Multi-surface biological sample imaging machine

Abstract

The utility model discloses a multi-surface biological sample imaging machine, which comprises a lens, a flow cell and a controller, and is used for imaging biological samples on a plurality of surfaces of a supporting structure. Such a machine can capture light emitted from nucleic acids disposed on first and second surfaces of the flow cell when the lens is located at first and second distances, respectively, from the flow cell. In such a machine, the lens may be submerged in a first fluid, and the first surface and the second surface of the flow cell may be separated by a second fluid. Further, in such a machine, when the lens is located at the first distance and the second distance from the flow cell, the difference between the edge ray and the axial ray in the field of view of the lens may be substantially equal.

Description

Technical Field

[0001] The disclosed technology generally relates to the field of imaging and evaluating analytical samples. More specifically, the disclosed technology relates to techniques for matching aberrations associated with imaging different surfaces of a multi-surface support structure. Background Art

[0002] There are more and more applications for imaging the analysis samples on the support structure. These support structures may include plates on which biological samples are present. For example, these plates may include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) probes, which are specific to nucleotide sequences in genes present in humans and other organisms. A single DNA or RNA probe may be attached to a specific position in a small geometric grid or array on the support structure. Depending on the technology used, the sample may be attached to a random, semi-random or predetermined position on the support structure. A test sample (such as from a known human or organism) may be exposed to an array or grid so that a complementary gene or fragment is hybridized with the probe at each site on the plate surface. In certain applications such as sequencing, a template or fragment of genetic material may be positioned at a site, and nucleotides or other molecules may be hybridized with the template to determine the nature or sequence of the template. These sites may then be examined by scanning light of a specific frequency at the site to identify which genes or fragments are present in the sample by the fluorescence of the site where the gene or fragment is hybridized.

[0003] These plates are sometimes referred to as microarrays, gene or genomic chips, DNA chips, gene arrays, etc., and can be used for expression profiling, monitoring expression levels, genotyping, sequencing, etc. For example, diagnostic uses can include evaluating the genetic makeup of a particular patient to determine whether a disease state exists or whether there is a susceptibility to a particular condition. The reading and evaluation of such plates are important aspects of their utility. Although microarrays allow the presentation of separate biological components for mass processing and single detection, the number of components that can be detected in a single experiment is limited by the resolution of the system. In addition, a large number of reagents used in some methods may be expensive, making it desirable to reduce volume. Although these problems can be solved by imaging multiple surfaces of a single flow cell to improve efficiency, multi-surface imaging may be associated with further complications caused by optical aberrations associated with each imaging surface. The disclosed technology provides methods and systems that can allow detection based on efficient multi-surface arrays, which have lower costs and complexity than currently used methods and systems. Other advantages are also provided, and these advantages will be apparent from the following description. Summary of the invention

[0004] The disclosed technology provides a novel method for analyzing sample imaging and evaluation, which reduces the complexity of imaging and evaluation subsystems with multiple surfaces supporting samples. The support structure can be, for example, a flow cell, and a reagent fluid is allowed to flow through the flow cell and interact with a biological sample. Excitation radiation from at least one radiation source can be used to excite biological samples on multiple surfaces. In this way, fluorescent radiation can be emitted from the biological sample and then captured and detected by detection optics and at least one detector. The returned radiation can then be used to generate image data. This imaging of multiple surfaces can be completed sequentially or simultaneously. In addition, the technology described herein can be used with any type of imaging system of various types. For example, both epifluorescence and total internal reflection (TIR) ​​methods can benefit from the disclosed technology. In addition, the imaged biological sample can be present on the surface of the support structure in a random position or pattern.

[0005] A specific implementation relates to a machine including a lens, a circulation cell and a controller. In such specific implementations, the lens can have a field of view and be immersed in a first fluid having a first refractive index. Similarly, in such specific implementations, the circulation cell can include a first surface and a second surface separated by a second fluid having a second refractive index. In addition, the controller can be used to move the lens from a first position having a first distance from the circulation cell to a second position having a second distance from the circulation cell. The controller can also be used to capture light emitted from a nucleic acid disposed on the first surface of the circulation cell using the lens when the lens is separated from the circulation cell by the first distance. The controller can also be used to capture light emitted from a nucleic acid disposed on the second surface of the circulation cell using the lens when the lens is separated from the circulation cell by the second distance. The controller can also be used to determine a nucleic acid sequence for a biological sample based on the emitted light. In such embodiments, the optical path difference between the marginal light and the axial light in the field of view of the lens when the lens is separated from the flow cell by the first distance can be substantially equal to the optical path difference between the marginal light and the axial light in the field of view of the lens when the lens is separated from the flow cell by the second distance.

