially and can be controlled by the same motor. The movement of the top stage relative to the stage can be performed in the sample plane (e.g., an x-y plane orthogonal to the z-axis). For each top stage, the x-axis can extend axially from the center of the top surface of the stage, and the y-axis can be orthogonal to both the x-axis and z-axis.Axis. For example, the top stage at the top of FIG38 can be moved relative to the sample stage along the y-axis and / or x-axis, such that different regions of the top stage can be moved relative to the optical system (e.g., objective lens) to a designated position for imaging. In some embodiments, the y-axis and x-axis corresponding to different top stages can be oriented in the x-y plane such that the y-axis is along the longest dimension of the flow cell device (e.g., along the radius of the top surface of the stage), while the x-axis is along the lateral direction of the flow cell device (e.g., along the tangential direction of the top surface of the stage).

[0069] Each top stage can be configured to hold a sample and its corresponding support thereon. The sample and its corresponding support can be fixed to the stage to move with the top stage. By fixing various samples to different top stages, specific sample regions can be rotated by rotating the stage, and / or linearly moved by linearly moving the top stage to image different samples in a temporal manner, such that the sample regions can be positioned relative to the optical system for imaging.

[0070] In some embodiments, the top stage includes a range of motion in the x-y plane sufficient to image a predetermined region of the sample. In some embodiments, the range of motion along the x-axis can be 0 to 50 mm, 0 to 40 mm, 0 to 30 mm, or 0 to 20 mm. In some embodiments, the range of motion along the y-axis can be 0 to 50 mm, 0 to 40 mm, 0 to 30 mm, 0 to 20 mm, 0 to 16 mm, or 0 to 10 mm. The resolution of movement along the x-axis or y-axis can be customized based on different samples or sequencing applications. In some embodiments, the resolution of movement along the x-axis or y-axis can be 1 μm to 40 μm, 1 μm to 30 μm, 1 μm to 20 μm, or 1 μm to 10 μm.

[0071] In some embodiments, the range of motion of each of the one or more top stages along a radius or orthogonal to the top surface of the substrate is greater than 15 mm and less than 80 mm. In some embodiments, the range of motion of each of the one or more top stages along a radius or orthogonal to the top surface of the base is greater than 25 mm and less than 100 mm.

[0072] The optical system may include one or more imaging heads, such as one or more optical components disclosed herein. Two imaging heads are shown in Figure 38. Having one imaging head can advantageously reduce the cost, size and complexity of the optical system, while having multiple imaging heads can advantageously increase imaging throughput and reduce the total imaging time for imaging a number of samples, but at the cost of increased cost, complexity and so on of the system hardware. In some embodiments, the sample stage further includes a first motor configured to actuate the sample stage (e.g., the base and the top stage) to a first resolution.Rotation of the sample stage. The rotation of the sample stage can be relative to an optical system, such as the optical axis of the optical system. The first resolution can be an angular resolution of less than 0.1 degrees, 0.2 degrees, 0.5 degrees, 1 degree, 2 degrees, 3 degrees, 4 degrees, 5 degrees, 10 degrees, 20 degrees, 30 degrees, or 50 degrees. The first resolution can be an angular resolution greater than 0.1 degrees, 0.2 degrees, 0.5 degrees, 1 degree, 2 degrees, 3 degrees, 4 degrees, 5 degrees, 10 degrees, 20 degrees, 30 degrees, or 50 degrees. In some embodiments, various actuation mechanisms can be used to enable the sample stage to rotate. For example, a gear mechanism or an induction motor can be used to actuate the movement of the sample stage. In some embodiments, the resolution of the rotational movement can be customized. For example, the resolution can be 0.1 degrees, 0.2 degrees, or 0.5 degrees. In some embodiments, the sample stage can rotate at least 10 to 360 degrees. In some embodiments, the sample stage can rotate any number of full revolutions. In some embodiments, the sample stage can rotate in one or both directions.

[0073] In some embodiments, the same mechanism as the base or a different actuation mechanism may be used to actuate the top stage for movement. In some embodiments, the sample stage further includes one or more second motors configured to independently actuate some of the one or more top stages relative to the base with a second resolution, while the remaining top stages do not move. In some embodiments, the sample stage further includes second motors configured to simultaneously actuate one or more top stages relative to the base with a second resolution. In some embodiments, the second resolution is less than 0.01mm, 0.015mm, 0.02mm, 0.025mm, 0.03mm, 0.05mm, 0.08mm, 0.1mm, 0.15mm, 0.2mm, or 0.5mm. In some embodiments, the second resolution is less than 0.01mm, 0.02mm, 0.05mm, 0.1mm, 0.2mm, 0.4mm, 0.8mm, 1mm, 2mm, or 5mm. In some embodiments, the second resolution is greater than 0.01 mm, 0.02 mm, 0.05 mm, 0.1 mm, 0.2 mm, 0.4 mm, 0.8 mm, 1 mm, 2 mm, or 5 mm. In some embodiments, the second resolution is greater than 0.01 mm, 0.015 mm, 0.02 mm, 0.025 mm, 0.03 mm, 0.05 mm, 0.08 mm, 0.1 mm, 0.15 mm, 0.2 mm, or 0.5 mm.

[0074] In some embodiments, the sample stage is coupled to one or more fluid control devices (e.g., 3830 in FIG. 38). The fluid control device may be in fluid communication with the sample stage (e.g., a flow cell device) and may be configured to hold, dispense, and collect various fluids used in the sequencing reaction within the flow cell device during a sequencing run. The fluid control device may be independent.The sample on its respective top stage is fluidly connected to the base. Figure 38 shows five different fluid control devices, each fluidly connected to the sample on its respective top stage. In some embodiments, the fluid control device may be fixed relative to the base. In some embodiments, the fluid control device may be fixed relative to the respective top stage. In some embodiments, each fluid control device may include a dispenser configured to dispense one or more reagents into the sample. For example, the dispenser may openly dispense reagents into the corresponding inlet of the flow cell. Another example is that the dispenser may be connected to the inlet of the flow cell device via a conduit, and the reagent may pass through the conduit to contact the sample in the flow cell device. In some embodiments, the fluid control device may include one or more pumps to facilitate the dispensing of fluid into and / or collection of fluid from the sample.

[0075] In some embodiments, compared to sequencing systems using multiple fluid control devices, multiple flow cell devices on a first top stage can share a single fluid control device to simplify the system, reduce system costs, and reduce waste of sequencing reagents. In such embodiments, different conduits may be used to provide fluid communication with different flow cell devices on different top stages. Such different conduits may be used for different reagents or the same reagent. Alternatively, different dispensing tips can be used (see page 10 / 54, CN 120936922 A) to allow fluid application to different flow cell devices on different platforms. In some embodiments, the same dispensing tip can be shared between different platforms, and reagent dispensing can be performed sequentially over time.

[0076] In some embodiments, the sample can be fixed to a solid support (e.g., a flow cell) for imaging using an optical system. A flow cell can include one or more lanes, each lane corresponding to a microfluidic channel that allows sequencing reagents or other fluids (e.g., wash buffer) to flow through during a sequencing run. In some flow cells with two lanes, the lanes are positioned parallel to each other. In some embodiments, the sample stage herein can utilize a flow cell with a lane orientation different from that of these flow cells. In some embodiments, the flow cell herein can include multiple lanes, and each pair of lanes can be positioned at an acute angle between their longitudinal directions such that they are not parallel to each other. For example, multiple lanes can be positioned along different radial axes of the sample stage (e.g., a platform with a predetermined angle between each pair of adjacent lanes). In such an embodiment, movement of the top stage relative to the base along the y-axis of the top stage can be eliminated. Instead, the base stage can be rotated at a predetermined angle to move to the next lane of the sample flow cell. Such embodiments with non-parallel lanes can advantageously eliminate the need to move the top stage along its corresponding x-axis, thereby simplifying the movement of the top stage relative to the base stage in the x-y plane.

[0077] In some embodiments, the top platform here may include a manifold that can securely hold one or more flow cell devices here. The manifold may include an open state in which the flow cell devices can be removed or installed in the manifold. The manifold may also include a closed state in which the flow cell devices are secured within the manifold, and a sealed fluid communication is formed between the flow cell devices (e.g., cleaning outlets) and the manifold. Further, in the closed state, the relative position of the flow cell devices with respect to the manifold is fixed. In some embodiments, a sealed fluid communication is formed between the manifold and a fluid control device.

[0078] In some embodiments, the fluid control device includes one or more sealed fluid passages leading to the manifold and the flow cell devices. In some embodiments, some of the sealed fluid passages are configured to seal the application of fluid to the flow cell devices. In some embodiments, some or all of the sealed fluid passages are configured to collect sealed fluid from the flow cell devices (e.g., cleaning fluid residue from the inlet of the flow cell devices).

[0079] In some embodiments, the sample stage, optical system, and optical components described herein advantageously eliminate the need for movement of the sample stage relative to the optical components or optical system described herein along the z-axis. Therefore, the problems and complexities that may arise from moving the sample stage and sample in the z-direction are also eliminated. Achieving z-axis movement of the focused sample can be done by moving a single lens element (e.g., a single lens element) relative to the housing of the imaging module, which is simpler, more convenient, and more accurate than some optical systems.

[0080] In some embodiments, methods are disclosed herein for sequencing multiple DNA samples positioned on a rotating sample stage for DNA sequencing using various sequencing methods, including but not limited to synthesis sequencing, affinity sequencing, and binding sequencing. Such methods can be repeated in one or more sequencing cycles during a sequencing run.

[0081] In some embodiments, the method for sequencing multiple DNA samples positioned on a rotating sample stage for DNA sequencing includes operations to obtain a sample stage comprising a stage and one or more top stages positioned on the top surface of the stage, wherein the stage is rotatable about the z-axis relative to the optical system or imaging module of the sequencing system.

[0082] In some embodiments, the method includes positioning and fixing a first flow cell device relative to a first top stage of one or more top stages.

[0083] The flow cell device may have a 2D or 3D sample embossed thereon. Flow cell devices can have different numbers of microfluidic channels, each having a channel surface on which a sample can be fixed. The flow cell device described herein can have two, three, four, or more channel surfaces. Multiple channel surfaces can be displaced relative to each other along the z-axis such that at least two, three...One or more channel surfaces are located at two, three or more different z-positions relative to the optical system. For example, the flow cell device specification 11 / 54 pages 18 CN 120936922 A may have two channels in the z direction, so that it has four surfaces at different z-positions.

[0084] The flow cell device may be fixed relative to the top platform such that there is no relative movement between the flow cell device and the corresponding top platform when the top platform moves. The flow cell device may be fixed by various fixing or fastening methods, including, but not limited to, mechanically clamping the flow cell device, fastening with magnetic or electromagnetic force, positioning the flow cell device in a suitable housing (e.g., manifold) fastened to the top platform, and connecting a pin or post of the top platform to a hole or slot of the flow cell device. For example, the flow cell device may be fixed in its corresponding manifold in a closed state, and a sealed fluid communication may be established between the flow cell device and the manifold in the closed state.

[0085] In some embodiments, the method further includes the operation of positioning and fixing a second flow cell device relative to a second top platform of one or more top platforms. The second stage may have the same or different fixing or fastening method as the first stage.

[0086] In some embodiments, the method further includes dispensing one or more sequencing reagents to a first flow cell device positioned on the first stage via a first fluid control device to enable sequencing of the sample. Such dispensing of sequencing reagents may be performed openly, for example, by dispensing a tip to an open area leading to a channel of the flow cell device. Alternatively, such dispensing of sequencing reagents may be performed via a closed conduit.

[0087] In some embodiments, the method further includes imaging a first sample region of the first flow cell device using the optical system of the sequencing system. The first sample region may include at least a portion of a first patch of the flow cell device. Such imaging may include collecting emitted light signals from the sample via an image sensor of an imaging module. Such imaging may also include automatically focusing the imaging module onto the sample using various autofocusing methods. Such imaging may also include generating excitation light propagating to the sample.

[0088] After completing the imaging operation of the first sample region of the first flow cell device, the method may include moving the first stage in the x-y plane relative to the optical system while preventing the second flow cell device from moving relative to the optical system. Such operations can enable the second sample region (e.g., at least a portion of the second patch) to be correctly positioned for imaging. This movement can occur within the sample plane (e.g., the x-y plane) along the x, y, or any other direction. During such movement of the first stage, the stage can remain stationary relative to the optical system.

[0089] In some embodiments, the method further includes the simultaneous movement of the first flow cell device relative to the optical system...The operation of moving the first fluid control device to keep the first fluid device (e.g., its dispensing tip) stationary relative to the first flow cell device. In some embodiments, for example, the first fluid control device does not need to move when a closed conduit contacts the first fluid control device with the first flow cell device while the first flow cell device is moving relative to the optical system. In some embodiments, the first top platform is actuated by a first motor configured to actuate the base. In some embodiments, the first top platform is actuated by a second motor configured to actuate one or more top platforms independently of the actuation of the base.

[0090] In some embodiments, the operation of moving the first top platform relative to the optical system in the x-y plane while preventing the second flow cell device from moving relative to the optical system includes: moving the first top platform relative to the optical system by a predetermined distance along the radius of the top surface of the base while preventing the second flow cell device from moving independently relative to the optical system.

[0091] In some embodiments, the operation of moving the first stage relative to the optical system in the x-y plane while preventing the second flow cell device from moving relative to the optical system includes: independently moving the first stage relative to the optical system at a predetermined distance along a direction orthogonal to the top surface of the stage while preventing the second flow cell device from moving relative to the optical system.

[0092] In some embodiments, the method further includes the operation of imaging a second sample region of the first flow cell device using the optical system of the sequencing system. Specification 12 / 54 pages 19 CN 120936922 A

[0093] After imaging the second sample region of the first flow cell device is completed, the method may further include the operation of moving the first stage and imaging other sample regions of the first flow cell device until all desired sample regions of the first flow cell device have been imaged.

[0094] In some embodiments, the method may further include the operation of rotating the sample stage at a predetermined angular resolution to position the second flow cell device at a predetermined position relative to the optical system. This operation may occur in the same sequencing cycle as the operation of imaging the sample region of the first flow cell device. Alternatively, this operation can occur in a different sequencing cycle than the operation of imaging the sample region of the first flow cell device. Angular resolution may be the first resolution disclosed herein.

[0095] After rotating the sample stage at a predetermined angular resolution to position the second flow cell device in a predetermined position relative to the optical system, in some embodiments, the method may further include dispensing one or more sequencing reagents to the second flow cell device via a first or second fluid control device such that the sample fixed on the second flow cell device can be subjected to a sequencing reaction. The one or more sequencing reagents dispensed to the second flow cell device may be different from those dispensed to the first flow cell device, for example, for different sequencing chemistry or applications. Dispensing sequencing reagents to the second flow cell device...The operation of the two flow cell devices can be optional, for example, if the same sequencing reagents are used and simultaneously dispensed into the first and second flow cell devices before imaging the first flow cell device.

[0096] In some embodiments, the method further includes: moving a first fluid control device or a second fluid control device to position the second flow cell device at a predetermined location relative to the first or second fluid control device.

[0097] In some embodiments, after rotating the sample stage at a predetermined angular resolution to position the second flow cell device at a predetermined location relative to the optical system, the method may further include imaging a first sample region of the second flow cell device (e.g., at least a portion of a first patch of the second flow cell device) using the optical system of the sequencing system. The imaging operation of the first sample region of the second flow cell device may be the same as the imaging operation of the sample region in the first flow cell device. For example, when the first flow cell device contains a 2D sample included on the first flow cell device and a 3D sample included on the second flow cell device such that the imaging operation may include imaging different z-levels of the 3D sample and imaging the 2D sample at a single z-level, the imaging operation may be different from the imaging operation of the first sample region of the second flow cell device.

[0098] In some embodiments, the rotating sample stage and the method of sequencing samples using the rotating sample stage can advantageously improve sequencing capability and system throughput by allowing sequencing and imaging of multiple flow cell devices using a single sample stage, and such multiple flow cell devices can contain different samples for different sequencing reactions. Furthermore, in some embodiments, the rotating sample stage and the sequencing method using the rotating sample stage can advantageously improve sequencing efficiency by allowing imaging of the first flow cell device while sequencing reagents are dispensed and flowed to the second flow cell device.

[0099] In some cases, the optical system may include a stage. This stage may be as described elsewhere herein (e.g., it may be configured to hold a flow cell device, a slide, or other similar solid support and place a sample thereon). The stage may lack movement along the optical axis of the system. Such movement may be relative to a non-movable housing of the optical components (e.g., the housing of the first or third element). For example, in a system configured to illuminate and image a sample (e.g., an in situ sample of cells or tissue) at multiple different z-levels along its z-axis, the stage may remain stationary relative to the immovable housing of the optical system (e.g., the housing of the first or third element of the optical assembly) along the z-axis during sample imaging. In another instance, the stage and optical assembly may not move relative to each other along the z-axis, yet still be able to focus the sample such that it lies within the focal plane of the optical assembly on the z-axis before or during imaging.The optical system can employ a focusing method that involves moving only one or more lens elements in the optical assembly (such as moving the stage or optical assembly to move the sample into the focal plane of the optical system). One or more lens elements of the optical assembly may exclude an objective lens. In some embodiments, the optical system herein may employ a focusing method disclosed herein that eliminates the need for a z-stage that responds to movement of the sample relative to an objective lens along the z-axis in another optical system.

[0100] A solid support (e.g., a flow cell) may include probes configured to bind nucleic acid molecules, proteins, peptides, etc. For example, probes complementary to nucleic acid molecules may be immobilized (e.g., bound) to a surface of the solid support.

[0101] A solid support (e.g., a flow cell) may include one or more samples immobilized thereon. For example, a solid support may include one or more probe molecules configured to bind one or more samples. A solid support may include two or more surfaces on which one or more samples are immobilized. For example, a solid support may include a first surface having a first sample immobilized thereon and a second surface having a second sample immobilized thereon. In some cases, the solid support comprises at least about two, about three, about four, about five, about six or more surfaces. In some cases, each surface of the solid support has a different sample fixed thereon, and each sample can be illuminated and imaged by the optical system. In some cases, two or more surfaces of the solid support can be axially displaced relative to each other along the optical axis (e.g., the z-axis) of the optical system. For example, the surfaces of the solid support can be stacked relative to the optical axis of the optical system.

[0102] The optical system may include a light source configured to illuminate the flow cell. The illumination may be used for at least a portion of the imaging operation (e.g., sequencing operation as described elsewhere herein). The irradiance of the illumination is at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 90, 100 or more milliwatts per square millimeter. In some cases, the light source may be a pulsed light source (e.g., a flash lamp, a pulsed laser, a pulsed light-emitting diode, etc.). In some cases, the light source can be a continuous light source (e.g., incandescent lamp, fluorescent lamp, light-emitting diode, continuous laser, etc.).

[0103] The optical system may include optical components. The optical components may be disposed within the optical path from the stage to the light source. The optical components may be configured to provide flow cell illumination greater than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 or more square millimeters. The optical components may be configured to provide at mostThe flow cell region is illuminated with approximately 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or smaller variations (e.g., peak-to-valley variation, standard deviation, variance, interquartile range, mean absolute deviation, coefficient of variation, etc.). The optical components can be configured to illuminate the flow cell region with up to 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or smaller variations (e.g., peak-to-valley variation, standard deviation, variance, interquartile range, mean absolute deviation, coefficient of variation, etc.). In some embodiments, such illumination variations may be variations in power over the flow cell region. In some embodiments, such illumination variations may be variations in irradiance over the flow cell region.

[0104] In some cases, the optical assembly does not include an objective lens (e.g., an objective lens assembly). For example, the optical assembly may not include an objective lens assembly. In some cases, the optical system does not include an objective lens. For example, the entire optical system may not include an objective lens at any point in the optical system. In another instance, the optical system may not include an objective lens in the optical path of the optical system. The optical assembly may not include a barrel lens. In some cases, the optical system may not include a barrel lens. For example, a barrel lens may not be present in the optical path of the optical system. Despite not having a barrel lens and / or an objective lens, the optical assembly or optical system is still able to achieve the large-area illumination described herein. For example, the optical system can achieve wide-range, uniform illumination without the use of a barrel lens or an objective lens.

[0105] The optical assembly, more specifically, the illumination system of the optical assembly (e.g., as shown in FIG. 1) may be configured to transmit illumination light from a light source to a stage and a sample fixed on the stage. For example, the illumination system can be positioned above the excitation dichroic filter, such as above 2770 as shown in FIG. 27, such that the excitation dichroic filter of the optical assembly can be configured to transmit illumination from the illumination system to the sample and stage via a first segment. In other words, the excitation dichroic filter and the optical elements of the optical assembly located between the excitation dichroic filter and the stage are located within the optical path between the light source and the stage, such as the first segments 2710 and 2770 in FIG. 27. In some embodiments, the excitation dichroic filter can also be configured to reflect emitted light from the sample to the detector. In other words, the excitation dichroic filter and the optical elements of the optical assembly, also located between the excitation dichroic filter and the stage, are located within the optical path between the stage and the detector, such as the first segments 2710 and 2770 in FIG. 27. The optical assembly can be configured to receive light from the flow...The emitted light from the flow cell (e.g., light generated by the interaction of illumination light with a marker in the sample within the flow cell). For example, optical components may be configured to receive the emitted light and transmit it to a detector. Non-limiting examples of detection include CCD or CMOS cameras. The optical components may have a numerical aperture of at least about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, or greater. The emitted light may have a wavelength of at least about 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000 nm or greater. The emitted light may have a wavelength of up to about 1,000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400 nm or less. The emitted light may have a wavelength within the range defined by any two of the aforementioned values. The optical assembly may have a working distance of at least about 1 mm to about 30 mm. The optical assembly may have a working distance of at least about 2 mm to about 25 mm.