[0006] In some implementations of the machine, such as described in the previous paragraph, the first refractive index and the second refractive index can be substantially equal.

[0007] In some implementations of the machine, such as that described in the preceding paragraph, the lens may include an optical device adapted to capture light emitted from the nucleic acid sequence disposed on the first surface of the flow cell and allow the biological sample to be imaged on the detector with diffraction-limited imaging quality. In some such implementations, the first refractive index and the second refractive index are substantially equal, meaning that spherical aberration caused by any difference between the first refractive index and the second refractive index is sufficiently low as not to prevent diffraction-limited imaging of the biological sample, the diffraction-limited imaging being based on: light emitted from the nucleic acid sequence disposed on the first surface of the flow cell; and light emitted from the nucleic acid sequence disposed on the second surface of the flow cell.

[0008] In some specific implementations of the machine, such as described in the third paragraph of the present disclosure, the first fluid and the second fluid may be the same fluid.

[0009] In some specific implementations of the machine, such as described in the third paragraph of the present disclosure, the first fluid and the second fluid may be different fluids.

[0010] In some specific implementations of the machine such as described in the second paragraph of the present invention, the first surface of the flow cell is the top surface of the flow cell, and the second surface of the flow cell is the bottom surface of the flow cell.

[0011] In some embodiments of the machine, such as described in the second paragraph of the present invention, the lens may include an optical device adapted to capture light emitted from the nucleic acid sequence disposed on the first surface of the flow cell and allow the biological sample to be imaged on the detector with diffraction-limited imaging quality. In some such embodiments, the optical path difference between marginal rays and axial rays in the field of view of the lens when the lens is separated from the flow cell by the first distance is substantially equal to the optical path difference between marginal rays and axial rays in the field of view of the lens when the lens is separated from the flow cell by the second distance, meaning that spherical aberration is low enough not to prevent diffraction-limited imaging of the biological sample, the diffraction-limited imaging being based on: light emitted from the nucleic acid sequence disposed on the first surface of the flow cell; and light emitted from the nucleic acid sequence disposed on the second surface of the flow cell.

[0012] Another specific implementation relates to a method, the method comprising capturing light emitted from a nucleic acid disposed on a first surface of a flow cell using a lens, the lens being at a first distance from the flow cell and immersed in a first fluid having a first refractive index. Such methods may also include moving the lens to a position at a second distance from the flow cell. Such methods may also include capturing light emitted from a nucleic acid disposed on a second surface of the flow cell using the lens immersed in the first fluid having the first refractive index when the lens is at the second distance from the flow cell, wherein the first surface of the flow cell is separated from the second surface of the flow cell by a second fluid having a second refractive index. Such methods may also include determining a nucleic acid sequence of a biological sample based on the light emitted from the nucleic acid disposed on the first surface of the flow cell and the light emitted from the nucleic acid disposed on the second surface of the flow cell. In such methods, the optical path difference between a marginal ray and an axial ray in the field of view of the lens when the lens is at the first distance from the flow cell is substantially equal to the optical path difference between a marginal ray and an axial ray in the field of view of the lens when the lens is at the second distance from the flow cell.

[0013] In some implementations of the method, such as described in the preceding paragraph, the first refractive index and the second refractive index can be substantially equal.

[0014] In some implementations of the method such as described in the previous paragraph, the lens can include an optical device that is suitable for capturing light emitted from the nucleic acid sequence disposed on the first surface of the flow cell and allowing the biological sample to be imaged on the detector with diffraction-limited imaging quality. In some such implementations, the first refractive index and the second refractive index are substantially equal, meaning that spherical aberration caused by any difference between the first refractive index and the second refractive index is sufficiently low to not prevent diffraction-limited imaging of the biological sample, the diffraction-limited imaging being based on: light emitted from the nucleic acid sequence disposed on the first surface of the flow cell; and light emitted from the nucleic acid sequence disposed on the second surface of the flow cell.

[0015] In some specific implementations of the method such as described in the tenth paragraph of the present disclosure, the first fluid and the second fluid may be the same fluid.

[0016] In some specific implementations of the method such as described in the tenth paragraph of the present disclosure, the first fluid and the second fluid may be different fluids.

[0017] In some specific implementations of the method such as described in the ninth paragraph of the present disclosure, the first surface of the flow cell is the top surface of the flow cell, and the second surface of the flow cell is the bottom surface of the flow cell.

[0018] In some specific implementations of the method such as described in the ninth paragraph of the present invention, the lens includes an optical device that is suitable for capturing light emitted from the nucleic acid sequence disposed on the first surface of the flow cell and allowing the biological sample to be imaged on the detector with diffraction-limited imaging quality. In some such specific implementations, the optical path difference between the marginal light and the axial light in the field of view of the lens when the lens is located at the first distance from the flow cell is substantially equal to the optical path difference between the marginal light and the axial light in the field of view of the lens when the lens is located at the second distance from the flow cell, meaning that the spherical aberration is low enough not to prevent diffraction-limited imaging of the biological sample, the diffraction-limited imaging being based on: light emitted from the nucleic acid sequence disposed on the first surface of the flow cell; and light emitted from the nucleic acid sequence disposed on the second surface of the flow cell.

[0019] Another specific implementation relates to a non-transitory computer-readable medium storing instructions that, when executed by a processor, cause a biological sample imaging system to perform an action. In some such specific implementations, the action may include capturing light emitted from a nucleic acid disposed on a first surface of a circulation cell using a lens located at a first distance from the circulation cell, the lens having a field of view and being immersed in a first fluid having a first refractive index. In some such specific implementations, the action may include moving the lens to a position at a second distance from the circulation cell. In some such specific implementations, the action may include capturing light emitted from a nucleic acid disposed on a second surface of the circulation cell using the lens immersed in the first fluid having the first refractive index when the lens is located at the second distance from the circulation cell, wherein the first surface of the circulation cell is separated from the second surface of the circulation cell by a second fluid having a second refractive index. In some such specific implementations, the action may include determining a nucleic acid sequence of a biological sample based on the light emitted from the nucleic acid disposed on the first surface of the circulation cell and the light emitted from the nucleic acid disposed on the second surface of the circulation cell. In some such embodiments, the optical path difference between the marginal light and the axial light in the field of view of the lens when the lens is located at the first distance from the flow cell can be substantially equal to the optical path difference between the marginal light and the axial light in the field of view of the lens when the lens is located at the second distance from the flow cell.

[0020] In some implementations, such as described in the preceding paragraph, the first refractive index and the second refractive index can be substantially equal.

[0021] In some implementations such as described in the previous paragraph, the lens can include an optical device that is suitable for capturing light emitted from the nucleic acid sequence disposed on the first surface of the flow cell and allowing the biological sample to be imaged on the detector with diffraction-limited imaging quality. In some such implementations, the first refractive index and the second refractive index are substantially equal, meaning that spherical aberration caused by any difference between the first refractive index and the second refractive index is sufficiently low to not prevent diffraction-limited imaging of the biological sample, the diffraction-limited imaging being based on: light emitted from the nucleic acid sequence disposed on the first surface of the flow cell; and light emitted from the nucleic acid sequence disposed on the second surface of the flow cell.

[0022] In some specific implementations such as those described in the sixteenth paragraph of the present invention, the first surface of the flow cell is the top surface of the flow cell, and the second surface of the flow cell is the bottom surface of the flow cell. In some specific implementations such as those described in the sixteenth paragraph of the present invention, the lens includes an optical device that is suitable for capturing light emitted from a nucleic acid sequence disposed on the first surface of the flow cell and allowing the biological sample to be imaged on a detector with diffraction-limited imaging quality. In some such specific implementations, the optical path difference between the marginal light and the axial light in the field of view of the lens when the lens is located at the first distance from the flow cell is substantially equal to the optical path difference between the marginal light and the axial light in the field of view of the lens when the lens is located at the second distance from the flow cell, meaning that the spherical aberration is low enough not to prevent diffraction-limited imaging of the sample, which is based on: light emitted from the nucleic acid sequence disposed on the first surface of the flow cell; and light emitted from the nucleic acid sequence disposed on the second surface of the flow cell.

[0023] In some specific implementations, such as described in the sixteenth paragraph of the present disclosure, the first fluid and the second fluid may be the same fluid.