[0106] In some cases, the optical assembly may include a motion coil housed within the optical assembly. The motion coil may be configured to move a focusing element within the optical path of the optical system. For example, the motion coil may be used to move the focusing element to change the focal point of the optical system without moving other parts of the system (e.g., the optical assembly, stage, etc.). Alternatives to the motion coil include, but are not limited to, piezoelectric actuators, motors (e.g., stepper motors, servo motors, etc.), electrostatic actuators, hydraulic actuators, pneumatic actuators, etc., or any combination thereof. In some cases, the motion coil (or other actuator) may be located outside the optical assembly or optical system and configured to move the focusing element along the optical axis. For example, a motor located outside the optical assembly may be operatively coupled to the focusing element and may adjust the position of the focusing element within the optical assembly. 2760 of Figure 27 illustrates an actuator integrated with the optical assembly. Examples of external actuators are shown in Figures 29A and 29B, where actuator 2901 is coupled to focusing element 2903 via coupling element 2902. In some cases, the actuator is coupled directly to the focusing element or the focusing element housing. For example, the actuator may be coupled without using a coupling element. In some embodiments, the focusing element includes one or more lenses of an optical assembly. In a particular embodiment shown in Figures 29A and 31, the focusing element is a single lens of an optical assembly. The focusing element may be mechanically coupled to the focusing element housing such that movement of the focusing element housing causes movement of the focusing element. The mechanical coupling between the focusing element and the focusing element housing can be performed in various ways. Such mechanical coupling may be direct and not in contact with a third element. Such mechanical coupling may be indirect and in contact with a third element therebetween. For example, as shown in Figure 29A, both ends of the focusing element 2903 are directly clamped to the focusing housing element 2904.

[0107] The optical system may have a wavefront compounding root-mean-square (RMS) error of up to about 0.2, 0.15, 0.14, 0.13, 0.12, 0.11, 0.1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1 or less for the light transmitted through the optical system. The optical components or system may have an illumination efficiency of at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or greater (e.g., the efficiency of the illumination light transmitted through the optical components or system). The optical system may be configured to image a solid support without moving an optical compensator into, out of, or into and out of the optical path between the solid support and the detector. For example, an image of the sample can be captured without moving the optical compensator into or out of the optical path between the sample and the detector. The optical assembly can be configured to produce one or more spatial contractions (e.g., one or more double beam waists in the light) transverse to the optical path of light traveling through it. These one or more spatial contractions can advantageously: enhance imaging resolution; enhance the depth of focus of the optical assembly; provide low illumination variation of the optical assembly at the sample stage; provide a wide field of view with low illumination variation; or a combination thereof. The optical assembly can be configured to produce at least one field curvature correction transverse to the optical path of light traveling through it. The at least one field curvature correction can similarly improve optical resolution and depth of focus. The optical assembly can be configured to produce more than two field curvature corrections transverse to the optical path of light traveling through it.

[0108] Figure 27 illustrates an example of an optical assembly according to some embodiments. A first segment 2710 may include a first housing containing a first plurality of lenses. A second segment 2720 may include a second housing. The third segment 2730 may include a third housing containing a second plurality of lenses. The first and third segments are optically aligned (e.g., aligned on the same optical axis). The first segment may be positioned between the third segment and a stage (e.g., a solid support 2740). The third segment may be positioned between the first segment and an image sensor (e.g., a detector) of the optical system 2750. One or more lens elements of the first plurality of lenses may be movable along the optical path of the optical system by at least approximately 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9.3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0 mm or larger. The first plurality of lenses can be moved along the optical path of the optical system by up to approximately 5.0, 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 4.0, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, or 0 mm. One or more lens elements of the first plurality of lenses can be moved along the optical path of the optical system by a distance within the range defined by any two of the aforementioned values. For example, a single lens element in the first plurality of lenses may be moved within a range of about 0 to about 25 mm along the optical path of the optical system in one or both directions along the optical axis. The optical assembly may include a dichroic filter 2770 configured to transmit illumination light from outside the optical assembly to a solid support.

[0109] In some cases, the first plurality of lenses includes one or more asymmetric biconvex lenses. In some cases, the second plurality of lenses includes one or more asymmetric biconcave lenses. The use of both biconvex and biconcave lenses, and the adjustment between the lenses, can be used to adjust the focal plane of the optical system (e.g., moving the focal plane to image the various surfaces of the solid support).

[0110] In some cases, an actuator (e.g., a motion coil) may be coupled to a portion of the optical assembly (e.g., a first, second, or third housing of the optical assembly), and a focusing element may be coupled to the portion of the optical assembly coupled to the actuator. In this way, the actuator can adjust the focus of the optical assembly. The optical assembly may be configured to produce at least one field curvature correction transverse to the optical path of the first, second, or third segment.

[0111] FIG30 illustrates an example of an optical element and associated focusing path of an optical assembly according to some embodiments. The optical assembly can be configured to focus light onto a solid substrate 3001 and a detector 3002, and between the two. For example, illumination light can pass through a dichroic filter 3003 and be focused onto the solid substrate 3001. Once the illumination light interacts with a sample on the solid support, the resulting sample light can be refocused onto the optical assembly, reflected from the dichroic filter, and transmitted through an internal focusing group 3004. The internal focusing group can be configured to adjust the focus of the optical assembly, thereby allowing multiple [projections] onto the solid support.Surface imaging is performed. The internal focusing group can be coupled to an actuator as described elsewhere herein. Signal light can pass through a multi-bandpass filter 3005. The multi-bandpass filter can have an optical density map as shown in the inset of FIG30. The multi-bandpass filter can be configured to transmit sample light from a marker in the sample while suppressing other light to reduce noise. Passing through a field leveler 3006, the signal light can be detected by a detector 3002 and further analyzed as described elsewhere herein.

[0112] Similarly, FIG31 shows an alternative lens configuration for illuminating and collecting light from a solid support (e.g., a flow cell, a glass slide, etc.) 3101. Lens group 3102 can be configured to focus incident illumination light (e.g., illumination light transmitted through a dichroic filter 3103) onto a solid substrate, while also focusing the signal light from the solid substrate through a notch filter 3104 and through a focusing element 3105 (part of lens group 3106). The focusing element can be as described elsewhere herein. Lens group 3017 can be configured to focus signal light onto detector 3108 for processing, as described elsewhere herein. Specification 16 / 54 pages 23 CN 120936922 A

[0113] Figure 24 shows a flowchart of a method 2400 for analyzing biomolecules according to some embodiments. In operation 2410, method 2400 may include providing a solid support containing biomolecules. The solid support may be a flow cell. The biomolecule may include a label. The biomolecule may include, for example, nucleic acid molecules, proteins, peptides, carbohydrates, lipids, etc. The label may vary depending on the characteristics of the biomolecule. For example, the labels for nucleic acids and proteins may be different so that they can bind to different molecules. In another instance, for nucleic acids, the label may hybridize with the nucleic acid.

[0114] The label may be an optical label (e.g., a fluorescent label, a luminescent label, a Raman label, a scattering label, a plasma label, etc.), a magnetic label, etc. In some cases, a probe is indispensable for the biomolecule. For example, a protein containing green fluorescent protein can be a biomolecule and a label. In some cases, prior to operation 2410, method 2400 may include binding a biomolecule to a probe in a solid support and / or conjugating a label to a biomolecule. For example, a biomolecule may flow into a flow cell containing a probe, bind to the probe, and then a fluorescent label may be conjugated to the biomolecule after binding to the probe. In another instance, a biomolecule containing a probe may flow into a flow cell and bind to the probe. As another example, a sample to be sequenced and imaged may be fixed on a flow cell device, and a label or probe may flow into the flow cell during various sequencing reactions. Examples of sequencing a sample will be described in detail below.

[0115] In another operation 2420, method 2400 may include using an optical system including a light source to provide a label containing a probe.Biomolecules provide illumination, thereby producing signal light or variations thereof. Illumination can be provided over an area of ​​the flow cell greater than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 square millimeters or larger. Illumination can have variations of up to about 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less (e.g., peak-to-valley variation, standard deviation, variance, interquartile range, mean absolute deviation, coefficient of variation, etc.).

[0116] The optical system may be as described elsewhere herein. For example, the optical system may not include an objective lens or a barrel lens. The solid support may not move along the optical axis of the optical system. For example, the flow cell may be fixed along the z-axis of the flow cell (e.g., the optical axis of the optical system) while being movable along the x-axis and y-axis of the flow cell. In some cases, multiple images of the flow cell may be acquired without moving the flow cell along the optical axis. For example, a first image at a first depth of focus may be acquired, and a second image at a second depth of focus may be acquired without moving the solid support.

[0117] Motion coils or other actuators, as described elsewhere herein, may be used within the optical system to move a focusing element within the optical path of the optical system, thereby changing the focus of the optical system on the solid support. For example, the actuator may move the focusing element along the optical path to change the parameters of the optical system, thereby changing the focus of the optical system from one side of the flow cell to the other. The light source may be a light source as described elsewhere herein (e.g., a pulsed light source). The optical system may have an illumination efficiency of at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or greater (e.g., the efficiency of illumination light transmitted through the optical system).

[0118] In another operation 2430, method 2400 may include detecting a signal light or a change thereof using a detector of the optical system. The signal light may have a wavelength of at least about 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000 nanometers or greater. The signal light may have a wavelength of up to about 1,000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400 nanometers or less. The signal light may have a wavelength within the range defined by any two values ​​mentioned above. The optical system may have a numerical aperture of at least about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, or greater. In some cases,The marker can be removed from the biomolecule. For example, a hybridization marker can be dehybridized from a nucleic acid molecule.

[0119] In another operation 2440, method 2400 may include at least partially processing the signal light or a variation thereof to analyze the biomolecule. The processing may be as described elsewhere herein (e.g., using the properties of the marker to determine a portion of the biomolecule, etc.). The image of the solid support may include a field of view larger than about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 square millimeters or more. The processing may include at least partially processing the signal light or a variation thereof to produce one or more images of the solid support, and analyzing one or more images of the solid support to produce a base interpretation of the sample.

[0120] In some cases, method 2400 may include repeating operations 2420 to 2440 on additional biomolecules coupled to additional surfaces of the solid support. For example, multiple biomolecules may be coupled to a solid support having multiple markers, each of which is attached to a multiple biomolecule, and an optical system may image each of the markers. In some cases, method 2400 may include repeating operations 2410 to 2440 on additional markers bound to another portion of the biomolecule. For example, a first marker may identify a first nucleotide of a nucleic acid, which may be removed from the nucleic acid, and a second marker may hybridize with the nucleic acid molecule at a second nucleotide. In this example, the second marker may be identified in a similar manner to the first marker, providing information related to the second nucleotide of the nucleic acid molecule.

[0121] Illumination System

[0122] The illumination system described herein may advantageously provide an ultrawide illumination field of not less than 10 mm², 20 mm², 30 mm², 40 mm², or 50 mm² at the sample plane. In some embodiments, the illumination system may advantageously provide an illumination field that is 2, 4, 5, 6, 8, 10, or 15 times larger than the illumination field produced by other illumination systems in the NGS optical system. The illumination system can advantageously provide an illumination power density of not less than 30, 40, 50, 60, or 70 milliwatts / mm² at the sample plane. In some embodiments, the illumination system is configured to generate an illumination field greater than 20, 30, 40, or 50 mm² at the sample stage, with a variance or standard deviation of the illumination power density of the entire illumination field of less than ±2%, 5%, 8%, 10%, or 12%. In some embodiments, the variance or standard deviation is measured as a percentage of the average power density, maximum power density, or median power density.

[0123] The illumination system is advantageously capable of imaging a wide FOV that is 2, 4, 5, 6, 8, 10, or 15 times larger than the maximum FOV provided by existing optical systems for NGS applications, thereby improving system throughput and flexibility in NGS applications.Because it can illuminate a wide field of view and image part or all of the wide illumination field, the illumination system and imaging module of this paper can advantageously eliminate the photon bleaching problem in unimaged areas associated with some optical systems.

[0124] In some embodiments, the illumination system can provide a power efficiency of not less than 65%, 70%, 75%, or 80%. In other words, the power loss within the illumination system can be less than 20%, 25%, 30%, or 35%. In some embodiments, the illumination subsystem, the beam transmission subsystem, or both can each provide a power efficiency of not less than 65%, 70%, 75%, 80%, 85%, or 90%. In some embodiments, one or more optical elements in the illumination system can each provide a power efficiency of not less than 65%, 70%, 75%, 80%, 85%, or 90%, for example, the power efficiency of optical fibers, lens arrays, etc. In some embodiments, the power efficiency can be determined as the ratio or percentage of power leaving the optical element to power entering the optical element.

[0125] The sample plane in this document can be the location where the sample is positioned and can be orthogonal to the z-axis or optical axis of the imaging module. In some embodiments, the sample plane overlaps with the focal plane of the objective lens of the imaging module.

[0126] In some embodiments, the illumination system includes an illumination subsystem and a beam transmission subsystem optically coupled to the illumination system.

[0127] Illumination Subsystem

[0128] The illumination subsystem may include a light source, which may be used alone or in combination with a speckle eliminator of the light source.

[0129] The light source may include one or more lasers. Lasers may be of various types. Some non-limiting examples of lasers include: gas lasers, solid-state lasers, fiber lasers, dye lasers, and semiconductor lasers (laser diodes). The one or more lasers may include one or more laser diodes. The one or more lasers may emit light of multiple wavelengths. In some embodiments, each laser or laser diode may emit light of a predetermined color (e.g., red, green, or blue). In some embodiments, each laser or laser diode may emit light within a wavelength range corresponding to the predetermined color (e.g., red, green, or blue). The wavelength range of the predetermined color may be less than 0.1Hz, 1Hz, 10Hz, 20Hz, 50Hz or more. In some embodiments, the one or more lasers or laser diodes may emit light of multiple colors or wavelength ranges. In some embodiments, each laser or laser diode may emit light of multiple colors, such as white light.

[0130] In some embodiments, the light source comprises one or more multicolor laser arrays. Each multicolor laser array may include lasers arranged in an array in any direction in the x-y plane, such as a 2D array. The lasers in the array may haveDifferent colors are available, allowing each laser to have a different color from the laser immediately adjacent to it in the array. In some embodiments, the multicolor laser array includes an array of laser diodes that emit 2, 3, 4, 5, or 6 wavelengths or wavelength ranges of 2, 3, 4, 5, or 6 wavelengths. Each wavelength or wavelength range may correspond to a different color. In some embodiments, the multicolor laser array includes lasers that emit light of at least 2, 3, or 4 color wavelengths or wavelength ranges in a direction orthogonal to the z-axis. Figure 2 shows an example of one embodiment of a multicolor laser array in which the laser diodes produce at least three different colors, such as blue, red, and green.

[0131] In some embodiments, the illumination subsystem may further include one or more optical fibers that may be coupled to a light source to transmit light from that source. In some embodiments, a single optical fiber is coupled to a corresponding laser or laser diode (multicolor or monochromatic). The optical fibers may have various fiber lengths. For example, the length of one or more optical fibers may be from 0.5 m to 5 m. In some embodiments, one or more optical fibers may include a fiber core. The fiber core may have a maximum dimension (e.g., diameter) of 50 μm to 2000 μm in its cross-section. The cross-section may be orthogonal to the longitudinal axis extending along the length of the fiber. In some embodiments, the cross-section of the fiber core may be circular or substantially circular. Figures 1, 3-4 and 7 illustrate examples of laser diodes and fiber coupling with laser diodes.

[0132] In some embodiments, the illumination subsystem further includes a single optical fiber as shown in Figure 2. The single optical fiber may be a multimode optical fiber. The multimode optical fiber is configured to transmit light of different colors, different wavelengths or different wavelength ranges in the same optical fiber. The single optical fiber may include a core having a maximum dimension (e.g., diameter) of 400 μm to 2000 μm in its cross-section orthogonal to the longitudinal axis of the fiber. The single optical fiber may include a core having a maximum dimension (e.g., diameter) of 600 μm to 1600 μm. The single optical fiber may include a core having a maximum dimension (e.g., diameter) of 800 μm to 1300 μm.

[0133] In some embodiments, the illumination subsystem may include multiple optical fibers. Each optical fiber can be optically coupled to one or more corresponding lasers of the light source. The one or more corresponding lasers can emit light of the same wavelength or wavelength range. The one or more corresponding lasers can emit light of the same color. In some embodiments, the illumination subsystem further includes one or more dichroic filters, optical lens elements, or both.

[0134] Figure 3 illustrates an example of an embodiment where the light source includes an array of red, green, and blue laser diodes. A single optical fiber is coupled to a laser, and various optical elements, such as dichroic filters and lens elements, can then be used to combine light of different colors from different optical fibers.

[0135] In some embodiments, the light source includes one or more beam combiners. Each beam combiner is configured to combine two different beams (e.g., different polarizations or other beam characteristics) into a combined beam. In some embodiments, the beam combiner may be a polarization beam combiner. In some embodiments, the light source may include two or more lasers emitting light of the same wavelength or the same wavelength range. Each beam combiner may combine light of the same wavelength or the same wavelength range emitted by such two or more lasers into a combined beam. In some embodiments, each beam combiner may combine light emitted by two or more lasers emitting light of the same color into a combined beam. In some embodiments, the beam combiner is configured to increase the power coupled to the fiber or sample plane by combining two beams into a combined beam. In some embodiments, the power of the combined beam is greater than the power of each individual beam before combination. Figure 5 illustrates an example of an embodiment that combines two beams of the same color from two lasers with a greater success rate than the combined beam of each individual beam. Specification 19 / 54 pages 26 CN 120936922 A

[0136] In some embodiments, the fiber coupled to a monochromatic or multicolor laser may include a core having a non-circular cross-section orthogonal to the z-axis. The non-circular cross-section can be of various shapes, such as oval, triangular, rhomboid, pentagonal, hexagonal, etc. For example, the fiber core may include a rectangular or square cross-section. Figure 7 shows an example of an embodiment of an optical fiber with a rectangular core, which can advantageously facilitate the transmission of more uniform optical power on a rectangular sample plane, thereby better matching the imaging FOV, which is also rectangular.

[0137] In some embodiments, the light source may be coupled to a liquid-core optical guide with a liquid core. In some embodiments, the one or more liquid-core optical guides are optically coupled to the light source in the absence of an optical fiber. In some embodiments, each laser may be coupled to the liquid-core optical guide. In other embodiments, multiple individual lasers may be coupled to a single liquid-core optical guide. Figure 8 shows an example of an embodiment of a liquid-core optical guide. In some embodiments, the liquid-core optical guide can facilitate the transmission of high optical power, wherein homogenization is achieved within a wide illumination field on the sample plane. In some embodiments, the one or more liquid-core optical guides include a liquid core. The liquid core may have a maximum size of 0.5 mm to 10 mm on a cross-section orthogonal to the z-axis. In some embodiments, the one or more liquid-core optical guides include a liquid core having a maximum dimension of 0.2 mm to 20 mm in a cross-section orthogonal to the z-axis. In some embodiments, the liquid core includes a circular cross-section. In some embodiments, the liquid core includes a non-circular cross-section. In some embodiments, the liquid core may include a cross-section having various non-circular shapes.

[0138] In some embodiments, the illumination subsystem further includes one or more coupling elements, such as optical lenses.One or more coupling lenses may be positioned between the light source and the optical fiber, for example, as shown in Figure 2. The coupling lens may be configured to couple laser light from the light source to an optical fiber, a liquid-core optical guide, or other optical element for transmitting light (e.g., a collimator). The coupling lens may include one or more of the following: an asymmetric biconvex lens, a convex-plano lens, a concave-plano lens, an asymmetric biconcave lens, and an asymmetric convex-concave lens.

[0139] Figures 26 and 27 illustrate non-limiting examples of the single-channel temporal color imaging module or optical assembly disclosed herein. The single-channel temporal color imaging module of this document can be advantageously used to image optical signals of different wavelengths, such that it is configured to image in the multi-color channels of a conventional system without the need for additional image sensors and other optical elements (such as associated dichroic beam splitters and excitation notch filters). Compared to systems in which signals are acquired through different channels, images of optical signals of different wavelengths can be acquired sequentially in a single channel. Compared to existing optical systems, the single-channel temporal color imaging module is advantageous for reducing system size, complexity, and cost.

[0140] Figure 26 is a perspective view of the imaging module, showing the housing of different segments (e.g., three segments) of the imaging module. Figure 27 is a cross-sectional view showing the different lens elements of the different segments and their relative positions to each other. The autofocusing beam and the excitation / illumination beam can be injected into the single-channel time-sequential color imaging module through the excitation dichroic filter in Figures 26 and 27.

[0141] The imaging module may have a “dual-waist” design, which includes at least two contractions in the optical path through the imaging module, for example, between the sample and the image sensor. For example, two contractions are shown in Figure 31, one of which occurs in the first segment, and the other may occur in the third segment. These contractions can induce overcorrection or backward bending components of field curvature correction compared to some imaging systems, thereby enabling flat-field imaging of the sample at a much wider size than allowed by these imaging systems. This wider field of view can advantageously translate into significantly improved image acquisition time to cover specific sample regions, thus providing the ability to build high-throughput sequencing systems. In this particular embodiment, the sample area or field of view that can be imaged within a single image is increased by 10, 13, 15, or 20 times compared to some imaging systems.

[0142] In some embodiments, a tri-notch or bi-notch filter (not shown) may be embedded in the collimation space between two lens elements (e.g., 5 and 7 in FIG. 28). The maximum angle of the optical path (with optical axis) at this location is controlled to be less than 10, 8, 6, or 5 degrees to achieve OD6 suppression of the excitation wavelength. In some embodiments, a bi-notch or tri-notch filter may be added at this location to suppress possible leakage from the excitation wavelength, for example, leakage from the illumination system to the sample. Example Description 20 / 54 pages 27 CN 120936922 AFor example, a triple or double notch filter may be located on top of 9 in FIG. 38, and the housing may provide an accessible hard aperture stop. The accessible hard aperture stop may be accessed from outside the imaging module.

[0143] In some embodiments, the imaging module herein is capable of autofocusing at least along the optical axis by moving one or more elements along the optical axis. In some embodiments, the lens elements may be longitudinally movable to achieve multi-surface imaging, wherein the z-motion range is 0 to 5 mm, 0 to 3 mm, 0 to 2 mm, 0 to 1 mm, 0 to 0.8 mm, 0 to 0.6 mm, 0 to 0.5 mm, or 0 to 0.4 mm. Movement of the internal lens elements may advantageously eliminate the z-stage assembly for moving the entire objective relative to the sample and the associated integration problems.

[0144] In some embodiments, the imaging module may include a lens element for aberration correction. The lens element may be aspherical. For example, 8 in FIG. 28 is a lens element for aberration correction. In some embodiments, the lens element may achieve spherical aberration correction to help improve both aberration correction and transmittance simultaneously. In some embodiments, the lens element or any other optical element may be fabricated using a glass type with low autofluorescence. In some embodiments, the lens element may include a global surface design.