[0024] Other features and aspects of the disclosed technology will become apparent from the following detailed description in conjunction with the accompanying drawings, which illustrate features of examples according to the disclosed technology by way of example. This utility model content is not intended to limit any scope of protection provided by this document or any related document, which is defined by the claims and equivalents of the corresponding documents.

[0025] It should be understood that all combinations of the aforementioned concepts (assuming such concepts are not mutually contradictory) are contemplated as being part of the inventive subject matter disclosed herein. In particular, all combinations of the claimed subject matter appearing at the end of this disclosure are contemplated as being part of the inventive subject matter disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is a graphical overview of biological sample imaging systems;

[0027] Figure 2 is a diagrammatic perspective view of an exemplary radiation line directed toward a surface of a support structure to semi-confocally irradiate a biological site and return the radiation semi-confocally to a detector;

[0028] Figure 3 is a cross-sectional view of an exemplary support structure, wherein excitation radiation is directed toward two surfaces of the support structure;

[0029] Figure 4 is a diagrammatic perspective view of an exemplary support structure having an array of biocomponent sites in a spatially ordered pattern;

[0030] Figure 5 is a diagrammatic perspective view of an exemplary support structure having biocomponent sites in a random spatial distribution;

[0031] Figure 6 is a cross-sectional view of an exemplary support structure wherein excitation radiation is directed toward multiple surfaces of the support structure;

[0032] Figure 7 shows exemplary dimensions between the objective lens and the support structure that may exist in some implementations;

[0033] Figure 8 shows an exemplary ray trace from a light source to a first lens of an objective for multi-surface imaging; and

[0034] Fig. 9 Illustrated are exemplary images expected for top and bottom surface imaging when the imaging system is optimized for top surface imaging. DETAILED DESCRIPTION

[0035] Turning now to the drawings, and first referring to Figure 1, a biological sample imaging system 10 is illustrated. The biological sample imaging system 10 is capable of imaging a plurality of biological components 12, 14 within a support structure 16. For example, in the illustrated embodiment, a first biological component 12 may be present on a first surface 18 of the support structure 16, and a second biological component 14 may be present on a second surface 20 of the support structure. The support structure 16 may be, for example, a flow cell having an array of biological components 12, 14 on inner surfaces 18, 20, the inner surfaces generally facing each other, and reagents, rinses, and other fluids may be introduced through the flow cell, such as sites for binding nucleotides or other molecules to the biological components 12, 14. Fluorescent labels bound to the molecules of the components may, for example, include dyes that fluoresce when excited by appropriate excitation radiation. Assays that include the use of fluorescent labels and that may be used in the apparatus or methods described herein include those described elsewhere herein, such as genotyping assays, gene expression analysis, methylation analysis, or nucleic acid sequencing analysis.

[0036] Those skilled in the art will recognize that the flow cell can be used with any of a variety of arrays known in the art to achieve similar results. Such arrays can be formed by placing the biological components of the sample randomly or in a predefined pattern on the surface of the support using various techniques. In a particular embodiment, the clustered array of nucleic acid colonies can be prepared as described in the following patents: U.S. Patent No. 7,115,400; U.S. Patent Application Publication No. 2005 / 0100900; PCT Publication No. WO 00 / 18957; or PCT Publication No. WO 98 / 44151, each of which is incorporated herein by reference. Such methods are referred to as bridge amplification or solid phase amplification, and are particularly useful for sequencing applications.

[0037] Other exemplary random arrays that can be used include, but are not limited to, random arrays of beads associated with a solid support, examples of which are described in U.S. Patent Nos. 6,355,431; 6,327,410; and US 6,770,441; U.S. Patent Application Publication Nos. 2004 / 0185483 and US2002 / 0102578; and PCT Publication No. WO 00 / 63437, each of which is incorporated herein by reference. The beads can be positioned at discrete locations on the solid support, such as wells, with each location housing a single bead.