[0145] In some embodiments, the imaging module or optical component described herein includes an illumination system. The illumination system may include an illumination subsystem and a beam delivery system. Figure 1 shows a non-limiting example of an illumination system with excitation / illumination light.

[0146] Speckle Eliminator

[0147] Due to the coherent characteristics of lasers, interference of light waves with the same frequency can occur, resulting in undesirable laser speckle noise. Speckle noise can cause inhomogeneity in the illumination field, thus leading to errors in DNA sequencing results. Thus, there is a need for an effective and easy-to-implement method to reduce speckle. Commercially available speckle eliminaters are expensive, for example, $1,000 per unit. The speckle eliminater described herein provides a low-cost, easy-to-implement, and effective way to reduce speckle noise from a light source.

[0148] In some embodiments, various laser-based illumination sources may be affected by speckle noise, and the speckle intensity may be controlled to minimize possible errors in image intensity-based sequencing analysis. Source coherence (e.g., speckle) can be reduced by using a predetermined fiber length and / or core size (e.g., fiber with a length of 1 to 3 m and a core size of 200 to 800 μm). In some embodiments, the illumination system may allow mode mixing to reduce speckle to a predetermined level. In some embodiments, a time-varying diffuser attenuation method may be integrated into the optical path to improve source coherence.

[0149] In some embodiments, a single fiber may be coupled to a corresponding laser or laser diode. In some embodiments, the characteristics of the fiber may be predetermined to reduce the speckle of the laser. Some non-limiting characteristics of the fiber may include:Fiber length, fiber bending radius, fiber core shape, fiber core size, and attachment of the fiber to the vibration source, etc.

[0150] In some embodiments, the speckle eliminator consists of an optical fiber that is optically coupled to a light source. In some embodiments, the speckle eliminator is coupled or associated with the optical fiber to reduce speckle from the light source. In some embodiments, the speckle eliminator is coupled or associated with the optical fiber such that a speckle reduction effect can be generated in a portion or all of the optical fiber.

[0151] In some embodiments, the speckle eliminator includes a mechanical vibration source, such as a vibration motor. The vibration source can generate vibration at a predetermined frequency or frequency range. In some embodiments, the mechanical vibration source is configured to vibrate at one or more frequencies in the audible sound frequency range, the ultrasonic frequency range, or both. In some embodiments, the mechanical vibration source is configured to vibrate at one or more frequencies between 10 and 500 Hz. For example, the vibration source can vibrate at a single frequency, wherein the standard deviation is less than 1%, 5%, or 10% of that single frequency. As another example, the vibration source can vibrate at a randomly selected frequency within a bandwidth (e.g., 80 to 90 Hz). In some embodiments, the mechanical vibration source is configured to generate one-dimensional, two-dimensional, or three-dimensional vibrational motion. In some embodiments, the mechanical vibration source is configured to generate vibrational motion including two-dimensional or three-dimensional translation, rotation, or both. In some embodiments, the mechanical vibration source is configured, but not limited to, to generate linear or nonlinear vibration, random or deterministic vibration, and / or undamped or damped vibration.

[0152] In some embodiments, at least a portion of the optical fiber is wound or coiled one or more times, as shown in Figures 9A to 9D. The wound or coiled portion may include a radius not less than the minimum bending radius of the optical fiber to avoid damaging the optical fiber. For example, the wound or coiled portion may include a radius of 60 mm, 65 mm, 70 mm, or larger, with a minimum bending radius of 60 mm. The wound or coiled portion may be, but is not necessarily, a perfect circle, as shown in Figures 9A to 9D. In some embodiments, the wound or coiled portion may include at least 2, 3, 4, 5, or more turns. In some embodiments, the number of turns of the wound or coiled portion may be limited by the length of the optical fiber and the minimum bending radius of the optical fiber to achieve the maximum possible number of turns. In some embodiments, the number of turns of the wound or coiled portion may be maximized based on the fiber length and the minimum bending radius of the fiber. In some embodiments, at least a portion of the fiber is not wound or coiled at or near its ends. The uncoiled portion of the fiber at each end may be less than 2%, 4%, 5%, 8%, or 10% of the total fiber length. The uncoiled portion of the fiber at each end may be less than 0.05m, 0.1m, 0.15m, 0.2m, or 0.3m.

[0153] In some embodiments, at least a portion of the fiber is loosely or fixedly attached to a mechanical vibration source. For example, light...The fiber can be attached to an off-the-shelf fan with tape, whether or not it is wrapped or coiled once or multiple times. The off-the-shelf fan is a cooling fan for a CPU or other component of the sequencing system described herein. As shown in Figures 9C to 9D, the fiber is wrapped or coiled on the off-the-shelf fan, or further attached to the fan with tape at some locations. As shown in Figure 9B, the fiber is coiled on a wheel, and the vibration source is fixedly attached to the center of the wheel at the bottom.

[0154] The mechanical vibration source can be a variety of machines capable of generating vibratory motion. The mechanical vibration source can be a variety of off-the-shelf machines that are more cost-effective than commercially available speckle eliminaters. As a non-limiting example, the mechanical vibration source may include one or more of the following: an eccentric rotating mass (ERM) vibrating motor, a linear resonant actuator, a coin-type vibrating motor, a mobile phone vibrating motor, an acoustic or ultrasonic vibrating motor (e.g., a vibrating motor such as one used in an electric toothbrush), a track gear or gear set, a track weight, etc.

[0155] In some embodiments, the speckle eliminator is physically isolated from other components of the imaging module, except for the optical fiber, to minimize the impact of speckle eliminator movement on sequencing response and / or imaging quality. For example, the speckle eliminator is not coupled to the optical fiber at or near either end of the fiber (e.g., less than 0.1 m). For example, the speckle eliminator is not positioned within a predetermined threshold distance (e.g., at least 0.1 m, 0.2 m, 0.5 m, or greater) from other components of the imaging module. In some embodiments, the speckle eliminator is physically isolated from the sample stage, objectives, and / or one or more image sensors, such that the mechanical movement of the speckle eliminator is independent of the sample stage, objectives, and one or more image sensors. For example, the speckle eliminator is located at least 0.1 m, 0.2 m, 0.5 m, or greater away from the sample stage, objectives, and / or one or more image sensors. In some embodiments, the speckle eliminator is configured to reduce speckle noise to no more than 4%, 4.5%, 5%, or 5.5%. In some embodiments, the speckle eliminator is configured to reduce speckle noise by at least 10%, 15%, 20%, 30%, 35%, 40% or more, such that the speckle noise after reduction by the speckle eliminator is less than 40%, 50%, 55%, 60%, 65%, 70% or 70% of the speckle noise before reduction.

[0156] Table 1 in Figure 10 shows the effect of different embodiments of the speckle eliminator of this invention on speckle noise in optical fibers. Two optical fibers, each coupled to a green and a red light source, were examined. By winding the optical fiber three times and vibrating at least the wound portion with an off-the-shelf fan, the speckle noise was reduced from 6.53% or 6.32% to less than 4.5%. The speckle reduction effect was more pronounced when the fan was kept upright rather than flat on a table or other horizontal surface. Figures 9C to 9D show the fan in upright or flat positions.

[0157] In some embodiments, a scale for determining the level of beam uniformity (e.g., the beam intensity at the sample plane) can be used.Various methods (quasi-deviation) can be used to calculate speckle noise. For example, the 2D beam profile within the imaging FOV can be divided into multiple regions, and the standard deviation of the intensity of each individual region can be determined, and the average standard deviation of all regions can be used as the speckle noise level.

[0158] In some embodiments, a speckle canceller is positioned in the optical path between the collimator and the objective of the imaging module, for example, as shown in FIG6. The speckle canceller is configured to generate micro-motion. A speckle canceller at such a large beam position can advantageously avoid high optical power density on the speckle canceller to prevent damage to the speckle canceller. In some embodiments, the speckle canceller herein may include a single speckle canceller. In some embodiments, the speckle canceller herein may include a combination of one or more first speckle cancellers associated with an optical fiber and one or more second large beam speckle cancellers positioned in the optical path after the collimator but before the objective or sample plane. In some embodiments, a large beam speckle eliminator is positioned where the maximum dimension (e.g., diameter or diagonal) of the beam at its cross-section (orthogonal to the z-axis) is greater than 1 mm, 2 mm, 5 mm, 8 mm, 10 mm, or 20 mm.

[0159] In one aspect, this disclosure provides an optical system. The optical system may include a stage configured to fix a solid support (e.g., a flow cell). The stage may be as described elsewhere herein. The optical system may include a light source configured to irradiate the solid support (as described elsewhere herein). The optical system may include a speckle eliminator optically coupled to the light source and disposed within an optical path from the light source to the stage.

[0160] The speckle eliminator may be as described elsewhere herein. The speckle eliminator may be configured to reduce speckle noise, act as a coupler to the light source, or a combination thereof. For example, the speckle eliminator may be configured to receive light from multiple light sources and combine the light from the multiple light sources into a single beam. In this example, various excitation wavelengths may be combined into a single optical path and speckle simultaneously. At least approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more light sources can be optically coupled to a single speckle eliminator. In some cases, each light source can be optically coupled to a different speckle eliminator. For example, four light sources can each be coupled to four speckle eliminators, thereby speckling the light from each light source.

[0161] The speckle eliminator may include a diffuse speckle eliminator (e.g., a speckle eliminator including diffusers such as fixed diffusers, rotating diffusers, etc.), a spatial light modulator, a phase speckle eliminator (e.g., a speckle eliminator configured to change the phase of light), a polarization speckle eliminator, a vibrational speckle eliminator (e.g., a speckle eliminator configured to vibrate one or more optical elements such as mirrors, optical fibers, lenses, etc.), and a tension speckle eliminator (e.g., a speckle eliminator configured to adjust the tension of an optical fiber to induce...).Speckle cancellers, etc., or any combination thereof. Speckle cancellers may be configured to reduce speckle noise in an optical system by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more. Speckle cancellers may be configured to reduce speckle noise in an optical system by at most about 25%, 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less. Using a speckle eliminator can improve the image quality of an optical system by reducing speckle noise generated in the optical system, enhancing contrast, or a combination thereof. Using a vibration speckle eliminator can provide unexpected benefits to an optical system, as vibration typically affects the resolution of the optical system and is therefore usually avoided. Conversely, by utilizing vibration (e.g., vibration already present in the system from fans, motors, etc.), the methods and systems of this disclosure can provide enhanced illumination distribution and imaging.

[0162] FIG25 illustrates a flowchart of a method 2500 for analyzing biological samples according to some embodiments. In operation 2510, method 2500 may include providing a solid support (e.g., a flow cell) containing a biological sample. The biological sample may include markers as described elsewhere herein. The biological sample may be as described elsewhere herein (e.g., a biological sample may include nucleic acid molecules, proteins, peptides, etc.). The marker may be an optical marker as described elsewhere herein. The biological sample may include a two-dimensional biological sample. The biological sample may include a three-dimensional biological sample.

[0163] In another operation 2520, method 2500 may include providing illumination to a biological sample containing a marker using an optical system including a light source, thereby generating signal light or a variation thereof. The optical system may be as described elsewhere herein. For example, the optical system may include a speckle eliminator. Illumination may be provided by a speckle eliminator oriented in the optical path of the optical system. The speckle eliminator may be as described elsewhere herein. For example, the speckle eliminator may be a vibrating speckle eliminator. In some cases, an additional light source may be used to illuminate the solid support. The additional light source may provide light of a different wavelength from the light source to the flow cell. For example, the light source may provide a first wavelength configured to excite a first marker, and the additional light source may provide a second wavelength configured to excite a second marker. The additional light source may be optically coupled to the speckle eliminator. For example, both the light source and the additional light source may be optically coupled to the same speckle eliminator, and the output of the speckle eliminator may include light from both light sources.

[0164] In another operation 2530, method 2500 may include detecting a signal light or a change thereof using a detector of an optical system. Detection may be as described elsewhere herein. For example, detection may include directing the signal light or a change thereof to the detector using optical components. In some cases, detection may include time-gated detection.

[0165] In another operation 2540, method 2500 may include processing the signal light or a change thereof at least partially to analyze a biological sample. Processing may include using one or more computer systems as described elsewhere herein. Processing may include generating one or more base readouts of the sample.

[0166] In some cases, method 2500 may include repeating operations 2520 through 2540 on additional biological samples coupled to additional surfaces of a solid support. For example, multiple biomolecules may be coupled to a solid support having multiple markers, each of which is attached to a multiple biomolecule, and an optical system may image each of the markers. In some cases, method 2500 may include repeating operations 2510 through 2540 on additional markers bound to another portion of a biomolecule. For example, a first marker can identify a first nucleotide of a nucleic acid, which can be removed from the nucleic acid, and a second marker can hybridize with the nucleic acid molecule at the second nucleotide. In this example, the second marker can be identified in a similar manner to the first marker, providing information related to the second nucleotide of the nucleic acid molecule.

[0167] Beam Transmission Subsystem

[0168] The beam transmission subsystem of this document may include one or more collimators and one or more optical lens elements. In some embodiments, the power in the beam transmission subsystem is greater than 5, 8, 10, 12, or 14 watts for one or more wavelengths or wavelength ranges. In some embodiments, the power at the sample plane is greater than 5, 8, 10, 12, or 14 watts for one or more wavelengths or wavelength ranges. In some embodiments, the power in the beam transmission subsystem is greater than 5, 8, 10, 12, or 14 watts for one or more colors. In some embodiments, the power at the sample plane is greater than 5, 8, 10, 12, or 14 watts for one or more colors.

[0169] In some embodiments, one or more collimators may be spaced apart along the z-axis or in the x-y plane. In some embodiments, the beam transmission system may include a single collimator. Figures 1 and 6 illustrate examples of embodiments of the beam transmission subsystem. As shown in Figure 1, one or more optical lens elements comprise one or more multi-lens arrays (e.g., MLA1 and MLA2). In some embodiments, each multi-lens array comprises one or more of the following: an asymmetric biconvex lens, a convex-plano lens, a concave-plano lens, an asymmetric biconcave lens, and an asymmetric convex-concave lens. Each multi-lens array may include a plurality of lens elements at least in a direction orthogonal to the z-axis. For example, the plurality of lens elements of the array may be along the x-axis or y-axis, or orthogonal to the z-axis.The distribution in any direction in the x-y plane (Figure 1). In some embodiments, one or more optical lens elements include: an asymmetric biconvex lens, a convex-plano lens, a concave-plano lens, an asymmetric biconcave lens, an asymmetric convex-concave lens, or a combination thereof.

[0170] In some embodiments, one or more optical lens elements include: a first multi-lens array (MLA1) and a second multi-lens array (MLA2), which are positioned along the z-axis between the collimator and the entrance pupil of the illumination system, as shown in Figure 1. In this particular embodiment, the illumination system includes a wide-field illumination module having fiber-coupled diode laser inputs, which are interlaced at an angle on adjacent columns of the first multi-element lens array pairs MLA1 and MLA2. Multiple images of the light source are generated externally or in the entrance pupil to form a uniform illumination field at the sample field (e.g., flow cell). The illumination system may include fiber-coupled laser diodes. The illuminator design is based on generating multiple source images on the entrance pupil plane of the imaging module. The output light of the fiber-coupled laser is connected to a collimator and then split in the pupil space by a plurality of multi-element lens arrays to form these secondary illumination sources. Imaging group G3 in Figure 1 is an alternative to the imaging module between the illumination system and the sample. Imaging group G3 may be shared by the imaging and illumination optical paths. In some embodiments, the illumination system is configured to generate overlapping secondary illumination sources on the sample stage or the sample positioned thereon, thereby averaging the individual intensities to provide intensity with improved uniformity compared to some methods.

[0171] In some embodiments, the various laser-based illumination sources may be affected by speckle and the speckle intensity may be controlled. Source coherence (e.g., speckle) can be mitigated by using optical fibers of length 1 to 3 m (with a core of 200 to 800 μm). In some embodiments, the illumination system may allow mode mixing to reduce speckle to a predetermined level. In some embodiments, a time-varying diffuser attenuation method may be integrated into the optical path to improve source coherence. Figures 3A to 3C show the intensity distribution at the sample using the illumination system described herein.

[0172] Sequencing System

[0173] FIG11 shows a block diagram of a system 100 for imaging a sequencing reaction of a sample on a flow cell according to one embodiment. The system 100 has a sequencing system 110, which may include a flow cell 112, a sequencer 114, an imager (e.g., an optical system) 116, a data storage 122, and a user interface 124. The sequencing system 110 may be connected to a cloud 130. The sequencing system 110 may include one or more of the following: a dedicated processor 118, a field-programmable gate array (FPGA) 120, and a computer system 126.

[0174] In some embodiments, the flow cell 112 is configured to capture DNA fragments and form a shape for sequencing on the flow cell.DNA sequence determined by base interpretation. Flow cell 112 may include the support disclosed herein. The support may be a solid support. As disclosed herein, the support may include a surface coating thereon. The surface coating may be a polymer coating as disclosed herein.

[0175] Flow cell 112 may include multiple patches or other imaging regions thereon, and each patch may be divided into a sub-patch grid. Each sub-patch may include multiple clusters or communities thereon. As a non-limiting example, the flow cell may have 424 patches, and each patch may be divided into a 6x9 grid, thus having 54 sub-patterns.

[0176] Flow cell images herein are images of a sample fixed to a support (e.g., a flow cell). Flow cell images as disclosed herein may be images of signals including multiple clusters or communities. Flow cell images may include one or more signal patches or one or more signal sub-patterns. In some embodiments, a flow cell image may be an image including all patches and approximately all signals thereon. Flow cell images may be acquired from channels using imager 116 during an imaging or sequencing cycle. In some embodiments, each patch may include millions of communities or clusters. As a non-limiting example, a patch may include about 1 to 10 million clusters or communities. Each community may be a collection of many copies of DNA fragments. Clusters or communities may be represented as bright spots extending from less than one pixel to several pixels.

[0177] Flow cell images may have various sizes or fields of view (FOV). In some embodiments, each flow cell image in the flow cell image includes a wide field of view (FOV) greater than 20 mm², 30 mm², 40 mm², or 50 mm². In some embodiments, each flow cell image in the flow cell image includes a field of view (FOV) that completely overlaps with or is contained within an illumination field generated by the illumination system on the sample plane. In some embodiments, each flow cell image in the flow cell image includes a field of view (FOV) that overlaps with at least 80%, 85%, 90%, or 95% of an illumination field generated by the illumination system at the sample plane. In some embodiments, each flow cell image in the flow cell image contains a field of view (FOV) of at least 80%, 85%, 90%, or 95% of the size of the illumination field generated by the illumination system at the sample plane.

[0178] In some embodiments, to capture such a wide FOV, the image sensor may have a wider size than other image sensors used in some NGS sequencing systems. In some embodiments, the image sensor may include a sensor size greater than 20 mm², 30 mm², 40 mm², or 50 mm². In some NGS systems, flow cell images are generally in the range of 1 mm² to 8 mm². Therefore, in these NGS systems, an illumination field greater than 10 mm² is not preferred to avoid or reduce the image FOV neighborhood.Undesirable photonic bleaching occurs in the region. The illumination system described herein produces an illumination field that is 2, 5, 10, or greater than the illumination field in these NGS systems. In some embodiments, imager 116 is capable of producing a flow cell image with a field of view (FOV) comparable to the size of the wide illumination field described herein. The FOV and illumination field can be customized such that they overlap or substantially overlap each other. Furthermore, the illumination field can be customized such that its shape matches the shape of the FOV to facilitate such overlap. Through such overlap, photonic bleaching in unimaged areas of the sample caused by an illumination field wider than the FOV of the flow cell image can be avoided or minimized.

[0179] Sequencing instrument 114 can be configured to flow a nucleotide mixture onto flow cell 112, cleave the blocking agent from the nucleotides between flow steps, and perform other steps for forming DNA sequences on flow cell 112. Nucleotides may have linked fluorescent elements that emit light or energy at wavelengths indicating the type of nucleotide. Each type of fluorescent element may correspond to a specific nucleotide base (e.g., A, G, C, T). The fluorescent elements may emit light at visible wavelengths.

[0180] For example, each nucleotide base may be assigned a color. In some cases, adenine may be red, cytosine may be blue, guanine may be green, and thymine may be yellow. The color or wavelength of the fluorescent element for each nucleotide may be selected such that the nucleotides can be distinguished from each other based on the wavelength of light emitted by the fluorescent element.

[0181] Imager 116 may be configured to capture an image of flow cell 112 after each flow step. Imager 116 may include one or more imaging modules disclosed herein. For example, the imager may include an optical system comprising four different imaging modules, each for capturing flow cell images from different color channels.

[0182] In one embodiment, imager 116 includes a camera, such as a CMOS or CCD camera, configured to capture digital images. The camera may be configured to capture images of the wavelength of the fluorescent element that binds to the nucleotide.

[0183] The resolution of imager 116 controls the level of detail in the flow cell image, including pixel size. This resolution is important in existing systems because it controls the accuracy of the point-finding algorithm in identifying cluster centers. One way to improve the accuracy of point finding is to improve the resolution of imager 116 or improve the processing of images captured by imager 116. The methods described herein can detect cluster centers in pixels other than those detected by the point finding algorithm. These methods allow for improved cluster center detection accuracy without increasing the resolution of imager 116. The resolution of the imager can even be lower than that of existing systems with comparable performance, which can reduce the cost of sequencing system 110.

[0184] In one embodiment, images from the flow cell can be captured in groups, wherein each image in the group is captured as a matched orThis includes images captured at the wavelength or spectrum of only one of the fluorescent elements. In another embodiment, the image may be captured as a single image that captures at least a portion (e.g., a portion, all, etc.) of the wavelength of the fluorescent element.

[0185] The sequencing system 100 may be configured to identify cluster locations on flow cell 112 based on flow cell images. Processing for cluster identification may be performed by a dedicated processor 118, one or more FPGAs 120, computing system 126, or a combination thereof. Identifying or determining cluster locations may involve performing conventional cluster lookup in conjunction with cluster lookup methods described more specifically herein.

[0186] General-purpose processors provide interfaces to run various programs in operating systems such as Windows™ or Linux™. Such operating systems can provide users with great flexibility.

[0187] In some embodiments, the dedicated processor 118 may be configured to perform operations of the cluster discovery methods described herein. The dedicated processor may not be a general-purpose processor, but rather a custom processor with specific hardware or instructions for performing these operations. The dedicated processor runs specific software directly without an operating system. The lack of an operating system reduces overhead, but at the cost of the processor's executable flexibility. Dedicated processors can use custom programming languages, which can be designed to operate more efficiently than software running on a general-purpose processor. This increases the speed of operation and allows for real-time processing.

[0188] In some embodiments, FPGA 120 can be configured to perform the cluster discovery method described herein. The FPGA is programmed to be hardware that will only perform specific tasks. Software operations can be translated into hardware components using a special programming language. Once the FPGA is programmed, the hardware directly processes the digital data provided to it without running software. Instead, the FPGA uses logic gates and registers to process digital data. Since the operating system does not require any overhead, FPGAs typically process data faster than general-purpose processors. Similar to dedicated processors, this comes at the cost of flexibility.

[0189] The lack of software overhead also allows FPGAs to operate faster than dedicated processors, although this will depend on the exact processing to be performed and the specific FPGA and dedicated processor.

[0190] A group of FPGAs 120 can be configured to perform these operations in parallel. For example, multiple FPGAs 120 can be configured to perform processing operations targeting cluster locations in an image, a set of images, or one or more images. Each FPGA 120 can execute its own portion of the processing operation simultaneously, thereby reducing the time required to process data. This allows processing steps to be completed in real time. Further discussion of the use of FPGAs is provided below.

[0191] Compared to methods that may require storing data before processing it (which may require more memory or access...)Compared to a computer system located in cloud 130, real-time execution of processing steps allows the system to use less memory because the data can be processed as it is received.

[0192] In some embodiments, data storage 122 is used to store information used in identifying cluster locations. This information may include the image itself or information derived from an image captured by imager 116. DNA sequences determined based on base interpretation may be stored in data storage 122. Parameters identifying cluster locations may also be stored in data storage 122.

[0193] User interface 124 may be used by a user to operate the sequencing system or access data stored in data storage 122 or computer system 126.

[0194] Computer system 126 may control the general operation of the sequencing system and may be coupled to user interface 124. It may also perform operations to identify cluster locations and base interpretation. In some embodiments, computer system 126 is a computer system. Computer system 126 may store information about the operation of sequencing system 110, such as configuration information, instructions for operating sequencing system 110, or user information. Computer system 126 may be configured to transfer information between sequencing system 110 and cloud 130.

[0195] As described above, sequencing system 110 may have a dedicated processor 118, FPGA 120, or computer system 126. The sequencing system may use one, two, or all of these elements to perform the necessary processing described above. In some embodiments, when these elements are present together, the processing tasks are separated between them. For example, one or more FPGAs 120 may be used to perform the cluster center finding method described herein, while computer system 126 may perform other processing functions for sequencing system 110. Various combinations of these elements allow for various system embodiments that balance the efficiency and speed of processing with the cost of the processing elements.

[0196] Cloud 130 may be a network, remote storage, or some other remote computing system separate from sequencing system 110. Connection to cloud 130 may allow access to data stored outside of sequencing system 110 or allow updates to software in sequencing system 110.

[0197] Support and Low Nonspecific Coating

[0198] In some embodiments, NGS sequencing compositions and methods (e.g., pairwise sequencing) employ a support containing a plurality of oligonucleotide surface primers immobilized thereon. In some embodiments, the support is passivated with a low nonspecific binding coating. The surface coating described herein exhibits very low nonspecific binding to reagents commonly used in nucleic acid capture, amplification, and sequencing workflows, such as dyes, nucleotides, enzymes, and nucleic acid primers. The surface coating exhibits a low background fluorescence signal or high contrast-to-noise (CNR) ratio compared to some other surface coatings. Specification 27 / 54 pages 34 CN 120936922 A

[0199] Low nonspecific binding coatings comprise one or more layers (Figure 11). In some embodiments, multiple surface primers are immobilized to the low nonspecific binding coating. In some embodiments, at least one surface primer is embedded within the low nonspecific binding coating. Low nonspecific binding coatings enable improved nucleic acid hybridization and amplification performance. Generally, a support comprises a substrate (or support structure), one or more layers of covalently or non-covalently attached low-binding chemically modified layers (such as silane layers or polymer films), and one or more covalently or non-covalently attached surface primers for tethering single-stranded nucleic acid library molecules to the support. In some embodiments, the formulation of the coating (e.g., the chemical composition of one or more layers, the coupling chemistry for crosslinking the one or more layers with the support and / or with each other, and the total number of layers) can be varied such that nonspecific binding of proteins, nucleic acid molecules, and other hybridization and amplification reaction components to the coating is minimized or reduced relative to a comparable monolayer. The formulation of the coatings described herein can be varied such that nonspecific hybridization on the coating is minimized or reduced relative to a comparable monolayer. The formulation of the coating can be varied such that nonspecific amplification on the coating is minimized or reduced relative to a comparable monolayer. The coating formulation can be varied to maximize the specific amplification rate and / or yield on the coating. In some cases disclosed herein, the amplification level suitable for detection is achieved in no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or more than 30 amplification cycles.

[0200] The support structure comprising the one or more chemically modified layers (e.g., layers of low nonspecific binding polymers) can be standalone or integrated into another structure or component. For example, in some embodiments, the support structure may include one or more surfaces within an integrated or assembled microfluidic flow cell. The support structure may include one or more surfaces within a microplate format (e.g., the bottom surface of a well in a microplate). In some embodiments, the support structure includes the inner surface of a capillary (such as an inner lumen surface). In some embodiments, the support structure includes the inner surface of a capillary etched into a planar chip (such as an inner lumen surface).

[0201] The attachment chemistry used to graft the first chemically modified layer onto the surface of the support will generally depend on both the material from which the surface is made and the chemical properties of the layer. In some embodiments, the first layer may be covalently attached to the surface. In some embodiments, the first layer may be non-covalently attached to the support (e.g., adsorbed onto the support) via non-covalent interactions (such as electrostatic interactions, hydrogen bonding, or van der Waals interactions) between the support and the molecular components of the first layer. In either case, the support may be treated prior to the attachment or deposition of the first layer. A variety of surface preparation techniques can be used to clean or...Surface treatment. For example, glass or silicon surfaces can be acid-washed using Piranha solution (a mixture of sulfuric acid (H2SO4) and hydrogen peroxide (H2O2), alkaline-treated with KOH and NaOH, and / or cleaned using oxygen plasma treatment methods.

[0202] Silane chemistry is a non-limiting method for covalently modifying silanol groups on glass or silicon surfaces to attach more reactive functional groups (e.g., amine or carboxyl groups), which can then be used to couple connector molecules (e.g., linear hydrocarbon molecules of various lengths (such as C6, C12, or C18 hydrocarbons) or linear polyethylene glycol (PEG) molecules) or layer molecules (e.g., branched PEG molecules or other polymers) to the surface. Examples of suitable silanes that can be used to generate any of the disclosed low-binding coatings include, but are not limited to: (3-aminopropyl)trimethoxysilane (APTMS), (3-aminopropyl)triethoxysilane (APTES), PEG-silanes (e.g., containing molecular weights of 1K, 2K, 5K, 10K, 20K, etc.), amino-PEG silanes (e.g., containing free amino functional groups), maleimide-PEG silanes, biotin-PEG silanes, etc.

[0203] A variety of molecules (including, but not limited to, amino acids, peptides, nucleotides, oligonucleotides, other monomers or polymers, or combinations thereof) can be used to generate the one or more chemically modified layers on the support, wherein the selection of the components used can be varied to alter one or more properties of the layer, such as the surface density of functional groups and / or tethered oligonucleotide primers, the hydrophilicity / hydrophobicity of the layer, or the three-dimensional properties of the layer (e.g., “thickness”). Examples of polymers that can be used to produce one or more layers of low-nonspecific binding material in any of the disclosed low-binding coatings include, but are not limited to: polyethylene glycol (PEG), streptavidin, polyacrylamide, polyester, dextran, polylysine and polylysine copolymers, or any combination thereof, of various molecular weights and branched structures. Examples of conjugation chemistry that can be used to graft one or more layers of material (e.g., polymer layers) onto a surface and / or crosslink the layers to each other include, but are not limited to: biotin-streptavidin interactions (or variants thereof), His-tag-Ni / NTA conjugation chemistry, methoxyether conjugation chemistry, carboxylic acid ester conjugation chemistry, amine conjugation chemistry, NHS esters, maleimide, thiols, epoxy resins, azides, hydrazides, alkynes, isocyanates, and silanes.

[0204] The low-nonspecific binding surface coating may be uniformly applied to the support. Alternatively, the surface coating may be patterned such that the chemically modified layer is confined to one or more discrete regions of the support. For example, photolithography can be used to pattern the coating to create an ordered array or random pattern of chemically modified regions on a support. Alternatively, or...In some embodiments, the coating can be patterned using techniques such as contact printing and / or inkjet printing. In some embodiments, the ordered array or random pattern of chemically modified regions may include at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10,000 or more discrete regions.

[0205] In some embodiments, the low nonspecific binding coating comprises a hydrophilic polymer that is nonspecifically adsorbed or covalently grafted onto a support. Passivation can be performed using poly(ethylene glycol) (PEG, also known as polyethylene oxide (PEO) or polyethylene oxide) or other hydrophilic polymers with different molecular weights and end groups chemically linked to the support, such as silanes. End groups distal to the surface may include, but are not limited to, biotin, methoxy ethers, carboxylic esters, amines, NHS esters, maleimides, and bissilanes. In some embodiments, two or more layers of a hydrophilic polymer (e.g., a linear polymer, a branched polymer, or a multibranched polymer) may be deposited on the surface. In some embodiments, the two or more layers may be covalently coupled to each other or internally crosslinked to improve the stability of the resulting coating. In some embodiments, surface primers (or other biomolecules, such as enzymes or antibodies) with different nucleotide sequences and / or base modifications may be tethered to the resulting layers at various surface densities. In some embodiments, for example, both the surface functional group density and the surface primer concentration may be varied to obtain a specified range of surface primer densities. Furthermore, the surface primer density can be controlled by diluting the surface primers with other molecules having the same functional groups. For example, amine-labeled surface primers can be diluted with amine-labeled polyethylene glycol in the reaction with an NHS-ester-coated surface to reduce the final primer density. Surface primers with different lengths of linkers between the hybridization region and the surface attachment functional groups can also be applied to control the surface density. Examples of suitable linkers include poly-T and poly-A chains (e.g., 0 to 20 bases) at the 5' end of the primer, PEG linkers (e.g., 3 to 20 monomer units), and carbon chains (e.g., C6, C12, C18, etc.). To measure primer density, fluorescently labeled primers can be tethered to the surface, and the fluorescence readings can then be compared with fluorescence readings of dye solutions of known concentrations.

[0206] In some embodiments, the low nonspecific binding coating comprises a functionalized polymer coating covalently bound to at least a portion of the support via chemical groups on the support, primers grafted onto the functionalized polymer coating, and primers on the primers.Water-soluble protective coatings and functionalized polymer coatings. In some embodiments, the functionalized polymer coating comprises poly(N-(5-azidoacetamidopentyl)acrylamide-co-acrylamide (PAZAM).

[0207] To scale primer surface density and add additional dimensions to hydrophilic or amphoteric coatings, supports comprising multilayer coatings of PEG and other hydrophilic polymers have been developed. Primer loading density on supports can be significantly increased by using hydrophilic and amphoteric surface layering methods (including, but not limited to, polymer / copolymer materials described below). Some PEG coating methods use monolayer primer deposition, which is generally reported for single-molecule applications but does not produce high copy numbers for nucleic acid amplification applications. As described herein, “layering” can be achieved using conventional crosslinking methods with any compatible polymer or monomer subunit, allowing surfaces comprising two or more highly crosslinked layers to be constructed sequentially. Examples of suitable polymers include, but are not limited to: streptavidin, polyacrylamide, polyester, dextran, polylysine, and polylysine and specification 29 / 54 pages 36 CN 120936922 A PEG copolymers. In some embodiments, different layers may be attached to each other via any of a variety of conjugation reactions (including, but not limited to, biotin-streptavidin binding, azide-alkyne click reaction, amine-NHS ester reaction, thiol-maleimide reaction, and ionic interactions between positively charged polymers and negatively charged polymers). In some embodiments, high primer density materials may be constructed in solution and subsequently layered onto a surface in multiple steps.

[0208] Examples of materials from which support structures may be fabricated include, but are not limited to, glass, fused silica, silicon, polymers (e.g., polystyrene (PS), macroporous polystyrene (MPPS), polymethyl methacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high-density polyethylene (HDPE), cyclic olefin polymers (COP), cyclic olefin copolymers (COC), polyethylene terephthalate (PET)) or any combination thereof. Various combinations of both glass and plastic support structures are contemplated.

[0209] The support structure can take on various geometries and sizes and can comprise a variety of materials. For example, the support structure can be partially planar (e.g., including a microscope slide or the surface of a microscope slide). Generally, the support structure can be cylindrical (e.g., including a capillary or the inner surface of a capillary), spherical (e.g., including the outer surface of a non-porous bead), or irregular (e.g., including the outer surface of an irregularly shaped non-porous bead or particle). In some embodiments, the surface of the support structure for nucleic acid hybridization and amplification can be a solid, non-porous surface. In some embodiments, the support structure for nucleic acid hybridization...The surface of the support structure for hybridization and amplification can be porous, allowing the coating described herein to penetrate the porous surface, and the nucleic acid hybridization and amplification reactions performed thereon to occur within the pores.

[0210] The support structure comprising one or more chemically modified layers (e.g., layers of low nonspecific binding polymers) can be standalone or integrated into another structure or component. For example, the support structure may comprise one or more surfaces within an integrated or assembled microfluidic flow cell. The support structure may comprise one or more surfaces within a microplate format (e.g., the bottom surface of a well in a microplate). In some embodiments, the support structure includes the inner surface of a capillary (such as an inner lumen surface). In some embodiments, the support structure includes the inner surface of a capillary etched into a planar chip (such as an inner lumen surface).

[0211] As noted, the low nonspecific binding supports of this disclosure exhibit reduced nonspecific binding to proteins, nucleic acids, and other components of hybridization and / or amplification formulations used for solid-phase nucleic acid amplification. The degree of nonspecific binding exhibited by a given support surface can be assessed qualitatively or quantitatively. For example, exposing a surface to a fluorescent dye (e.g., anthocyanins (such as Cy3 or Cy5), fluorescein, coumarin, rhodamine, or other dyes disclosed herein) under a standardized set of conditions, fluorescently labeled nucleotides, fluorescently labeled oligonucleotides, and / or fluorescently labeled proteins (e.g., polymerases), followed by a specified rinsing protocol and fluorescence imaging, can be used as a qualitative tool for comparing nonspecific binding on supports containing different surface formulations. In some embodiments, exposing a surface to a fluorescent dye, fluorescently labeled nucleotides, fluorescently labeled oligonucleotides, and / or fluorescently labeled proteins (e.g., polymerases) under a standardized set of conditions, followed by a specified rinsing protocol and fluorescence imaging, can be used as a quantitative tool for comparing nonspecific binding on supports containing different surface formulations. Care should be taken to ensure that fluorescence imaging is performed under conditions where the fluorescence signal is linearly correlated (or predictably correlated) with the number of fluorophores on the support surface and using suitable calibration standards (e.g., where signal saturation and / or fluorophore self-quenching are not problematic). In some embodiments, other techniques, such as radioisotope labeling and counting methods, may be used to quantitatively assess the degree of nonspecific binding exhibited by different support surface formulations of this disclosure.

[0212] Some surfaces disclosed herein exhibit at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, or greater than 100, or any intermediate value covered by the scope herein. (30 / 54 pages, 37 CN)120936922 A The ratio of specific to nonspecific binding of fluorophores (such as Cy3). Some surfaces disclosed herein exhibit a ratio of specific to nonspecific fluorescence of fluorophores (such as Cy3) of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, or greater than 100, or any intermediate value covered by the scope herein.

[0213] The degree of nonspecific binding exhibited by the disclosed low-binding supports can be evaluated using a standardized protocol for contacting a surface with labeled proteins (e.g., bovine serum albumin (BSA), streptavidin, DNA polymerase, reverse transcriptase, helicase, single-stranded binding protein (SSB), etc., or any combination thereof), labeled nucleotides, labeled oligonucleotides, etc., under a standardized set of incubation and rinsing conditions, followed by detection of the amount of label remaining on the surface and comparison of the resulting signal with an appropriate calibration standard. In some embodiments, the label may include a fluorescent label. In some embodiments, the label may include a radioisotope. In some embodiments, the label may include any other detectable label. In some embodiments, the degree of nonspecific binding exhibited by a given support surface formulation can therefore be evaluated based on the number of nonspecifically bound protein molecules (or nucleic acid molecules or other molecules) per unit area. In some embodiments, the low-binding supports of this disclosure may exhibit nonspecific protein binding (or nonspecific binding of other specified molecules (e.g., anthocyanins such as Cy3 or Cy5, fluorescein, coumarin, rhodamine, or other dyes disclosed herein) at locations falling within this range. In some embodiments, the low-binding supports of this disclosure may exhibit nonspecific binding at locations falling within this range (e.g., less than 86 molecules / μm²). For example, some of the modified surfaces disclosed herein exhibited nonspecific protein binding of less than 0.5 molecules / μm² after contacting with 1 μM Cy3-labeled streptavidin (GE Amersham) solution in phosphate-buffered saline (PBS) buffer for 15 minutes, followed by rinsing three times with deionized water. Some of the modified surfaces disclosed herein also exhibited nonspecific binding of Cy3 dye molecules of less than 0.25 molecules / μm². In independent nonspecific binding assays, 1 μM labeled Cy3 SA (ThermoFisher) and 1 μM Cy5 SA...Dyes (ThermoFisher), 10 μM aminoallyl-dUTP-ATTO-647N (Jena Biosciences), 10 μM aminoallyl-dUTP-ATTO-Rhol 1 (Jena Biosciences), 10 μM aminoallyl-dUTP-ATTO-Rhol 1 (Jena Biosciences), 10 μM 7-propyneamino-7-deazona-dGTP-Cy5 (Jena Biosciences), and 10 μM 7-propyneamino-7-deazona-dGTP-Cy3 (Jena Biosciences) were incubated in 384-well plates on low-binding coated supports at 37°C for 15 min. Each well was washed 2 to 3 times with 50 μL of deionized RNase-free / DNase-free water and 2 to 3 times with 25 mM MACES buffer (pH 7.4). Image the 384-well plate on a GE Typhoon instrument using a Cy3, AF555, or Cy5 filter set as specified by the manufacturer (depending on the dye test performed) at a PMT gain setting of 800 and a resolution of 50 to 100 μm. For higher resolution imaging, images were acquired on an Olympus IX83 microscope (e.g., an inverted fluorescence microscope) with a total internal reflection fluorescence (TIRF) objective (100×, 1.5NA, Olympus), a CCD camera (e.g., an Olympus EM-CCD monochrome camera, an Olympus XM-10 monochrome camera, or an Olympus DP80 color and monochrome camera), an illumination source (e.g., an Olympus 100W mercury lamp, an Olympus 75W xenon lamp, or an Olympus U-HGLGPS fluorescence source), and an excitation wavelength of 532 nm or 635 nm. Images were also acquired on an Olympus IX83 microscope (e.g., an inverted fluorescence microscope) (Olympus Corp., Center Valley, Pa.). Dichroic mirrors were purchased from Semrock (IDEX Health & Science, LLC, Rochester, NY), such as 405nm, 488nm, 532nm, or 633nm dichroic mirrors / beam splitters, and bandpass filters were selected as 532LP or 645LP integrated with appropriate excitation wavelengths. Some of the modified surfaces disclosed herein exhibit nonspecific binding of dye molecules less than 0.25 molecules / μm². In some embodiments, the coated support is immersed in a buffer solution (e.g., 25 mMACES, pH 7.4) while images are acquired.

[0214] In some embodiments, the surfaces disclosed herein exhibit at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, ... (Specification 31 / 54 pages 38 CN)120936922 A A ratio of specific to nonspecific binding to a fluorophore (such as Cy3) of 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, or greater than 100, or any intermediate value covered herein. In some embodiments, the surfaces disclosed herein exhibit a ratio of specific to nonspecific fluorescence signal for a fluorophore (such as Cy3) of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, or greater than 100, or any intermediate value covered herein.

[0215] Low background surfaces conforming to the disclosure herein may exhibit a specific dye attachment (e.g., Cy3 attachment) to nonspecific dye adsorption (e.g., Cy3 dye adsorption) ratio of at least 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 30:1, 40:1, 50:1, or more than 50 specific dye molecules attached to each nonspecifically adsorbed molecule. Similarly, when subjected to excitation energy, a low-background surface with attached fluorophores (e.g., Cy3) conforming to the disclosure herein may exhibit a ratio of at least 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 30:1, 40:1, 50:1, or greater than 50:1 to a specific fluorescence signal (e.g., generated from Cy3-labeled oligonucleotides attached to the surface) to a fluorescence signal from a non-specifically adsorbed dye.

[0216] In some embodiments, the degree of hydrophilicity (or “wetability” with an aqueous solution) of the disclosed support surface may be assessed, for example, by measuring the water contact angle, wherein a small drop of water is placed on the surface and its contact angle with the surface is measured using, for example, an optical tensiometer. In some embodiments, a static contact angle may be determined. In some embodiments, a forward or backward contact angle may be determined. In some embodiments, the water contact angle disclosed herein for a low-binding hydrophilic support surface may be in the range of about 0 degrees to about 30 degrees. In some embodiments, the water contact angles disclosed herein for hydrophilic, low-binding support surfaces may not exceed 50 degrees, 40 degrees, 30 degrees, 25 degrees, 20 degrees, 18 degrees, 16 degrees, 14 degrees, 12 degrees, 10 degrees, 8 degrees, 6 degrees, 4 degrees, 2 degrees, or 1 degree. In many cases, the contact angle does not exceed 40 degrees. A given hydrophilic, low-binding support surface of this disclosure may exhibit a water contact angle with values ​​at any location within this range.

[0217] In some embodiments, the hydrophilic surfaces disclosed herein contribute to reducing washing time for bioassays, typically due to reduced nonspecific binding of biomolecules to low-binding surfaces. In some embodiments, smallA sufficient washing step is performed for 60, 50, 40, 30, 20, 15, 10 seconds or less. For example, a sufficient washing step may be performed for less than 30 seconds.