[0038] Any of a variety of other arrays or methods for making such arrays known in the art may be used. Commercially available microarrays that may be used include, for example, VLSIPS TMOther microarrays synthesized using Very Large Scale Immobilized Polymer Synthesis (Very Large Scale Immobilized Polymer Synthesis) techniques, such as, for example, U.S. Pat. Nos. 5,324,633; 5,744,305; 5,451,683; 5,482,867; 5,491,074; 5,624,711; 5,795,716; 5,831,070; 5,856,101; 5,858,659; 5,874,219; 5,968,740; 5,974 ,164; 5,981,185; 5,981,956; 6,025,601; 6,033,860; 6,090,555; 6,136,269; 6,022,963; 6,083,697; 6,291,183; 6,309,831; 6,416,949; 6,428,752; and 6,482,591, each of which is incorporated herein by reference. Spotted microarrays can also be used. An exemplary spotted microarray is the CodeLink TM Another microarray that can be used is one that uses an inkjet printing method (such as SurePrint from Agilent Technologies). TM Microarrays fabricated using PCR technology.

[0039] The sites or features of the array are typically discrete and spaced apart from each other. The size of the sites and / or the spacing between the sites can vary. For example, an array that can be used in conjunction with the disclosed technology can have sites that are spaced less than 100 μm, 50 μm, 10 μm, 5 μm, 1 μm, or 0.5 μm (e.g., 350 nm or 10 nm). The apparatus or method implemented based on the present disclosure can be used to image the array with a resolution sufficient to distinguish sites of the above-mentioned density or density range.

[0040] As exemplified herein, the surfaces used in the devices or methods based on the present disclosure are generally manufactured surfaces. Natural surfaces or surfaces of natural support structures may also be used; however, the disclosed technology may be applied to embodiments where the surface is neither a natural material nor a surface of a natural support structure. Thus, components of biological samples may be removed from their native environment and attached to manufactured surfaces.

[0041] Any of the various biological components may be present on the surface. Exemplary components include, but are not limited to, nucleic acids (such as DNA or RNA), proteins (such as enzymes or receptors), polypeptides, nucleotides, amino acids, sugars, cofactors, metabolites or derivatives of these natural components. Although the devices and methods described herein are exemplified with respect to components of biological samples, it should be understood that other samples or components may also be used. For example, synthetic samples may be used, such as combinatorial libraries, or compound libraries with known or suspected species of desired structures or functions. Therefore, the device or method may be used to synthesize a batch of compounds and / or screen a batch of compounds for desired structures or functions.

[0042] Return to Figure 1 In an exemplary system, the biological sample imaging system 10 may include at least a first radiation source 22, but may also include a second radiation source 24 (or an additional source). The radiation sources 22 and 24 may be lasers operating at different wavelengths. The choice of the wavelength of the laser generally depends on the fluorescent properties of the dye used to image the component site. The multiple different wavelengths of the laser used can allow the dyes at various sites within the support structure 16 to be distinguished, and imaging can be performed by continuously acquiring a series of images so that the molecules at the component sites can be identified according to the image processing and reading logic generally known in the art. In other specific implementations, light emitting diodes (LEDs) can be used as radiation sources 22 and 24. Other radiation sources known in the art can be used, including, for example, arc lamps or quartz halogen lamps. Particularly useful radiation sources are those that generate electromagnetic radiation in the ultraviolet (UV) range (about 200nm to 390nm), the visible (VIS) range (about 390nm to 770nm), the infrared (IR) range (about 0.77 microns to 25 microns), or other ranges of the electromagnetic spectrum.

[0043] For ease of description, the embodiment utilizing fluorescence-based detection is used as an example. However, it should be understood that other detection methods can be used in combination with the apparatus and methods described herein. For example, a variety of different emission types can be detected, such as fluorescence, luminescence or chemiluminescence. Therefore, the component to be detected can be marked with a compound or part of fluorescence, luminescence or chemiluminescence. It is also possible to use a device and method similar to those illustrated herein to detect signals other than optical signals from multiple surfaces except for the specific physical properties of the signal to be detected, which is modified to adapt to the signal to be detected.

[0044] The output from the radiation sources 22, 24 can be directed through conditioning optics 26, 28 for filtering and shaping the beam. For example, in a currently contemplated embodiment, conditioning optics 26, 28 can generate a substantially linear radiation beam and combine beams from multiple lasers, for example, as described in U.S. Pat. No. 7,329,860. The laser module may also include a measurement component for recording the power of each laser. The power measurement can be used as a feedback mechanism to control the length of time an image is recorded so as to obtain a uniform exposure and, therefore, a more easily comparable signal. In other specific implementations, conditioning optics 26, 28 can form a straight radiation beam, such as a square or rectangular shape.