[0218] Some low-binding surfaces of this disclosure exhibit significant improvements in stability or durability with prolonged exposure to solvents and elevated temperatures or with repeated cycles of solvent exposure or temperature changes. For example, the stability of the disclosed surfaces can be tested by fluorescently labeling functional groups on the surface or tethered biomolecules (e.g., oligonucleotide primers) on the surface, and monitoring the fluorescence signal before, during, and after prolonged exposure to solvents and elevated temperatures or repeated cycles of solvent exposure or temperature changes. In some embodiments, the degree of fluorescence change used to assess surface quality may be less than 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours, 50 hours, or 100 hours (or any combination of these percentages as measured within these time periods) over a period of 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 hours, 10 hours, 15%, 20% or 25% of the fluorescence after exposure to solvents and / or elevated temperatures. In some embodiments, the degree of change in fluorescence used to assess surface quality may be less than 1%, 2%, 3%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1,000 cycles of repeated exposure to solvent variations and / or temperature variations (or any combination of these percentages as measured by the range of cycles). Specification 32 / 54 pages 39 CN 120936922 A

[0219] In some embodiments, the surfaces disclosed herein may exhibit a high ratio of specific signals to non-specific signals or other background. For example, when used for nucleic acid amplification, some surfaces may exhibit amplification signals that are at least 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 75, 100, or greater than 100 times greater than those in adjacent unpopulated regions. Similarly, some surfaces exhibit amplification signals that are significantly greater than those in adjacent unpopulated regions.The signal of the adjacent amplified nucleic acid population region is amplified by a factor of at least 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 75, 100 or greater than 100.

[0220] In some embodiments, when used for nucleic acid hybridization or amplification applications to generate communities of hybridized or clonal amplified nucleic acid molecules (e.g., directly or indirectly labeled with fluorophores), the disclosed low background surface fluorescence image exhibits a contrast-to-noise ratio (CNR) of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 210, 220, 230, 240, 250 or greater than 250.

[0221] One or more types of primers may be attached or tethered to the surface of the support. In some embodiments, the one or more types of adaptors or primers may include: spacer sequences, adaptor sequences for hybridizing with target library nucleic acid sequences linked to the adaptor, forward amplification primers, reverse amplification primers, sequencing primers and / or molecular barcode sequences, or any combination thereof. In some embodiments, one primer or adaptor sequence may be tethered to at least one layer of the surface. In some embodiments, at least two, three, four, five, six, seven, eight, nine, ten, or more than ten different primer or adaptor sequences may be tethered to at least one layer of the surface.

[0222] In some embodiments, the length of the tethered adaptor and / or primer sequences may range from about 10 nucleotides to about 100 nucleotides. In some embodiments, the length of the tethered adaptor and / or primer sequence may be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 nucleotides. In some embodiments, the length of the tethered adaptor and / or primer sequence may be at most 100, at most 90, at most 80, at most 70, at most 60, at most 50, at most 40, at most 30, at most 20, or at most 10 nucleotides. Any of the lower and upper limits described in this paragraph may be combined to form a range included within this disclosure; for example, in some embodiments, the length of the tethered adaptor and / or primer sequence may be in the range of about 20 nucleotides to about 80 nucleotides. The length of the tethered adaptor and / or primer sequence may have any value within this range, for example, about 24 nucleotides.

[0223] In some embodiments, the resulting surface density of primers (e.g., capture primers) on the low-binding support surface of this disclosure can be in the range of about 100 primer molecules / μm² to about 100,000 primer molecules / μm². In some embodimentsIn this disclosure, the resulting surface density of primers on the low-binding support surface can range from about 1,000 primer molecules / μm² to about 1,000,000 primer molecules / μm². In some embodiments, the primer surface density can be at least 1,000, at least 10,000, at least 100,000, or at least 1,000,000 molecules / μm². In some embodiments, the primer surface density can be at most 1,000,000, at most 100,000, at most 10,000, or at most 1,000 molecules / μm². Any of the lower and upper limits described in this paragraph can be combined to form a range included in this disclosure; for example, in some embodiments, the primer surface density can range from about 10,000 molecules / μm² to about 100,000 molecules / μm². The surface density of primer molecules can have any value within this range, for example, about 455,000 molecules / μm2. In some embodiments, the surface density of the target library nucleic acid sequence initially hybridized with the adaptor or primer sequence on the support surface may be less than or equal to the surface density indicated by the surface density of the tethered primer. In some embodiments, the surface density of the target library nucleic acid sequence clonedly amplified with the adaptor or primer sequence on the support surface may span the same range as indicated by the surface density of the tethered primer.

[0224] The local densities listed above do not exclude variations in density across the surface, such that the surface may include regions having oligonucleotide densities such as 500,000 / μm2, as described on pages 33 / 54 of the specification (CN 120936922 A), while also including at least a second region having substantially different local densities.

[0225] Contrast-to-Noise Ratio (CNR)

[0226] In some embodiments, fluorescence imaging techniques can be used to evaluate the performance of nucleic acid hybridization, amplification reactions, or combinations thereof, using the disclosed reaction formulations and low-binding supports, wherein the contrast-to-noise ratio (CNR) of the image provides a key metric for evaluating amplification specificity and non-specific binding on the support. CNR is defined as: CNR = (Signal - Background) / Noise. The background term is considered as the signal measured for the gap region around a specific feature (diffraction limited spot, DLS) in a specified region of interest (ROI). While signal-to-noise ratio (SNR) is generally considered a benchmark for overall signal quality, it has been demonstrated that in applications requiring rapid image capture (e.g., sequencing applications where cycle time must be minimized), an improved CNR can provide a significant advantage over SNR as a benchmark for signal quality, as illustrated in the examples below. At high CNR, even slight improvements in CNR can significantly reduce the need for accurate differentiation (in order to achieve a high signal quality).And therefore, the imaging time required for accurate base interpretation in sequencing applications. Improved CNR in imaging data regarding imaging integration time provides a method for more accurate detection of features such as clonal amplified nucleic acid colonies on support surfaces.

[0227] In some ensemble-based sequencing methods, background terms can be measured as signals associated with “gap” regions. In addition to the “gap” background (B-gap), the “intrastitial” background (B-endoplasm) exists within regions occupied by amplified DNA colonies. The combination of these two background signals determines the achievable CNR and subsequently directly affects optical instrumentation requirements, architecture costs, reagent costs, runtime, cost / genome, and ultimately accuracy and data quality for cyclic array-based sequencing applications. B-gap background signals have a variety of sources; some examples include: autofluorescence from consumable flow cells, nonspecific adsorption of detection molecules that produce false fluorescence signals (which may mask signals from ROI), and the presence of nonspecific DNA amplification products (e.g., those generated from primer dimers). In some next-generation sequencing (NGS) applications, this background signal in the current field of view (FOV) is averaged and subtracted over time. Signals generated from individual DNA colonies (e.g., (signal)-B (gap) in the FOV) produce classifiable features. In some embodiments, the endoplasmic background (B (endoplasm)) can contribute a mixed fluorescent signal that is not specific to the target of interest but is present in the same ROI, thus making averaging and subtraction much more difficult.

[0228] Nucleic acid amplification on the low-binding coated support described herein can reduce the B (gap) background signal by reducing nonspecific binding, leading to improvements in specific nucleic acid amplification and reductions in nonspecific amplification (which can affect the background signal generated from both the gap and endoplasmic regions). Compared to some methods, the disclosed low-binding coated support (optionally used in combination with the disclosed hybridization and / or amplification reaction formulations) can cause improvements in CNR by 2-fold, 5-fold, 10-fold, 100-fold, 250-fold, 500-fold, or 1000-fold. Although described herein in the context of using fluorescence imaging as a readout or detection mode, the same principles apply to the use of the disclosed low-binding coated supports and nucleic acid hybridization and amplification formulations for other detection modes, including both optical and non-optical detection modes.

[0229] Methods for Sequencing

[0230] This disclosure provides methods for using an autofocusing optical system for sequencing immobilized or non-immobilized template nucleotide molecules. In some embodiments, the immobilized template molecule comprises a plurality of nucleic acid template molecules having one copy of a target sequence of interest. In some embodiments, the nucleic acid template molecule having one copy of a target sequence of interest can be...The linear library molecules are used for bridge amplification to generate the sequence. In some embodiments, the immobilized template molecule comprises a plurality of nucleic acid template molecules, each having two or more tandem copies (e.g., tandem copies) of the target sequence of interest. In some embodiments, the nucleic acid template molecule containing the tandem copy molecule can be generated by rolling circle amplification of the circularized linear library molecules. In some embodiments, the non-immobilized template molecule comprises a circular molecule. In some embodiments, the method for sequencing employs a soluble (e.g., non-immobilized) sequencing polymerase or a sequencing polymerase immobilized to a support.

[0231] In some embodiments, the sequencing reaction employs a detectably labeled nucleotide analog. In some embodiments, the sequencing reaction employs a two-stage sequencing reaction, including binding a detectably labeled multivalent molecule and incorporating a nucleotide analog. In some embodiments, the sequencing reaction employs an unlabeled nucleotide analog. In some embodiments, the sequencing reaction employs a phosphate ester chain labeled nucleotide.

[0232] Multivalent molecule

[0233] This disclosure provides a method for an autofocusing optics system for sequencing template nucleic acid molecules. In some embodiments, a sample fixed or otherwise positioned on a support may include at least one multivalent molecule. In some embodiments, a sample used for autofocusing an optical system may include at least one multivalent molecule. In some embodiments, a sequencing method using an optical system for imaging may employ at least one multivalent molecule. In some embodiments, a sequencing method using an optical system for imaging may include autofocusing the optical system prior to imaging one or more surfaces during a flow cycle of a sequencing run.

[0234] In some embodiments, the multivalent molecule comprises a plurality of nucleotide arms attached to a nucleus and having any conformation, including starburst, spiral ladder, or bottle brush conformations (e.g., FIG. 12). The multivalent molecule comprises: (1) a nucleus; and (2) a plurality of nucleotide arms comprising: (i) a nucleus attachment portion; (ii) a spacer comprising a PEG portion; (iii) a linker; and (iv) a nucleotide unit, wherein the nucleus is attached to the plurality of nucleotide arms, wherein the spacer is attached to the linker, and wherein the linker is attached to the nucleotide unit. In some embodiments, the nucleotide unit comprises a base, a sugar, and at least one phosphate ester group, and the linker is attached to the nucleotide unit via a base. In some embodiments, the linker comprises an aliphatic chain or an oligoethylene glycol chain, wherein both linker chains have 2 to 6 subunits. In some embodiments, the linker further comprises an aromatic moiety. An example of a nucleotide arm is shown in Figure 16. Examples of multivalent molecules are shown in Figures 12 to 15. An example of a spacer (top) is shown in Figure 17, and examples of linkers are shown in Figures 17 (bottom) and 18. Nucleosides attached to the linker are shown in Figures 19 to 22.Examples of acids. An example of a biotinylated nucleotide arm is shown in Figure 23.

[0235] In some embodiments, the multivalent molecule comprises a core attached to a plurality of nucleotide arms, wherein said plurality of nucleotide arms have the same type of nucleotide unit selected from the group consisting of: dATP, dGTP, dCTP, dTTP, and dUTP.

[0236] In some embodiments, the multivalent molecule comprises a core attached to a plurality of nucleotide arms, wherein each arm comprises a nucleotide unit. The nucleotide unit comprises an aromatic base, a pentose sugar (e.g., ribose or deoxyribose), and one or more phosphate groups (e.g., 1 to 10 phosphate groups). The plurality of multivalent molecules may comprise a type of multivalent molecule having a type of nucleotide unit selected from the group consisting of: dATP, dGTP, dCTP, dTTP, and dUTP. The plurality of multivalent molecules may comprise a mixture of any combination of two or more types of multivalent molecules, wherein the individual multivalent molecules in the mixture comprise nucleotide units selected from the group consisting of: dATP, dGTP, dCTP, dTTP, dUTP, or combinations thereof.

[0237] In some embodiments, the nucleotide unit comprises a chain of one, two, or three phosphorus atoms, wherein the chain is attached to the 5' carbon of the sugar moiety via an ester bond or a phosphoramide bond. In some embodiments, at least one nucleotide unit is a nucleotide analog having a phosphorus chain, wherein the phosphorus atoms are linked together by an intermediate O, S, NH, methylene, or ethylene group. In some embodiments, the phosphorus atom in the chain comprises a substituted side group (including O, S, or BH3). In some embodiments, the chain comprises a phosphate ester group substituted with an analog, which comprises a phosphoramide, a thiophosphate, a dithiophosphate, and an O-methylphosphoramide group.

[0238] In some embodiments, the multivalent molecule comprises a core linked to a plurality of nucleotide arms, and wherein each nucleotide arm comprises a nucleotide unit having a chain termination portion (e.g., a blocking portion) at the 2' position of the sugar, at the 3' position of the sugar, or at the 2' and 3' positions of the sugar. In some embodiments, the nucleotide unit includes a chain termination portion (e.g., a blocking portion) at the 2' position of the sugar, at the 3' position of the sugar, or at both the 2' and 3' positions of the sugar. In some embodiments, the chain termination portion may inhibit polymerase-catalyzed incorporation of subsequent nucleotide units or free nucleotides into the nascent chain during primer extension reactions. In some embodiments, the chain termination portion is attached to the 3' sugar position, wherein the sugar comprises a ribose or deoxyribose portion. In some embodiments, the chain termination portion may be removed / cleaved from the 3' sugar position to produce a nucleotide having a 3'OH sugar group, which may be extended with subsequent nucleotides in a polymerase-catalyzed nucleotide incorporation reaction. In some embodiments, the chain termination portionThe chain terminator comprises alkyl, alkenyl, alkynyl, allyl, aryl, benzyl, azide, amine, amide, ketone, isocyanate, phosphate, thio, disulfide, carbonate, urea, or silyl groups. In some embodiments, the chain terminator may be cleaved / removed from the nucleotide unit, for example, by reacting the chain terminator with a chemical agent, pH change, light, or heat. In some embodiments, the alkyl, alkenyl, alkynyl, and allyl chain terminators may be cleaved with tetrakis(triphenylphosphine)palladium(O) (Pd(PPh3)4), piperidine, or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). In some embodiments, the aryl and benzyl chain terminators may be cleaved with H2 Pd / C. In some embodiments, the amine, amide, ketone, isocyanate, phosphate, sulfur, and disulfide chain terminators may be cleaved with phosphine or with thiol groups (including β-mercaptoethanol or dithiothreitol (DTT)). In some embodiments, the chain-terminating carbonate portion can be cleaved using potassium carbonate (K2CO3) in MeOH, triethylamine in pyridine, or Zn in acetic acid (AcOH). In some embodiments, the chain-terminating urea and silyl groups can be cleaved using tetrabutylammonium fluoride, pyridine-HF, ammonium fluoride, or triethylamine trihydrofluoride.

[0239] In some embodiments, the nucleotide unit comprises a chain-terminating portion (e.g., a blocking portion) at the 2' position of the sugar, at the 3' position of the sugar, or at both the 2' and 3' positions of the sugar. In some embodiments, the chain-terminating portion comprises an azide, an azide group, or an azide methyl group. In some embodiments, the chain-terminating portion comprises a 3'-O-azido group or a 3'-O-azido methyl group. In some embodiments, the chain-terminating azide, azide group, and an azide methyl group can be cleaved / removed using a phosphine compound. In some embodiments, the phosphine compound comprises a derived trialkylphosphine portion or a derived triarylphosphine portion. In some embodiments, the phosphine compound includes: tris(2-carboxyethyl)phosphine (TCEP), or bissulfotriphenylphosphine (BS-TPP), or tris(hydroxypropyl)phosphine (THPP). In some embodiments, the cleavage agent includes 4-dimethylaminopyridine (4-DMAP).

[0240] In some embodiments, the nucleotide unit includes a chain termination portion selected from the group consisting of: 3'-deoxynucleotide, 2',3'-dideoxynucleotide, 3'-methyl, 3'-azido, 3'-azidomethyl, 3'-O-azidoalkyl, 3'-O-ethynyl, 3'-O-aminoalkyl, 3'-O-fluoroalkyl, 3'-fluoromethyl, 3'-difluoromethyl, 3'-trifluoromethyl, 3'-sulfonyl, 3'-malonyl, 3'-amino, 3'-O-amino, 3'-mercapto, 3'-aminomethyl, 3'-ethyl, 3'-butyl, 3'-tert-butyl,3'-fluorenylmethoxycarbonyl, 3'-tert-butoxycarbonyl, 3'-O-alkylhydroxyamino, 3'-thiophosphate, and 3-O-benzyl, and their derivatives.

[0241] In some embodiments, the multivalent molecule includes a core attached to a plurality of nucleotide arms, wherein the nucleotide arms include spacers, linkers, and nucleotide units, and wherein the core, linkers, and / or nucleotide units are labeled with a detectable reporter gene portion. In some embodiments, the detectable reporter gene portion includes a fluorophore. In some embodiments, a specific detectable reporter gene portion (e.g., a fluorophore) attached to the multivalent molecule may correspond to a base of a nucleotide unit (e.g., dATP, dGTP, dCTP, dTTP, or dUTP) to allow detection and identification of nucleotide bases.

[0242] In some embodiments, at least one nucleotide arm of the multivalent molecule has a nucleotide unit attached to a detectable reporter gene portion. In some embodiments, the detectable reporter gene portion is attached to a nucleotide base. In some embodiments, the detectable reporter gene portion includes a fluorophore. In some embodiments, a specific detectable gene portion (e.g., a fluorophore) attached to the multivalent molecule may correspond to a base of a nucleotide unit (e.g., dATP, dGTP, dCTP, dTTP, or dUTP) to allow detection and identification of nucleotide bases.

[0243] In some embodiments, the core of the multivalent molecule comprises an avidin-like or streptavidin-like portion, and the core attachment portion comprises biotin. In some embodiments, the core comprises a streptavidin-type or avidin-type portion (containing avidin protein), and any derivative, analogue, and other non-natural forms of avidin that can bind to at least one biotin portion. Other forms of the avidin portion include natural and recombinant avidin and streptavidin, as well as derived molecules, such as non-glycosylated avidin and truncated streptavidin. For example, the avidin moiety includes a deglycosylated form of avidin, bacterial streptavidin produced by Streptomyces (e.g., Streptomyces averdin), and derived forms such as N-acylavidin, such as N-acetyl, N-phthalyl, and N-succinylavidin, as well as commercially available products EXTRAVIDIN, CAPTAVIDIN, NEUTRAVIDIN, and NEUTRAALITE AVIDIN.

[0244] In some embodiments, any method for sequencing the nucleic acid molecules described herein may include forming a binding complex comprising (i) a polymerase, a primer-double-helicalized nucleic acid template molecule, and nucleotides, or the binding complex comprising (ii) a polymerase, a primer-double-helicalized nucleic acid template molecule, and nucleotides of a multivalent molecule.Unit. In some embodiments, the binding complex has a residence time greater than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 second. The binding complex has a residence time greater than about 0.1 to 0.25 seconds, or about 0.25 to 0.5 seconds, or about 0.5 to 0.75 seconds, or about 0.75 to 1 second, or about 1 to 2 seconds, or about 2 to 3 seconds, or about 3 to 4 seconds, or about 4 to 5 seconds, and / or the method is performed at or may be performed at temperatures of: 15°C or higher, 20°C or higher, 25°C or higher, 35°C or higher, 37°C or higher, 42°C or higher, 55°C or higher, 60°C or higher, 72°C or higher, or 80°C, or within the range defined by any of the foregoing. The binding complex (e.g., a ternary complex) remains stable before being subjected to conditions that cause dissociation between the polymerase, template molecule, primer, and / or nucleotide unit or any of the nucleotides. For example, dissociation conditions include contacting the binding complex with any of or any combination of detergent, EDTA, and / or water. In some embodiments, this disclosure provides a method in which the binding complex is deposited onto, attached to, or hybridized to a surface that exhibits a contrast-to-noise ratio greater than 20 in a detection step. In some embodiments, this disclosure provides a method in which contact is performed under conditions that stabilize the binding complex when the nucleotide or nucleotide unit is complementary to the next base of the template nucleic acid and destabilize the binding complex when the nucleotide or nucleotide unit is not complementary to the next base of the template nucleic acid.

[0245] Methods for sequencing using phosphate-labeled nucleotides

[0246] In some embodiments, the methods herein can be used for autofocusing of an optical system that can be used for sequencing using an immobilized sequencing polymerase that binds to a non-immobilized template molecule. This disclosure provides a method for sequencing using an immobilized sequencing polymerase bound to a non-immobilized template molecule, wherein a sequencing reaction is performed with phosphate-labeled nucleotides. In some embodiments, the sequencing method includes the action (a): providing a support to which a plurality of sequencing polymerases are immobilized. In some embodiments, the sequencing polymerase includes a persistent DNA polymerase. In some embodiments, the sequencing polymerase comprises a wild-type or mutant DNA polymerase, including, for example, Phi29 DNA polymerase. In some embodiments, the support comprises a plurality of individual compartments, and the sequencing polymerase is immobilized to the bottom of the compartments. In some embodiments, the individual compartments comprise a silica bottom that is light-permeable. In some embodiments, the individual compartments comprise a silica bottom configured with a nanophotonic confinement structure comprising pores in a metal-coated membrane (e.g., an aluminum-coated membrane). In some embodiments, the metal-coated membrane...The pores have small apertures, for example, about 70 nm. In some embodiments, the height of the nanophotonic confinement structure is about 100 nm. In some embodiments, the nanophotonic confinement structure includes a zero-mode waveguide (ZMW). In some embodiments, the nanophotonic confinement structure contains a liquid. Specification 37 / 54 pages 44 CN 120936922 A

[0247] In some embodiments, the sequencing method further includes operation (b): contacting multiple immobilized sequencing polymerases with multiple single-stranded circular nucleic acid template molecules and multiple oligonucleotide sequencing primers under conditions suitable for binding of individual immobilized sequencing polymerases to single-stranded circular template molecules and suitable for hybridization of individual sequencing primers to individual single-stranded circular template molecules, thereby generating multiple polymerase / template / primer complexes. In some embodiments, individual sequencing primers hybridize to universal sequencing primer binding sites on single-stranded circular template molecules.

[0248] In some embodiments, the sequencing method further includes the operation (c): contacting a plurality of polymerase / template / primer complexes with a plurality of phosphate-ester-tagged nucleotides, each phosphate-ester-tagged nucleotide comprising an aromatic base, a pentose sugar (e.g., ribose or deoxyribose), and a phosphate ester chain comprising 3 to 20 phosphate groups, wherein the terminal phosphate group is linked to a detectable reporter gene moiety (e.g., a fluorophore). The first, second, and third phosphate groups may be referred to as α, β, and γ phosphate groups. In some embodiments, the specific detectable reporter gene moiety attached to the terminal phosphate group corresponds to a nucleotide base (e.g., dATP, dGTP, dCTP, dTTP, or dUTP) to allow detection and identification of nucleotides. In some embodiments, the plurality of polymerase / template / primer complexes are contacted with a plurality of phosphate-ester-tagged nucleotides under conditions suitable for polymerase-catalyzed nucleotide incorporation. In some embodiments, the sequencing polymerase is capable of binding to a complementary phosphate-ester-tagged nucleotide and incorporating a complementary nucleotide to a nucleotide in the template molecule. In some embodiments, the polymerase-catalyzed nucleotide incorporation reaction involves cleavage between an α-phosphate group and a β-phosphate group, thereby releasing a polyphosphate chain linked to the fluorophore.

[0249] In some embodiments, the sequencing method further includes operation (d): detecting a fluorescent signal emitted by a phosphate-labeled nucleotide, which is bound by a sequencing polymerase and incorporated into the end of a sequencing primer. In some embodiments, operation (d) further includes identifying the phosphate-labeled nucleotide bound by the sequencing polymerase and incorporated into the end of the sequencing primer.

[0250] In some embodiments, the sequencing method further includes operation (e): repeating steps (c) through (d) at least once. In some embodiments, the sequencing method using phosphate-labeled nucleotides is based on U.S. Patent No. 7,170.The methods described in 7,050; 7,302,146; and / or 7,405,281, each of which is incorporated herein by reference in its entirety.

[0251] The headings provided herein are not intended to limit the individual aspects of this disclosure, which can be understood by referring to the specification as a whole.

[0252] Certain Terms

[0253] Unless otherwise defined, the technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art, unless otherwise defined. Generally, terms related to the techniques of molecular biology, nucleic acid chemistry, protein chemistry, genetics, microbiology, transgenic cell production, and hybridization described herein are terms well-known and commonly used in the art. The techniques and procedures described herein are generally performed according to conventional methods well-known in the art and as described in the various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 2000). See also Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992). The nomenclature used in conjunction with the laboratory procedures and techniques described herein is well-known and commonly used in the art.

[0254] Unless the context otherwise requires, singular terms shall include plurals, and plural terms shall include singulars. Unless explicitly and definitively limited to one referent, the singular forms “a / an” and “the” and any singular use of the word encompass multiple referents.

[0255] It should be understood that the use of alternative terms (e.g., “or”) is considered to mean one or both of the alternatives or any combination thereof.

[0256] The term “and / or” as used herein shall be considered to mean each of the specified features or components with or without the other in a specific disclosure. For example, the term "and / or" as used in phrases such as "A and / or B" herein is intended to include: "A and B"; "A or B"; "A" (A alone); and "B" (B alone). Similarly, the term "and / or" as used in phrases such as "A, B and / or C" herein is intended to cover each of the following: "A, B and C"; "A, B or C"; "A or C"; "A"or B"; "B or C"; "A and B"; "B and C"; "A and C"; "A" (A alone); "B" (B alone); and "C" (C alone).

[0257] As used herein and in the appended claims, the terms "comprising," "including," "having," and "containing," and their grammatical variations, are intended to be non-limiting, such that one or more items in the list do not exclude other items that may be substituted for or added to the listed items. It should be understood that whenever an aspect is described herein with the language "comprising," other similar aspects described as "consisting of" and / or "substantially consisting of" are also provided.

[0258] As used herein, the terms “about,” “approximately,” and “substantially” mean a value or composition within an acceptable range of error for a particular value or composition, as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, according to practice in the art, “about” or “substantially” may mean within one or more standard deviations. Alternatively, “about” or “approximately” may mean a range of up to 10% (i.e., ±10%) or greater, depending on the limitations of the measurement system. For example, about 5 mg may include any number between 4.5 mg and 5.5 mg. Furthermore, specifically with respect to biological systems or processes, the term may mean up to an order of magnitude or up to 5 times the value. When a particular value or composition is provided in this disclosure, unless otherwise stated, the meaning of “about,” “approximately,” and “substantially” should be assumed to be within an acceptable range of error for said particular value or composition. Furthermore, in the case of providing ranges and / or subranges of values, the range and / or subranges may include the endpoints of the range and / or subranges.

[0259] As used herein, the term "polony" refers to a nucleic acid library molecule that can be clonally amplified in solution or on a support to produce an amplicon that can serve as a template molecule for sequencing. In some embodiments, linear library molecules may be circularized to produce circularized library molecules, and circularized library molecules may be clonally amplified in solution or on a support to produce tandem molecules. In some embodiments, tandem molecules may serve as nucleic acid template molecules that can be sequenced. Tandem molecules are sometimes referred to as polony. In some embodiments, a polony comprises nucleotide chains.

[0260] References herein to "an embodiment," "an example embodiment," "some embodiments," or similar phrases indicate that the described embodiments may include specific features, structures, or characteristics, but not every embodiment necessarily includes specific features, structures, or characteristics. Furthermore, such phrases do not necessarily refer to the same embodiment. In addition, when a specific feature, structure, or characteristic is described in relation to an embodiment, incorporating such feature, structure, or characteristic into other embodiments (whether expressly mentioned or described herein) will be within the knowledge of those skilled in the art.

[0261] It should be understood that the Detailed Description section, and not any other section, is intended to interpret the claims. Other sections may set forth one or more exemplary embodiments contemplated by the inventors, but not all exemplary embodiments, and therefore are not intended to limit this disclosure or the appended claims in any way.

[0262] Although this disclosure describes exemplary embodiments of some fields and applications, it should be understood that this disclosure is not limited thereto. Other embodiments and modifications thereof are possible and within the scope and spirit of this disclosure. For example, and without limiting the generality of this paragraph, the embodiments are not limited to the software, hardware, firmware, and / or entities shown in the figures and / or described herein. Furthermore, the embodiments (whether or not explicitly described herein) have significant utility in fields and applications beyond those described herein.

[0263] Embodiments have been described herein by means of functional building blocks that illustrate implementations of specified functions and their relationships. For ease of description, the boundaries of these functional building blocks have been arbitrarily defined herein. Alternative boundaries can be defined by properly performing the functions and relationships (or their equivalents) specified in the specification (pages 39 / 54, 46 CN 120936922 A). Similarly, alternative embodiments may use a different order of execution for functional blocks, steps, operations, methods, etc. than those described herein.

[0264] Examples

[0265] These examples are provided for illustrative purposes only and are not intended to limit the scope of the claims provided herein.

[0266] Example 1 – Optical System Efficiency

[0267] Figures 32A and 32B provide the diffraction modulation transfer function (MTF) of optical systems according to some embodiments. Figure 32A shows the MTF of an objective-based system with p = 480 nm, while Figure 32B shows the MTF of the optical system of this disclosure with p = 500 nm. As can be seen from the figures, the optical system of this disclosure has higher overall efficiency at all spatial frequencies than the objective-based system, and the system of this disclosure provides a much larger field of view (9 mm, compared to 2 mm for the objective-based system). Figures 33A and 33B illustrate wavefront analysis calculations of the optical system of this disclosure according to some embodiments. In both cases, the optical system exhibits low compound root mean square errors of approximately 24 mλ and 26 mλ, respectively, within a wide field of view (e.g., 9 mm).

[0268] Figures 34 and 35 show the optical performance curves of the top and bottom surfaces according to some embodiments. In both cases, longitudinal spherical aberration, astigmatism curves, and distortion are all low, indicating improved optical (e.g., imaging) performance. Figure 36 shows a plot of the MTF of the optical system according to some embodiments. The MTF plot can show a wide depth of field (e.g., ±1 μm) with low field curvature, which can indicate the optical system's ability to provide wide-area, large depth-of-field imaging, capable of simultaneously imaging on a solid support.Multiple samples are imaged. In some cases, wide depth of field enables multi-surface imaging (e.g., simultaneous imaging of multiple surfaces of a solid support). Figure 37 shows a cumulative probability plot of achieving a given wavefront error according to some embodiments. This plot shows data on low wavefront errors achievable with the system of this disclosure.

[0269] Embodiments of the Numbered Parts of this Disclosure

[0270] 1. An optical system comprising:

[0271] a stage configured to hold a solid support;

[0272] a light source configured to illuminate the solid support; and

[0273] an optical assembly at least partially disposed in an optical path from the stage to the light source, wherein the optical assembly is configured to provide illumination over an area of ​​the solid support greater than about 20 square millimeters (mm2), wherein the peak-to-valley variation is at most about 5%.

[0274] 2. The optical system according to any of the preceding embodiments, wherein the optical assembly does not include an objective lens.

[0275] 3. The optical system according to any one of the foregoing embodiments, wherein the optical system does not include the objective lens.

[0276] 4. The optical system according to any one of the foregoing embodiments, wherein the optical component does not include a barrel lens.

[0277] 5. The optical system according to any one of the foregoing embodiments, wherein the optical system does not include the barrel lens.

[0278] 6. The optical system according to any one of the foregoing embodiments, wherein the stage is not adjusted on the optical axis of the system.

[0279] 7. The optical system according to any one of the foregoing embodiments, wherein the irradiance of the illumination is at least about 40 milliwatts per square millimeter.

[0280] 8. The optical system according to any one of the foregoing embodiments, wherein the optical component is configured to receive emitted light from the solid support.

[0281] 9. The optical system according to any one of the foregoing embodiments, wherein the numerical aperture (NA) of the optical component is at least about 0.3.

[0282] 10. The optical system according to any one of the foregoing embodiments, wherein the wavelength of the emitted light is about 500 nanometers to about 750 nanometers.

[0283] 11. The optical system according to any one of the foregoing embodiments, wherein the working distance of the optical component is at least about 1 mm to 25 mm.

[0284] 12. The optical system according to any one of the foregoing embodiments, the optical system further comprising a movable coil housed within the optical component, the movable coil being configured to move a focusing element within the optical path of the optical system.

[0285] 13. The optical system according to any one of the foregoing embodiments, wherein the electrical...The motor is configured to move the focusing element along the optical axis in one or two directions.

[0286] 14. The optical system according to any one of the preceding embodiments, wherein the motor is directly coupled to a portion of the first, second, or third housing of the optical component, and a portion of the first, second, or third housing of the optical component is directly coupled to the focusing element.

[0287] 15. The optical system according to any one of the preceding embodiments, wherein the light source is a pulsed light source.

[0288] 16. The optical system according to any one of the preceding embodiments, wherein the composite root mean square error of the optical system is less than about 0.05.

[0289] 17. The optical system according to any one of the preceding embodiments, wherein the illumination efficiency of the optical component is at least about 90%.

[0290] 18. The optical system according to any one of the preceding embodiments, wherein the area is greater than 30 mm².

[0291] 19. The optical system according to any one of the preceding embodiments, wherein the area is greater than 50 mm² or 60 mm².

[0292] 20. The optical system according to any one of the preceding embodiments, wherein the optical system further includes the solid support within the stage.

[0293] 21. The optical system according to any one of the foregoing embodiments, wherein the solid support comprises two or more surfaces having one or more samples imaged by the optical system fixed thereon.

[0294] 22. The optical system according to any one of the foregoing embodiments, wherein the solid support comprises three or more surfaces having one or more samples imaged by the optical system fixed thereon.

[0295] 23. The optical system according to any one of the foregoing embodiments, wherein the three or more surfaces are axially displaced from each other at least along the optical axis of the optical system.

[0296] 24. The optical system according to any one of the foregoing embodiments, wherein the solid support comprises a probe configured to bind nucleic acid molecules.

[0297] 25. The optical system according to any one of the foregoing embodiments, wherein the probe is bound to the surface of the solid support.

[0298] 26. The optical system according to any one of the foregoing embodiments, wherein the light source is a laser light source.

[0299] 27. The optical system according to any one of the foregoing embodiments, wherein the optical component comprises a dichroic filter configured to transmit the illumination.

[0300] 28. An optical system according to any one of the foregoing embodiments, wherein the optical component comprises: a first segment including a first housing, the first housing including a first plurality of lenses; a second segment including a second housing; and a third segment including a third housing, the third housing including a second plurality of lenses. Specification 41 / 54 pages 48 CN120936922 A

[0301] 29. The optical system according to any one of the preceding embodiments, wherein the first segment is optically aligned with the third segment.

[0302] 30. The optical system according to any one of the preceding embodiments, wherein the first segment is positioned between the third segment and the stage.

[0303] 31. The optical system according to any one of the preceding embodiments, wherein the third segment is positioned between the first segment and the image sensor of the optical system.

[0304] 32. The optical system according to any one of the preceding embodiments, wherein the first plurality of lenses are movable along the optical path in a range of about 0 to about 2 mm.

[0305] 33. The optical system according to any one of the preceding embodiments, wherein the first plurality of lenses comprises an asymmetric biconvex lens.

[0306] 34. The optical system according to any one of the preceding embodiments, wherein the second plurality of lenses comprises an asymmetric biconcave lens.

[0307] 35. The optical system according to any one of the preceding embodiments, wherein the asymmetric biconcave lens is an aspherical asymmetric biconcave lens.

[0308] 36. The optical system according to any one of the foregoing embodiments, wherein the optical system is configured to acquire an image of the solid support without moving the optical compensator into the optical path between the solid support and the detector of the optical system.

[0309] 37. The optical system according to any one of the foregoing embodiments, wherein the optical system is configured to acquire an image of the solid support without removing the optical compensator from the optical path between the sample and the detector of the optical system.

[0310] 38. The optical system according to any one of the foregoing embodiments, wherein the solid support is a flow cell.

[0311] 39. The optical system according to any one of the foregoing embodiments, wherein the optical component is configured to generate one or more spatial contractions transverse to the optical path through which light travels.

[0312] 40. The optical system according to any one of the foregoing embodiments, wherein the optical component is configured to generate one or more field curvature corrections transverse to the optical path through which light travels.

[0313] 41. An optical system according to any one of the foregoing embodiments, wherein the optical components are configured to generate at least one field curvature correction in a first, second, or third segment transverse to the optical path through which light travels.

[0314] 42. A method for analyzing biomolecules, the method comprising:

[0315] (a) providing a solid support comprising the biomolecule containing a marker;

[0316] (b) illuminating the biomolecule containing the marker using an optical system including a light source, thereby...The method generates signal light or a variation thereof, wherein the illumination is provided over an area of ​​the solid support greater than about 20 square millimeters (mm2), wherein the peak-to-valley variation is at most about 5%;

[0317] (c) detects the signal light or the variation thereof using a detector of the optical system; and

[0318] (d) processes the signal light or the variation thereof at least partially to analyze the biomolecule.

[0319] 43. The method according to any one of the preceding embodiments, wherein the biomolecule is a nucleic acid molecule, a protein, or a polypeptide.

[0320] 44. The method according to any one of the preceding embodiments, wherein the biomolecule is a nucleic acid.

[0321] 45. The method according to any one of the preceding embodiments, further comprising, prior to (a), binding the biomolecule to a probe bound to the solid support, and conjugating the label to the biomolecule. Specification 42 / 54 pages 49 CN 120936922 A

[0322] 46. The method according to any one of the preceding embodiments, wherein the label is conjugated to the biomolecule by hybridization.

[0323] 47. The method according to any one of the foregoing embodiments, wherein the optical system does not include an objective lens.

[0324] 48. The method according to any one of the foregoing embodiments, wherein the solid support is not moved along the optical axis of the optical system.

[0325] 49. The method according to any one of the foregoing embodiments, wherein multiple images of the solid support are acquired without moving the solid support along the optical axis.

[0326] 50. The method according to any one of the foregoing embodiments, wherein the irradiance of the illumination is at least about 40 milliwatts per square millimeter.

[0327] 51. The method according to any one of the foregoing embodiments, wherein the wavelength of the signal light is from 500 nanometers to about 750 nanometers.

[0328] 52. The method according to any one of the foregoing embodiments, wherein the detection in (c) is performed using an optical element with a numerical aperture of at least about 0.3.

[0329] 53. The method according to any one of the foregoing embodiments, further comprising, in (b), using a motion coil within the optical system to move a focusing element within the optical path of the optical system, thereby changing the focal point of the optical system on the solid support.

[0330] 54. The method according to any one of the foregoing embodiments, wherein the light source is a pulsed light source.

[0331] 55. The method according to any one of the foregoing embodiments, wherein the illumination is provided with an efficiency of at least about 90%.

[0332] 56. The method according to any one of the foregoing embodiments, further comprising repeating (b)-(d) on additional biomolecules coupled to another surface of the solid support.

[0333] 57. The method according to any one of the foregoing embodiments, the method further comprising, after (c), removing the marker from the biomolecule.

[0334] 58. The method according to any one of the foregoing embodiments, the method further comprising repeating (a)-(d) on an additional marker bound to another portion of the biomolecule.

[0335] 59. The method according to any one of the foregoing embodiments, wherein the optical component is configured to generate one or more spatial contractions transverse to the optical path of light traveling through it.

[0336] 60. The method according to any one of the foregoing embodiments, wherein the optical component is configured to generate one or more field curvature corrections transverse to the optical path of light traveling through it.

[0337] 61. The method according to any one of the foregoing embodiments, wherein the optical component is configured to generate at least one field curvature correction transverse to the optical path of light traveling through it in a first segment, a second segment, or a third segment.

[0338] 62. The method according to any one of the preceding embodiments, wherein (d) includes at least partially processing the signal light or the said variation thereof to generate one or more solid support images, and analyzing the one or more solid support images to generate a base interpretation of the sample.

[0339] 63. The method according to any one of the preceding embodiments, wherein each solid support image in the solid support images includes a field of view (FOV) greater than 20 square millimeters (mm2).

[0340] 64. The method according to any one of the preceding embodiments, wherein the solid support is a flow cell.

[0341] 65. An optical system comprising:

[0342] a stage configured to hold a solid support;

[0343] a light source configured to illuminate the solid support; and

[0344] a speckle eliminator optically coupled to the light source and disposed in an optical path from the light source to the stage.

[0345] 66. The optical system according to any one of the foregoing embodiments, the optical system further comprising an additional light source optically coupled to the speckle eliminator.

[0346] 67. The optical system according to any one of the foregoing embodiments, wherein light from the additional light source is configured to illuminate the solid support with light of a different wavelength from the light source.

[0347] 68. The optical system according to any one of the foregoing embodiments, wherein at least about four light sources are coupled to the speckle eliminator.

[0348] 69. The optical system according to any one of the foregoing embodiments, wherein the speckle eliminator is a vibratory speckle eliminator.

[0349] 70. The optical system according to any one of the foregoing embodiments, wherein the speckle eliminator is a passive speckle eliminator.71. An optical system according to any one of the foregoing embodiments, wherein the passive speckle eliminator comprises a diffuse scattering plate.

[0351] 72. An optical system according to any one of the foregoing embodiments, wherein the speckle eliminator is a tension speckle eliminator.

[0352] 73. An optical system according to any one of the foregoing embodiments, wherein the speckle eliminator is configured to reduce speckle noise to at most about 5%.

[0353] 74. An optical system according to any one of the foregoing embodiments, wherein the solid support is a flow cell.

[0354] 75. A method for analyzing biomolecules, the method comprising:

[0355] (a) providing a solid support comprising a biological sample containing a marker;

[0356] (b) illuminating the biological sample containing the marker using an optical system including a light source to generate a signal light or a variation thereof, wherein the illumination is provided by a speckle eliminator in the optical path of the optical system;

[0357] (c) detecting the signal light or the variation thereof using a detector of the optical system; and

[0358] (d) processing the signal light or the variation thereof at least partially to analyze the biomolecules.

[0359] 76. The method according to any of the preceding embodiments, the method further comprising repeating (b)-(d) on an additional biological sample coupled to another surface of the solid support.

[0360] 77. The method according to any of the preceding embodiments, the method further comprising, after (c), removing the marker from the biological sample.

[0361] 78. The method according to any one of the foregoing embodiments, further comprising repeating steps (a)-(d) on an additional marker bound to the biological sample.

[0362] 79. The method according to any one of the foregoing embodiments, wherein the speckle eliminator uses vibration to perform speckle elimination on the illumination.

[0363] 80. The method according to any one of the foregoing embodiments, further comprising illuminating the solid support using an additional light source.

[0364] 81. The method according to any one of the foregoing embodiments, wherein the additional light source provides light of a different wavelength to the solid support.

[0365] 82. The method according to any one of the foregoing embodiments, wherein the additional light source is optically coupled to the speckle eliminator.

[0366] 83. The method according to any one of the foregoing embodiments, wherein the biological sample comprises nucleic acid molecules, proteins, or polypeptides.

[0367] 84. The method according to any one of the foregoing embodiments, wherein the biological sample comprises nucleic acids.

[0368] 85. An illumination system for a multichannel fluorescence imaging module, the illumination system comprising:

[0369] an illumination subsystem comprising:

[0370] a light source;

[0371] a speckle eliminator; and

[0372] a beam delivery subsystem optically coupled to the illumination system, comprising:

[0373] a collimator; and

[0374] one or more optical lens elements.

[0375] 86. The illumination system according to any of the preceding embodiments, wherein the illumination system is configured to provide an illumination field of not less than 10 mm², 20 mm², 30 mm², 40 mm², or 50 mm² at a sample plane.

[0376] 87. The illumination system according to any of the preceding embodiments, wherein the illumination system is configured to provide a power density of not less than 40, 50, or 60 milliwatts / mm² at a sample plane.

[0377] 88. The illumination system according to any of the preceding embodiments, wherein the sample plane is orthogonal to the z-axis.

[0378] 89. The lighting system according to any one of the foregoing embodiments, wherein the light source comprises one or more lasers.

[0379] 90. The lighting system according to any one of the foregoing embodiments, wherein the one or more lasers comprises one or more laser diodes.

[0380] 91. The lighting system according to any one of the foregoing embodiments, wherein the one or more lasers emit light of multiple wavelengths.

[0381] 92. The lighting system according to any one of the foregoing embodiments, wherein the lighting subsystem further comprises one or more optical fibers.