[0045] After passing through the conditioning optics 26, 28, the beam can be directed toward a guiding optic 30 that redirects the beam from the radiation source 22, 24 toward a focusing optic 32. The guiding optic 30 can include a dichroic mirror configured to redirect the beam toward the focusing optic 32 while also allowing certain wavelengths of the retrobeam to pass. The focusing optic 32 can direct the radiation confocally to one or more surfaces 18, 20 of the support structure 16 on which the individual biological components 12, 14 are positioned. For example, the focusing optic 32 can include a microscope objective that confocally directs and focuses the radiation source 22, 24 along a line to the surface 18, 20 of the support structure 16. In some implementations, the focusing optic 32 can focus the radiation source 22, 24 above or below the surface 18, 20 so that the concentration is below the light saturation threshold of the component to be excited and / or the threshold for reducing damage to the sample. In such implementations, the focusing optics 32 can also maintain the focal plane of the detector 36 at the plane of the surfaces 18, 20 when the excitation radiation is defocused relative to the plane of the surfaces 18, 20. In some implementations, the focusing optics 32 can be movable relative to the surfaces 18, 20. For example, the focusing optics can be mounted to a z-stage, such as a voice coil or a piezoelectric component, to move the focusing optics 32 through an axis perpendicular to the plane of the surfaces 18, 20. In some implementations, the focusing optics 32 can be movable relative to the surfaces 18, 20 along the x-axis and / or the y-axis.

[0046] The biological component sites on the support structure 16 can use one or more reagents, which respond to the excitation light beam and fluoresce at a specific wavelength, and thus return radiation for imaging. For example, the fluorescent component can be generated by fluorescently labeled nucleotides, which use polymerase to hybridize with the complementary nucleotides of the sample. In other specific implementations, the fluorescently labeled nucleotides can be temporarily associated with the complementary nucleotides of the sample and then removed. In a further specific implementation, fluorescently labeled oligonucleotides can be used. As mentioned above, the fluorescence properties of these components can be changed by introducing reagents into the support structure 16 (for example, by cutting dyes from molecules, blocking the attachment of additional molecules, adding quenching agents, adding receptors for energy transfer, etc.). As will be understood by those skilled in the art, the wavelength of the dye that excites the sample and the wavelength of its fluorescence will depend on the absorption and emission spectra of the specific dye. Such returned radiation can be propagated back by guiding optical device 30. The reverse beam can usually be directed toward detection optical device 34, which can filter the beam to separate the different wavelengths in the reverse beam, and direct the reverse beam toward at least one detector 36.

[0047] The detector 36 may be based on any suitable technology and may be, for example, a charged coupled device (CCD) sensor that generates pixelated image data based on photons striking locations in the device. However, it should be understood that any of a variety of other detectors may also be used, including but not limited to detector arrays configured for time delay integration (TDI) operation, complementary metal oxide semiconductor (CMOS) detectors, avalanche photodiode (APD) detectors, Geiger mode photon counters, or any other suitable detector. TDI mode detection may be coupled with line scanning, as described in U.S. Pat. No. 7,329,860.

[0048] The detector 36 can generate image data, for example, with a resolution between 0.1 microns and 50 microns, which is then forwarded to the control / processing system 38. Generally speaking, the control / processing system 38 can perform various operations, such as analog-to-digital conversion, scaling, filtering, and multi-frame data association, to appropriately and accurately image multiple sites at specific locations on the sample. The control / processing system 38 can store the image data and can ultimately forward the image data to a post-processing system (not shown) for analyzing the data. Depending on the type of sample, the reagents used, and the processing performed, the image data can be used in many different ways. For example, nucleotide sequence data can be derived from the image data, or the data can be used to determine the presence of a specific gene, characterize one or more molecules at a component site, etc. Figure 1The operation of the various components shown in the figure may also be coordinated with the control / processing system 38. In actual applications, the control / processing system 38 may include hardware, firmware, and software designed to control the operation of the radiation sources 22, 24, the movement and focusing of the focusing optics 32, the translation system 40, and the detection optics 34, and the acquisition and processing of the signals from the detector 36. The control / processing system 38 may thus store the processed data and further process the data for use in generating a reconstructed image of the irradiated sites that fluoresce within the support structure 16. The image data may be analyzed by the system itself, or may be stored for analysis by other systems and at a different time after imaging.