[0382] 93. The lighting system according to any one of the foregoing embodiments, wherein at least one of the optical fibers has a length of 0.5 m to 5 m.

[0383] 94. The lighting system according to any one of the foregoing embodiments, wherein at least one of the optical fibers comprises a fiber core, the maximum cross-sectional dimension of which is 50 μm to 1500 μm.

[0384] 95. The lighting system according to any one of the foregoing embodiments, wherein the cross-section of the fiber core is circular.

[0385] 96. The lighting system according to any one of the foregoing embodiments, wherein the power efficiency of the lighting system is not less than 65%, 70%, 75%, or 80%.

[0386] 97. The lighting system according to any one of the foregoing embodiments, wherein the lighting field is rectangular or square.

[0387] 98. The lighting system according to any one of the foregoing embodiments, wherein the one or more optical lens elements comprise one or more multi-lens arrays.

[0388] 99. The lighting system according to any one of the foregoing embodiments, wherein each of the one or more multi-lens arrays comprises one or more of the following: an asymmetric biconvex lens, a convex plano lens, a concave plano lens, an asymmetric biconcave lens, and an asymmetric convex-concave lens.

[0389] 100. The lighting system according to any one of the foregoing embodiments, wherein each of the one or more multi-lens arrays includes at least a plurality of lens elements in a direction orthogonal to the z-axis. Specification 45 / 54 pages 52 CN 120936922 A

[0390] 101. The lighting system according to any one of the foregoing embodiments, wherein the lighting subsystem further includes an optical fiber optically coupled to a laser diode and reducing light source speckle.

[0391] 102. The lighting system according to any one of the foregoing embodiments, wherein the speckle canceller includes an optical fiber optically coupled to a laser diode and reducing light source speckle.

[0392] 103. The lighting system according to any one of the foregoing embodiments, wherein the one or more optical lens elements include:

[0393] an asymmetric biconvex lens, a convex-plano lens, a concave-plano lens, an asymmetric biconcave lens, an asymmetric convex-concave lens, or a combination thereof. 104. The illumination system according to any one of the preceding embodiments, wherein the one or more optical lens elements comprise:

[0394] a first multi-lens array and a second multi-lens array, positioned along the z-axis between the collimator and the entrance pupil of the illumination system.

[0395] 105. The illumination system according to any one of the preceding embodiments, wherein the illumination system is configured to

[0396] generate an illumination field greater than 50 mm² at the sample stage, wherein the illumination power density variation of the entire illumination field is less than ±2%, 5%, 8%, 10%, or 12%.

[0397] 106. The illumination system according to any one of the preceding embodiments, wherein the speckle eliminator comprises a mechanical vibration source.

[0398] 107. The illumination system according to any one of the preceding embodiments, wherein the speckle eliminator comprises a vibration source that generates vibrations within a predetermined frequency range.

[0399] 108. The illumination system according to any one of the preceding embodiments, wherein the mechanical vibration source is configured to vibrate at one or more frequencies within an audible range, an ultrasonic range, or both.

[0400] 109. The illumination system according to any one of the foregoing embodiments, wherein the mechanical vibration source is configured to generate one-dimensional, two-dimensional, or three-dimensional vibrational movement.

[0401] 110. The illumination system according to any one of the foregoing embodiments, wherein at least a portion of each of the optical fibers or single optical fibers is wound or coiled one or more times.

[0402] 111. The illumination system according to any one of the foregoing embodiments, wherein at least a portion of each of the optical fibers or single optical fibers is fixedly or loosely attached to the mechanical vibration source.

[0403] 112. The illumination system according to any one of the foregoing embodiments, wherein the speckle eliminator is integrated with the sample stage, objective lens, and

[0404] The one or more image sensors are physically isolated, such that the mechanical movement of the speckle eliminator is independent of the sample stage, the objective lens, and the one or more image sensors.

[0405] 113. The illumination system according to any one of the preceding embodiments, wherein the speckle eliminator is configured to reduce speckle noise to no more than 4%, 4.5%, 5%, or 5.5%.

[0406] 114. The illumination system according to any one of the preceding embodiments, wherein the light source comprises a multicolor laser array.

[0407] 115. The illumination system according to any one of the preceding embodiments, wherein the multicolor laser array comprises a laser diode array

[0408] that emits laser light of 2, 3, 4, 5, or 6 wavelengths, or emits laser light within a wavelength range of 2, 3, 4, 5, or 6 wavelengths. Specification 46 / 54 pages 53 CN 120936922 A

[0409] 116. The lighting system according to any one of the foregoing embodiments, wherein the multicolor laser array comprises a laser that emits light of at least 2, 3, or 4 color wavelengths or wavelength ranges in a direction orthogonal to the z-axis.

[0410] 117. The lighting system according to any one of the foregoing embodiments, wherein the lighting subsystem further comprises one or more coupling lenses.

[0411] 118. The lighting system according to any one of the foregoing embodiments, wherein the lighting subsystem comprises a single optical fiber.

[0412] 119. The lighting system according to any one of the foregoing embodiments, wherein the single optical fiber comprises a fiber core having a maximum cross-sectional dimension of 500 μm to 1500 μm.

[0413] 120. The lighting system according to any one of the foregoing embodiments, wherein the power in the beam transmission subsystem is greater than 5, 8, 10, 12, or 14 watts for one or more wavelengths or wavelength ranges.

[0414] 121. The lighting system according to any one of the foregoing embodiments, wherein the power at the sample plane is greater than 5, 8, 10, 12, or 14 watts for one or more wavelengths or wavelength ranges.

[0415] 122. The lighting system according to any one of the foregoing embodiments, wherein the lighting subsystem further comprises multiple optical fibers, each optical fiber being optically coupled to one or more corresponding lasers of the light source, wherein the one or more corresponding lasers emit light of the same wavelength or wavelength range as the light source.

[0417] 123. The lighting system according to any one of the foregoing embodiments, wherein the lighting subsystem further comprises one or more dichroic filters.

[0418] 124. The lighting system according to any one of the foregoing embodiments, wherein the light source comprises multiple beam combiners.

[0419] 125. The lighting system according to any one of the foregoing embodiments, wherein the light source comprises multiple polarized beam combiners.

[0420] 126. The illumination system according to any one of the preceding embodiments, wherein the light source comprises two or more lasers emitting light of the same wavelength or the same wavelength range.

[0421] 127. The illumination system according to any one of the preceding embodiments, wherein each polarization beam combiner in the polarization beam combiner is configured to combine light of the same wavelength or the same wavelength range from two or more lasers.

[0422] 128. The illumination system according to any one of the preceding embodiments, wherein the speckle eliminator is positioned within the optical path between the collimator and the sample plane.

[0423] 129. The illumination system according to any one of the preceding embodiments, wherein the speckle eliminator is positioned at a position where the beam diameter is greater than 5 mm, 10 mm, or 20 mm, and wherein the diameter of the beam is orthogonal to the z-axis.

[0424] 130. The illumination system according to any one of the preceding embodiments, wherein the optical fiber comprises a core having a rectangular or square cross-section.

[0425] 131. The illumination system according to any one of the preceding embodiments, wherein the optical fiber comprises a core having a non-circular cross-section.

[0426] 132. The lighting system according to any one of the foregoing embodiments, wherein the lighting subsystem further comprises one or more liquid light guides.

[0427] 133. The lighting system according to any one of the foregoing embodiments, wherein the one or more liquid light guides are optically coupled to a light source in the absence of an optical fiber. Specification 47 / 54 pages 54 CN 120936922 A

[0428] 134. The lighting system according to any one of the foregoing embodiments, wherein the one or more liquid light guides comprise a liquid core, wherein the maximum dimension of the cross-section of the liquid core is 0.5 mm to 10 mm, and wherein the cross-section is orthogonal to the z-axis.

[0429] 135. The lighting system according to any one of the foregoing embodiments, wherein the liquid core comprises a circular cross-section.

[0430] 136. The lighting system according to any one of the foregoing embodiments, wherein the liquid core comprises a non-circular cross-section.

[0431] 137. An imaging module for multi-channel fluorescence imaging, the imaging module comprising:

[0432] an illumination system according to any one of the preceding embodiments; and

[0433] an image acquisition system configured to acquire flow cell images of a sample fixed on a sample stage at a sample plane.

[0434] 138. The imaging module according to any one of the preceding embodiments, wherein each flow cell image in the flow cell image includes a field of view (FOV) greater than 20 mm², 30 mm², 40 mm², or 50 mm².

[0435] 139. The imaging module according to any one of the preceding embodiments, wherein each flow cell image in the flow cell image includes a field of view (FOV) greater than 20 mm², 30 mm², 40 mm², or 50 mm².The flow cell image includes a field of view (FOV) that overlaps with the illumination field generated by the illumination system at the sample plane.

[0436] 140. The imaging module according to any of the preceding embodiments, wherein each flow cell image in the flow cell image

[0437] includes a field of view (FOV) that overlaps with at least 80%, 85%, 90%, or 95% of the illumination field generated by the illumination system at the sample plane.

[0438] 141. The imaging module according to any of the preceding embodiments, wherein each flow cell image in the flow cell image

[0439] includes a field of view (FOV) of at least 80%, 85%, 90%, or 95% of the illumination field generated by the illumination system at the sample plane.

[0440] 142. The imaging module according to any of the preceding embodiments, wherein the imaging module includes:

[0441] one or more image sensors; and

[0442] an objective lens.

[0443] 143. An imaging module according to any of the preceding embodiments, wherein the numerical aperture (NA) of the imaging module is greater than 0.4, 0.5, or 0.6.

[0444] 144. An imaging module according to any of the preceding embodiments, wherein the imaging module includes an optical axis parallel to the z-axis. 145. An imaging module according to any of the preceding embodiments, wherein the flow cell image is an image of a sample fixed to one or more surfaces of a solid support.

[0445] 146. An imaging module according to any of the preceding embodiments, wherein the one or more surfaces include at least two or more surfaces axially displaced from each other along the z-axis.

[0446] 147. An imaging module according to any of the preceding embodiments, wherein the flow cell image is acquired without moving an optical compensator into the optical path between the objective lens and the one or more image sensors.

[0447] 148. An imaging module according to any of the preceding embodiments, wherein the flow cell image of one or more surfaces is acquired without removing an optical compensator from the optical path between the objective lens and the at least one image sensor.

[0448] 149. The imaging module according to any one of the foregoing embodiments, wherein each flow cell image in the flow cell image

[0449] includes a contrast-to-noise ratio (CNR) of at least 5 when the following conditions are met: nucleic acid polones set on three or more surfaces are labeled with cyanine dye 3 (Cy3); the dichroic mirror and bandpass filter group are optimized for Cy3 emission; and the flow cell image is acquired by the optical system under non-signal saturation conditions when one or more of the surfaces are immersed in 25 mMACES, pH 7.4 buffer.

[0450] 150. The imaging module according to any one of the preceding embodiments, wherein the imaging module is configured to determine the nucleotides of nucleic acid molecules in a sample.

[0451] 151. The imaging module according to any one of the preceding embodiments, wherein the imaging module is configured to perform affinity-based sequencing, nucleotide base pairing-based sequencing, nucleotide binding-based sequencing, or nucleotide incorporation-based sequencing reactions on at least one of the one or more surfaces.

[0452] 152. The imaging module according to any one of the preceding embodiments, wherein each of the one or more surfaces

[0454] includes a plurality of induced target nucleic acid sequences coupled thereto, wherein the induced target nucleic acid sequences of the plurality of induced target nucleic acid sequences have a polymerase intended to bind.

[0455] 153. A method for sequencing nucleic acid molecules, the method comprising:

[0456] providing a flow cell comprising one or more surfaces, wherein each surface comprises:

[0457] at least one hydrophilic polymer coating;

[0458] a plurality of oligonucleotide molecules attached to the at least one hydrophilic polymer coating; and

[0459] at least one discrete region of each surface comprising a plurality of cloned and amplified sample nucleic acid molecules immobilized to the plurality of attached oligonucleotide molecules;

[0460] causing the plurality of cloned and amplified sample nucleic acid molecules in an illumination field to fluoresce in different colors during on and off events by means of an illumination system; and

[0461] detecting the on and off events in one or more color channels by means of the one or more image sensors when the plurality of cloned and amplified sample nucleic acid molecules undergo the on and off events to determine the nucleotide identity of the cloned and amplified sample nucleic acid molecules.

[0462] 154. The method according to any one of the preceding embodiments, wherein the method further comprises:

[0463] adjusting the NA of an imaging module.

[0464] 155. The method according to any one of the foregoing embodiments, wherein the method further comprises:

[0465] changing the NA of the imaging module in the range of 0.4 to 0.6 by changing the adjustable optical aperture size.

[0466] 156. The method according to any one of the foregoing embodiments, wherein the illumination system is configured to provide an illumination field of not less than 10 mm², 20 mm², 30 mm², 40 mm², or 50 mm² at the sample plane.

[0467] 157. The method according to any one of the foregoing embodiments, wherein the illumination system is configured to provide a power density of not less than 40, 50, or 60 milliwatts / mm² at the sample plane.

[0468] 158. The method according to any one of the foregoing embodiments, wherein the illumination system comprises:

[0469] an illumination subsystem comprising:

[0470] A light source;

[0471] a speckle eliminator; and

[0472] a beam delivery subsystem optically coupled to the illumination system, comprising:

[0473] a collimator; and

[0474] one or more optical lens elements. Specification 49 / 54 pages 56 CN 120936922 A

[0475] 159. The method according to any one of the foregoing embodiments, wherein the sample plane is orthogonal to the z-axis.

[0476] 160. The method according to any one of the foregoing embodiments, wherein the light source comprises one or more lasers.

[0477] 161. The method according to any one of the foregoing embodiments, wherein the one or more lasers comprises one or more laser diodes.

[0478] 162. The method according to any one of the foregoing embodiments, wherein the one or more lasers emit light of multiple wavelengths. 163. The method according to any one of the foregoing embodiments, wherein the illumination subsystem further comprises one or more optical fibers.

[0479] 164. The method according to any one of the foregoing embodiments, wherein at least one of the optical fibers has a length of 0.5 m to 5 m.

[0480] 165. The method according to any one of the foregoing embodiments, wherein at least one of the optical fibers comprises a core, the maximum cross-sectional dimension of which is 50 μm to 1500 μm.

[0481] 166. The method according to any one of the foregoing embodiments, wherein the cross-section of the core is circular.

[0482] 167. The method according to any one of the foregoing embodiments, wherein the power efficiency of the lighting system is not less than 65%, 70%, 75%, or 80%.

[0483] 168. The method according to any one of the foregoing embodiments, wherein the lighting field is rectangular or square.

[0484] 169. The method according to any one of the foregoing embodiments, wherein the one or more optical lens elements comprise one or more multi-lens arrays.

[0485] 170. The method according to any one of the foregoing embodiments, wherein each multi-lens array comprises one or more of the following: an asymmetric biconvex lens, a convex-plano lens, a concave-plano lens, an asymmetric biconcave lens, and an asymmetric convex-concave lens.

[0486] 171. The method according to any one of the foregoing embodiments, wherein each of the multi-lens arrays in the multi-lens array includes a plurality of lens elements in at least a direction orthogonal to the z-axis.

[0487] 172. The method according to any one of the foregoing embodiments, wherein the illumination subsystem further includes an optical fiber optically coupled to a laser diode and reducing speckle from the light source.

[0488] 173. The method according to any one of the foregoing embodiments, wherein the speckle eliminator is composed of an optical fiber optically coupled to a laser diode and reducing speckle from the light source.

[0489] 174. The method according to any one of the preceding embodiments, wherein the one or more optical lens elements comprise: an asymmetric biconvex lens, a convex-plano lens, a concave-plano lens, an asymmetric biconcave lens, an asymmetric convex-concave lens, or a combination thereof. 175. The method according to any one of the preceding embodiments, wherein the one or more optical lens elements comprise: a first multi-lens array and a second multi-lens array, positioned along the z-axis between the collimator of the illumination system and the entrance pupil.

[0492] 176. The method according to any one of the preceding embodiments, wherein the illumination system is configured to generate an illumination field

[0493] greater than 50 mm² at the sample stage, the illumination power density difference of the entire illumination field being less than ±2%, 5%, 8%, 10%, or 12%.

[0494] 177. The method according to any one of the preceding embodiments, wherein the speckle eliminator comprises a mechanical vibration source. Specification page 50 / 54 57 CN 120936922 A

[0495] 178. The method according to any one of the foregoing embodiments, wherein the speckle eliminate includes a vibration source that generates vibrations within a predetermined frequency range.

[0496] 179. The method according to any one of the foregoing embodiments, wherein the mechanical vibration source is configured to vibrate at one or more frequencies in the audible sound range, the ultrasonic range, or both.

[0497] 180. The method according to any one of the foregoing embodiments, wherein the mechanical vibration source is configured to generate vibrational motion in one, two, or three dimensions.

[0498] 181. The method according to any one of the foregoing embodiments, wherein at least a portion of each optical fiber or single optical fiber is wound or coiled one or more times.

[0499] 182. The method according to any one of the foregoing embodiments, wherein at least a portion of each optical fiber or single optical fiber is fixedly or loosely attached to the mechanical vibration source.

[0500] 183. The method according to any one of the foregoing embodiments, wherein the speckle eliminator is physically isolated from the sample stage, the objective lens, and the one or more image sensors, such that the mechanical movement of the speckle eliminator is independent of the sample stage, the objective lens, and the one or more image sensors.

[0502] 184. The method according to any one of the foregoing embodiments, wherein the speckle eliminator is configured to reduce speckle noise to no more than 4%, 4.5%, 5%, or 5.5%.

[0503] 185. The method according to any one of the foregoing embodiments, wherein the light source comprises a multicolor laser array.

[0504] 186. The method according to any one of the foregoing embodiments, wherein the multicolor laser array comprises an emission 2,187. A laser diode array of 3, 4, 5, or 6 wavelengths or 2, 3, 4, 5, or 6 wavelength ranges.

[0505] 188. The method according to any one of the preceding embodiments, wherein the multicolor laser array comprises a laser that emits light of 2, 3, or 4 color wavelengths or wavelength ranges in a direction orthogonal to the z-axis.

[0506] 189. The method according to any one of the preceding embodiments, wherein the illumination subsystem further comprises one or more coupling lenses.

[0507] 180. The method according to any one of the preceding embodiments, wherein the illumination subsystem comprises a single optical fiber.

[0508] 191. The method according to any one of the preceding embodiments, wherein the single optical fiber comprises a fiber core having a maximum cross-sectional dimension of 500 μm to 1500 μm.

[0509] 192. The method according to any one of the preceding embodiments, wherein the power in the beam transmission subsystem is greater than 5, 8, 10, 12, or 14 watts for one or more wavelengths or wavelength ranges.

[0510] 192. The method according to any one of the foregoing embodiments, wherein the power at the sample plane is greater than 5, 8, 10, 12, or 14 watts for one or more wavelengths or wavelength ranges.

[0511] 193. The method according to any one of the foregoing embodiments, wherein the illumination subsystem further comprises a plurality of optical fibers, each optical fiber being optically coupled to one or more corresponding lasers of the light source, the one or more corresponding lasers emitting light of the same wavelength or wavelength range.

[0512] 194. The method according to any one of the foregoing embodiments, wherein the illumination subsystem further comprises one or more dichroic filters.

[0513] 195. The method according to any one of the foregoing embodiments, wherein the light source comprises a plurality of beam combiners.

[0514] 196. The method according to any one of the foregoing embodiments, wherein the light source comprises a plurality of polarized beam combiners.

[0515] 197. The method according to any one of the foregoing embodiments, wherein the light source comprises two or more lasers emitting light of the same wavelength or wavelength range.

[0516] 198. The method according to any one of the foregoing embodiments, wherein each polarized beam

[0517] combiner in the polarized beam combiner is configured to combine light from two or more lasers of the same wavelength or the same wavelength range.

[0518] 199. The method according to any one of the foregoing embodiments, wherein the speckle eliminator is positioned in the optical path between the collimator and the sample plane.

[0519] 200. The method according to any one of the foregoing embodiments, wherein the speckle eliminator is positioned in the optical path between the collimator and the sample plane.

[0520] 201. The method according to any one of the preceding embodiments, wherein the optical fiber comprises a core having a rectangular or square cross-section.

[0521] 202. The method according to any one of the preceding embodiments, wherein the optical fiber comprises a core having a non-circular cross-section.

[0522] 203. The method according to any one of the preceding embodiments, wherein the illumination subsystem further comprises one or more liquid light guides.

[0523] 204. The method according to any one of the preceding embodiments, wherein the one or more liquid light guides are optically coupled to the light source in the absence of an optical fiber.

[0524] 205. The method according to any one of the preceding embodiments, wherein the one or more liquid light guides comprise a liquid core having a maximum cross-sectional dimension of 0.5 mm to 10 mm, and wherein the cross-section is orthogonal to the z-axis.

[0525] 206. The method according to any one of the preceding embodiments, wherein the liquid core comprises a circular cross-section.

[0526] 207. The method according to any one of the preceding embodiments, wherein the liquid fiber core comprises a non-circular cross-section.

[0527] 208. A sample stage for holding a DNA sample for DNA sequencing reaction and imaging, the sample stage comprising:

[0528] a stage including a top surface, wherein the stage is rotatable about a z-axis relative to an optical system of a sequencing system;

[0529] one or more top stages positioned on the top surface of the stage, wherein each of the one or more top stages is configured to receive and fix one or more flow cell devices thereon, and wherein each of the one or more top stages is movable relative to the stage;

[0530] a first motor configured to actuate the stage to rotate at a first resolution.

[0531] 209. The sample stage according to any one of the preceding embodiments, wherein the top surface is circular.

[0532] 210. The sample stage according to any one of the foregoing embodiments, wherein the first resolution is an angular resolution, and is less than 0.1 degrees, 0.2 degrees, 0.5 degrees, 1 degree, 2 degrees, 3 degrees, 4 degrees, 5 degrees, 10 degrees, 20 degrees, 30 degrees, or 50 degrees.

[0533] 211. The sample stage according to any one of the foregoing embodiments, wherein each flow cell device in the flow cell apparatus includes one or more samples to be sequenced fixed thereon.

[0534] 212. The sample stage according to any one of the foregoing embodiments, wherein at least one flow cell device in the flow cell apparatus includes an in-situ sample fixed thereon.

[0535] 213. The sample stage according to any one of the foregoing embodiments, wherein the sample stage further includes one or more samples to be sequenced.Pages 52 / 54, 59 CN 120936922 A

[0536] Two motors, the second motor being configured to individually actuate the one or more top stages relative to the base station with a second resolution.

[0537] 214. A sample stage according to any of the preceding embodiments, wherein the sample stage further includes a second motor, the second motor being configured to simultaneously actuate the one or more top stages relative to the base station with a second resolution.

[0538] 215. A sample stage according to any of the preceding embodiments, wherein the second resolution is less than 0.01 mm, 0.015 mm, 0.02 mm, 0.03 mm, 0.04 mm, 0.05 mm, 0.08 mm, 0.1 mm, 0.2 mm, or 1 mm.

[0539] 216. A sample stage according to any of the preceding embodiments, wherein the sequencing system includes a fluid control device in fluid communication with the flow cell device positioned on the sample stage.

[0540] 217. The sample stage according to any one of the foregoing embodiments, wherein each of the one or more top stages is movable relative to the base in the sample plane.

[0541] 218. The sample stage according to any one of the foregoing embodiments, wherein a first top stage of the one or more top stages is movable independently relative to a second top stage of the one or more top stages.

[0542] 219. The sample stage according to any one of the foregoing embodiments, wherein the first top stage of the one or more top stages and the second top stage of the one or more top stages are movable simultaneously relative to the base.

[0543] 220. The sample stage according to any one of the foregoing embodiments, wherein each of the one or more top stages is movable relative to the base along a radius of the top surface of the base.

[0544] 221. The sample stage according to any one of the foregoing embodiments, wherein each of the one or more top stages is movable relative to the base along a radius orthogonal to the top surface of the base.

[0545] 222. A method for sequencing multiple DNA samples positioned on a rotating sample stage, the method comprising:

[0546] obtaining a sample stage, the sample stage including a base and one or more top stages positioned on a top surface of the base, wherein the base is rotatable about a z-axis relative to an optical system of a sequencing system;

[0547] positioning and fixing a first flow cell device relative to a first top stage of the one or more top stages; positioning and fixing a second flow cell device relative to a second top stage of the one or more top stages; dispensing one or more sequencing reagents into the first flow cell device using a first fluid control device; and imaging a first sample region of the first flow cell device using the optical system of the sequencing system;

[0548] While preventing the second flow cell device from moving relative to the optical system, the first top stage is moved relative to the optical system in the x-y plane;

[0549] the second sample region of the first flow cell device is imaged using the optical system of the sequencing system;

[0550] the sample stage is rotated at a predetermined angular resolution to position the second flow cell device relative to the optical system at a predetermined position; and

[0551] the first sample region of the second flow cell device is imaged using the optical system of the sequencing system. 223. The method according to any one of the foregoing embodiments, wherein moving the first top stage relative to the optical system in the x-y plane while preventing the second flow cell device from moving relative to the optical system comprises:

[0553] moving the first top stage independently relative to the optical system at a predetermined distance along the radius of the top surface of the stage while preventing the second flow cell device from moving relative to the optical system. Specification page 53 / 54 60 CN 120936922 A

[0554] 224. The method according to any one of the foregoing embodiments, wherein moving the first top stage relative to the optical system in the x-y plane while preventing the second flow cell device from moving relative to the

[0555] optical system comprises:

[0556] moving the first top stage relative to the optical system at a predetermined distance along a direction orthogonal to the radius of the top surface of the base while preventing the second flow cell device from moving relative to the optical system.

[0557] 225. The method according to any one of the foregoing embodiments, wherein the method further comprises:

[0558] moving the first fluid control device or the second fluid control device to position the second flow cell device relative to the first fluid control device or the second fluid control device at a predetermined position.

[0559] 226. The method according to any one of the foregoing embodiments, wherein the first sample area or the second sample area comprises a block.

[0560] 227. The method according to any one of the foregoing embodiments, wherein each of the one or more top platforms includes

[0561] a range of motion greater than 15 mm and less than 80 mm along the radius of the top surface of the base or orthogonal to the radius of the top surface of the base.

[0562] 228. The method according to any one of the foregoing embodiments, wherein the range of motion of each of the one or more top platforms along the radius of the top surface of the base or orthogonal to the radius of the top surface of the base is greater than 25 mm and less than 100 mm.

[0563] Although preferred embodiments of the inventive concept have been shown and described herein, those skilled in the art will appreciate the following.It will be apparent to those skilled in the art that such embodiments are provided by way of example only. Various changes, modifications, and substitutions will now occur to those skilled in the art without departing from the inventive concept. It should be understood that various alternatives to the embodiments of the inventive concept described herein can be used to practice these inventive concepts. The following claims are intended to define the scope of the inventive concept and cover the methods and structures within the scope of these claims and their equivalents. Instruction manual, page 54 / 54, page 61, CN 120936922 A, Figure 1, Figure 2; Instruction manual, Figure 1 / 35, page 62, CN 120936922 A, Figure 3A; Instruction manual, Figure 2 / 35, page 63, CN 120936922 A, Figure 3B; Instruction manual, Figure 3 / 35, page 64, CN 120936922 A, Figure 3C, Figure 4; Instruction manual, Figure 4 / 35, page 65, CN 120936922 A, Figure 5, Figure 6; Instruction manual, Figure 5 / 35, page 66, CN 120936922 A, Figure 7; Instruction manual, Figure 6 / 35, page 67, CN 120936922 A, Figure 8; Instruction manual, Figure 7 / 35, page 68, CN 120936922 A, Figure 9A, Figure 9B; Instruction manual, Figure 8 / 35, page 69, CN 120936922 A, Figure 9C; Instruction manual, Figure 9 / 35, page 70, CN Figure 9D of 120936922 A, Figure 10 of the instruction manual, page 10 / 35, 71 CN; Figure 11 of 120936922 A, Figure 12 of 120936922 A, Figure 13 of 120936922 A, Figure 14 of 120936922 A, Figure 15 of 120936922 A, Figure 16 of 120936922 A, Figure 17 of 120936922 A, Figure 18 of 120936922 A, Figure 19 of 120936922 A, Figure 20 of 120936922 A, Figure 18 of 120936922 A, Figure 19 of 120936922 A, Figure 20 of 120936922 A, Figure 18 of 120936922 A, 79 CN 120936922 A Figure 21 Instruction Manual Drawing, Page 19 / 35, 80 CN 120936922 A Figure 22 Instruction Manual Drawing, Page 20 / 35, 81 CN 120936922 A Figure 23 DescriptionFigure 24 of the book, page 21 / 35, CN 120936922 A; Figure 25 of the instruction manual, page 22 / 35, CN 120936922 A; Figure 26 of the instruction manual, page 23 / 35, CN 120936922 A; Figure 27 of the instruction manual, page 24 / 35, CN 120936922 A; Figure 28 of the instruction manual, page 25 / 35, CN 120936922 A; Figure 30 of the instruction manual, page 27 / 35, CN 120936922 A; Figure 31 of the instruction manual, page 28 / 35, CN 120936922 A; Figure 32A of the instruction manual, CN 120936922 A; Figure 32B of the instruction manual, page 29 / 35, CN 120936922 A; Figure 33A of the instruction manual, CN 120936922 A; Figure 33B of the instruction manual, CN 120936922 A. Figure 34 of the instruction manual, on pages 30 / 35, CN 120936922 A; Figure 35 of the instruction manual, on pages 31 / 35, CN 120936922 A; Figure 36 of the instruction manual, on pages 32 / 35, CN 120936922 A; Figure 37 of the instruction manual, on pages 34 / 35, CN 120936922 A; Figure 38 of the instruction manual, on pages 35 / 35, CN 120936922 A.

Claims

1. An optical system, the optical system comprising: A stage, configured to hold a solid support; A light source configured to illuminate the solid support; as well as An optical component, at least partially disposed within the optical path from the stage to the light source, wherein the optical component is configured to be larger than approximately 20 square millimeters (mm) on the solid support. 2 Lighting is provided in the area where the peak-to-valley variation is at most about 5%.

2. The optical system of claim 1, wherein the optical components do not include an objective lens.

3. The optical system of claim 2, wherein the optical system does not include the objective lens.

4. The optical system of claim 1, wherein the optical components do not include a barrel lens.

5. The optical system of claim 4, wherein the optical system does not include the barrel lens.

6. The optical system of claim 1, wherein the stage is not adjusted on the optical axis of the system.

7. The optical system of claim 1, wherein the irradiance of the illumination is at least about 40 milliwatts per square millimeter.

8. The optical system of claim 1, wherein the optical component is configured to receive emitted light from the solid support.

9. The optical system of claim 8, wherein the numerical aperture (NA) of the optical component is at least about 0.

3.

10. The optical system of claim 8, wherein the wavelength of the emitted light is about 500 nanometers to about 750 nanometers.

11. The optical system of claim 1, wherein the working distance of the optical component is at least about 1 mm to 25 mm.

12. The optical system of claim 1, further comprising a motion coil housed within the optical component, the motion coil being configured to move a focusing element within the optical path of the optical system.

13. The optical system of claim 1, wherein a motor located outside the optical system is configured to move the focusing element along the optical axis in one or both directions.

14. The optical system of claim 13, wherein the motor is directly coupled to a portion of the first, second, or third housing of the optical assembly, and the portion of the first, second, or third housing of the optical assembly is directly coupled to the focusing element.

15. The optical system according to claim 1, wherein the light source is a pulsed light source.

16. The optical system of claim 1, wherein the composite root mean square error of the optical system is less than about 0.

05.

17. The optical system of claim 1, wherein the illumination efficiency of said optical component is at least about 90%.

18. The optical system of claim 1, wherein the region is greater than 30 mm 2 .

19. The optical system of claim 1, wherein the region is greater than 50 mm 2 Or 60mm 2 .

20. The optical system of claim 1, further comprising the solid support within the stage.

21. The optical system of claim 20, wherein the solid support comprises two or more surfaces having one or more samples imaged by the optical system fixed thereon.

22. The optical system of claim 21, wherein the solid support comprises three or more surfaces having one or more samples imaged by the optical system fixed thereon.

23. The optical system of claim 22, wherein the three or more surfaces are axially displaced from each other at least along the optical axis of the optical system.

24. The optical system of claim 20, wherein the solid support comprises a probe configured to bind nucleic acid molecules.

25. The optical system of claim 24, wherein the probe is attached to the surface of the solid support.

26. The optical system according to claim 1, wherein the light source is a laser light source.

27. The optical system of claim 1, wherein the optical component includes a dichroic filter configured to transmit the illumination.

28. The optical system of claim 1, wherein the optical components comprise: The first segment includes a first housing, the first housing including a first plurality of lenses; The second section includes a second housing. And the third segment, which includes a third housing, the third housing including a second plurality of lenses.

29. The optical system of claim 28, wherein the first segment is optically aligned with the third segment.

30. The optical system of claim 28, wherein the first segment is positioned between the third segment and the stage.

31. The optical system of claim 28, wherein the third segment is positioned between the first segment and the image sensor of the optical system.

32. The optical system of claim 28, wherein the first plurality of lenses are movable along the optical path ranging from about 0 to about 2 millimeters.

33. The optical system of claim 28, wherein the first plurality of lenses comprises an asymmetric biconvex lens.

34. The optical system of claim 28, wherein the second plurality of lenses comprises an asymmetric biconcave lens.

35. The optical system according to claim 34, wherein the asymmetric biconcave lens is an aspherical asymmetric biconcave lens.

36. The optical system of claim 1, wherein the optical system is configured to acquire an image of the solid support without moving the optical compensator into the optical path between the solid support and the detector of the optical system.

37. The optical system of claim 1, wherein the optical system is configured to acquire an image of the solid support without removing the optical compensator from the optical path between the sample and the detector of the optical system.

38. The optical system of claim 1, wherein the solid support is a flow cell.

39. The optical system of claim 1, wherein the optical components are configured to produce one or more spatial contractions transverse to the optical path of light traveling through it.

40. The optical system of claim 1, wherein the optical components are configured to generate one or more field curvature corrections transverse to the optical path of light traveling through it.

41. The optical system of claim 1, wherein the optical component is configured to generate at least one field curvature correction in the first, second, or third segment, transverse to the optical path of light traveling through it.

42. A method for analyzing biomolecules, the method comprising: (a) Providing a solid support comprising the biomolecule containing the marker; (b) Illuminating the biomolecule containing the marker using an optical system including a light source, thereby generating signal light or a change thereof, wherein the solid support is larger than about 20 square millimeters (mm²). 2 The lighting is provided over the area where the peak-to-valley variation is at most about 5%; (c) Detecting the signal light or any changes thereof using the detector of the optical system; and (d) Process the signal light or its changes at least partially to analyze the biomolecules.

43. The method according to claim 42, wherein the biomolecule is a nucleic acid molecule, a protein, or a polypeptide.

44. The method according to claim 43, wherein the biomolecule is a nucleic acid.

45. The method of claim 42, further comprising, prior to (a), binding the biomolecule to a probe bound to the solid support and coupling the marker to the biomolecule.

46. ​​The method of claim 42, wherein the marker is coupled to the biomolecule via hybridization.

47. The method of claim 42, wherein the optical system does not include an objective lens.

48. The method of claim 42, wherein the solid support is not moved along the optical axis of the optical system.

49. The method of claim 48, wherein multiple images of the solid support are acquired without moving the solid support along the optical axis.

50. The method of claim 42, wherein the irradiance of the illumination is at least about 40 milliwatts per square millimeter.

51. The method of claim 42, wherein the wavelength of the signal light is about 500 nanometers to about 750 nanometers.

52. The method of claim 42, wherein the detection in (c) is performed using an optical element with a numerical aperture of at least about 0.

3.

53. The method of claim 42, further comprising (b) using a motion coil within the optical system to move a focusing element within the optical path of the optical system, thereby changing the focal point of the optical system on the solid support.

54. The method according to claim 42, wherein the light source is a pulsed light source.

55. The method of claim 42, wherein the illumination is provided with an efficiency of at least about 90%.

56. The method of claim 42, the method further comprising repeating (b)-(d) additional biomolecules coupled to additional surfaces of the solid support.

57. The method of claim 42, further comprising, after (c), removing the marker from the biomolecule.

58. The method of claim 57, further comprising repeating (a)-(d) on an additional marker that binds to another portion of the biomolecule.

59. The method of claim 42, wherein the optical components are configured to produce one or more spatial contractions transverse to the optical path through which light travels.

60. The method of claim 42, wherein the optical components are configured to generate one or more field curvature corrections transverse to the optical path of light traveling through it.

61. The method of claim 42, wherein the optical components are configured to generate at least one field curvature correction in the first, second, or third segment, transverse to the optical path through which the light travels.

62. The method of claim 42, wherein (d) comprises at least partially processing the signal light or the said variation thereof to generate one or more solid support images, and analyzing the one or more solid support images to generate a base interpretation of the sample.

63. The method of claim 62, wherein each solid support image in the solid support images comprises more than 20 square millimeters (mm²). 2 ) field of view (FOV).

64. The method of claim 42, wherein the solid support is a flow cell.

65. An optical system, the optical system comprising: A stage, configured to hold a solid support; A light source configured to illuminate the solid support; as well as A speckle eliminater, which is optically coupled to the light source and disposed within the optical path from the light source to the stage.

66. The optical system of claim 65, further comprising an additional light source optically coupled to the speckle eliminator.

67. The optical system of claim 66, wherein light from the additional light source is configured to illuminate the solid support with light of a different wavelength than that of the light source.

68. The optical system of claim 66, wherein at least about four light sources are coupled to the speckle eliminator.

69. The optical system of claim 65, wherein the speckle eliminator is a vibration speckle eliminator.

70. The optical system of claim 65, wherein the speckle eliminator is a passive speckle eliminator.

71. The optical system of claim 70, wherein the passive speckle eliminator comprises a diffuse scattering plate.

72. The optical system of claim 65, wherein the speckle eliminator is a tension speckle eliminator.

73. The optical system of claim 65, wherein the speckle canceller is configured to reduce speckle noise to at most about 5%.

74. The optical system of claim 65, wherein the solid support is a flow cell.

75. A method for analyzing biomolecules, the method comprising: (a) Providing a solid support, said solid support comprising a biological sample containing a marker; (b) Illuminating the biological sample containing the marker using an optical system including a light source to generate signal light or a variation thereof, wherein the illumination is provided by a speckle eliminator in the optical path of the optical system; (c) Detecting the signal light or the changes thereof using the detector of the optical system; as well as (d) Process the signal light or its changes at least partially to analyze the biomolecules.

76. The method of claim 75, further comprising repeating (b)-(d) on additional biological samples coupled to additional surfaces of the solid support.

77. The method of claim 75, further comprising, after (c), removing the marker from the biological sample.

78. The method of claim 77, further comprising repeating (a)-(d) on an additional marker bound to the biological sample.

79. The method of claim 75, wherein the speckle eliminator uses vibration to eliminate speckle on the illumination.

80. The method of claim 75, further comprising illuminating the solid support using an additional light source.

81. The method of claim 80, wherein the additional light source provides light of a different wavelength to the solid support.

82. The method of claim 80, wherein the additional light source is optically coupled to the speckle eliminator.

83. The method of claim 75, wherein the biological sample comprises nucleic acid molecules, proteins, or polypeptides.

84. The method of claim 83, wherein the biological sample comprises nucleic acid.

85. The optical system of claim 1, wherein the optical components are at least partially disposed within the optical path from the stage to the detector of the optical system.

86. The optical system of claim 1, wherein the illumination system of the optical component is disposed within the optical path from the stage to the detector of the optical system.

87. A sample stage for holding a DNA sample for DNA sequencing reactions and imaging, the sample stage comprising: A platform, including a top surface, wherein the platform is rotatable about the z-axis relative to the optical system of the sequencing system; One or more top platforms are positioned on the top surface of the base, wherein each of the one or more top platforms is configured to receive and fix one or more flow cell devices thereon, and wherein each of the one or more top platforms is movable relative to the base. A first motor is configured to actuate the base to rotate at a first resolution. The sample stage according to any one of the preceding claims, wherein the top surface is circular.

88. The sample stage according to any one of the preceding claims, wherein the first resolution is an angular resolution and is less than 0.1 degrees, 0.2 degrees, 0.5 degrees, 1 degree, 2 degrees, 3 degrees, 4 degrees, 5 degrees, 10 degrees, 20 degrees, 30 degrees or 50 degrees.

89. The sample stage according to any one of the preceding claims, wherein each flow cell device in the flow cell device includes one or more samples to be sequenced fixed thereon.

90. The sample stage according to any one of the preceding claims, wherein at least one flow cell device in the flow cell device includes an in-situ sample fixed thereon.

91. The sample stage according to any one of the preceding claims, wherein the sample stage further comprises one or more second motors configured to individually actuate the one or more top stages relative to the base stage with a second resolution.

92. The sample stage according to any one of the preceding claims, wherein the sample stage further comprises a second motor configured to simultaneously actuate the one or more top stages relative to the base stage with a second resolution.

93. The sample stage according to any one of the preceding claims, wherein the second resolution is less than 0.01 mm, 0.015 mm, 0.02 mm, 0.03 mm, 0.04 mm, 0.05 mm, 0.08 mm, 0.1 mm, 0.2 mm or 1 mm.

94. The sample stage according to any one of the preceding claims, wherein the sequencing system includes a fluid control device in fluid communication with the flow cell device positioned on the sample stage.

95. The sample stage according to any one of the preceding claims, wherein each of the one or more top stages is movable in the sample plane relative to the base stage.

96. The sample stage according to any one of the preceding claims, wherein the first of the one or more top stages is movable independently relative to the second of the one or more top stages.

97. The sample stage according to any one of the preceding claims, wherein the first of the one or more top stages is movable simultaneously relative to the base stage and the second of the one or more top stages.

98. The sample stage according to any one of the preceding claims, wherein each of the one or more top stages is movable relative to the base stage along the radius of the top surface of the base stage.

99. The sample stage according to any one of the preceding claims, wherein each of the one or more top stages is movable relative to the base with a radius orthogonal to the top surface of the base.

100. A method for sequencing multiple DNA samples positioned on a rotating sample stage, the method comprising: A sample stage is obtained, the sample stage including a base and one or more top stages positioned on the top surface of the base, wherein the base is rotatable about the z-axis relative to the optical system of the sequencing system. The first flow pool device is positioned and fixed relative to the first of the one or more top platforms; The second flow cell device is positioned and fixed relative to the second of the one or more top platforms; One or more sequencing reagents are dispensed into the first flow cell device using a first fluid control device; The first sample region of the first flow cell device is imaged using the optical system of the sequencing system. While preventing the second flow cell device from moving relative to the optical system, the first top platform is moved in the xy plane relative to the optical system; The second sample region of the first flow cell device is imaged using the optical system of the sequencing system. The sample stage is rotated with a predetermined angular resolution to position the second flow cell device relative to the optical system at a predetermined position. as well as The first sample region of the second flow cell device is imaged using the optical system of the sequencing system.

101. The method according to any one of the preceding claims, wherein moving the first top stage relative to the optical system in the xy plane while preventing the second flow cell device from moving relative to the optical system comprises: While preventing the second flow cell device from moving relative to the optical system, the first top stage is moved independently relative to the optical system at a predetermined distance along the radius of the top surface of the base.

102. The method according to any one of the preceding claims, wherein moving the first top stage relative to the optical system in the xy plane while preventing the second flow cell device from moving relative to the optical system comprises: While preventing the second flow cell device from moving relative to the optical system, the first top platform is moved independently relative to the optical system at a predetermined distance in a direction orthogonal to the radius of the top surface of the base.

103. The method according to any one of the preceding claims, wherein the method further comprises: Move the first fluid control device or the second fluid control device to position the second fluid pool device relative to the first fluid control device or the second fluid control device at a predetermined position.

104. The method according to any one of the preceding claims, wherein the first sample region or the second sample region comprises a pattern.

105. The method according to any one of the preceding claims, wherein each of the one or more top platforms includes a range of motion greater than 15 mm and less than 80 mm along the radius of the top surface of the base or orthogonal to the radius of the top surface of the base.

106. The method according to any one of the preceding claims, wherein each of the one or more top platforms includes a range of motion greater than 25 mm and less than 100 mm along the radius of the top surface of the base or orthogonal to the radius of the top surface of the base.

Images