[0049] The support structure 16 can be supported on a translation system 40 that allows the support structure 16 to be focused and moved before and during imaging. The platform can be configured to move the support structure 16, thereby changing the relative position of the radiation source 22, 24 and the detector 36 relative to the biological components bound to the surface for progressive scanning. The movement of the translation system 40 can be in one or more dimensions, including, for example, one or two dimensions in the dimension orthogonal to the propagation direction of the excitation radiation line, usually expressed as X and Y dimensions. In a specific embodiment, the translation system 40 can be configured to move in a direction perpendicular to the scanning axis of the detector array. The translation system 40 can be further configured to move in the dimension (usually expressed as the Z dimension) along which the excitation radiation line propagates. Movement in the Z dimension can also be used for focusing.

[0050] Figure 2 4 is a diagrammatic representation of an exemplary semi-confocal line scanning method for imaging the support structure 16. In the illustrated embodiment, the support structure 16 includes an upper plate 42 and a lower plate 44, with an internal volume 46 between the upper plate 42 and the lower plate 44. The upper plate 42 and the lower plate 44 can be made of any of a variety of materials, but can preferably be made of a substrate material that is substantially transparent at the wavelength of the excitation radiation and the fluorescent reverse beam, thereby allowing the excitation radiation and the returned fluorescent emission to pass without significant loss of signal quality. In addition, when used in an epifluorescence imaging arrangement as shown, one of the surfaces through which the radiation passes can be substantially transparent at the relevant wavelength, while the other surface (which is not passed by the radiation) can be less transparent, translucent, or even opaque or reflective. The upper plate 42 and the lower plate 44 can include a polymer material or other intermediate component, and can further include biological components 12, 14 on their respective inwardly facing surfaces 18, 20. As discussed above, the internal volume 46 can, for example, include one or more internal channels of a flow cell, and a reagent fluid can flow through the channel.

[0051] Reactive elements associated with the support structure 16, such as fluorinated nucleotides, can be irradiated by excitation radiation 48 along a radiation line 50. The radiation line 50 can be formed by excitation radiation 48 from the radiation sources 22, 24, which is directed by the guide optics 30 through the focusing optics 32. The radiation sources 22, 24 can generate a beam of light that is processed and shaped to provide a linear profile or radiation line that includes radiation of multiple wavelengths for causing fluorescence of corresponding different wavelengths from the biological components 12, 14 depending on the specific dye used. The focusing optics 32 can then semi-confocally direct the excitation radiation 48 toward the first surface 18 of the support structure 16 to irradiate sites of fluorinated nucleotides associated with the biological component 12 along the radiation line 50. In addition, the support structure 16, the guide optics 30, the focusing optics 32, or some combination thereof can be slowly translated so that the resulting radiation line 50 progressively irradiates the component as indicated by the arrow 52. This translation results in a continuous scan of the area 54, which allows for the gradual irradiation of the fluorinated nucleotides associated with the biological component 12 throughout the first surface 18 of the support structure 16. As will be discussed in more detail below, the same process can also be used to gradually irradiate the second surface 20 of the support structure 16. In fact, this method can be used for multiple surfaces within the support structure 16.

[0052] An exemplary method and apparatus for line scanning is described in U.S. Pat. No. 7,329,860, which is incorporated herein by reference, and which describes a line scanning apparatus having a detector array configured to achieve confocality on the scanning axis by limiting the scanning axis size of the detector array. More specifically, the scanning device can be configured so that the detector array has rectangular dimensions, so that the shorter dimension of the detector is in the scanning axis size, and the imaging optics are placed to direct a rectangular image of the sample area to the detector array so that the shorter dimension of the image is also in the scanning axis size. In this way, semi-confocality can be achieved because confocality occurs on a single axis (i.e., the scanning axis). Therefore, detection is specific to features on the surface of the substrate portion, thereby rejecting signals that may be generated by the solution around the feature. The apparatus and method described in U.S. Pat. No. 7,329,860 can be modified so that two or more surfaces of the support are scanned according to the description herein.

[0053] Detection devices and methods other than line scanning can also be used. For example, point scanning can be used as described below or in U.S. Patent No. 5,646,411 (which is incorporated herein by reference). Wide-angle area detection can be used with or without scanning motion. As further described elsewhere in this article, TIR methods can also be used.

[0054] Roughly like Figure 2 As shown in , the radiation line 50 used to image the sites of the biological components 12, 14 can be a continuous or discontinuous line. Therefore, some embodiments can include a discontinuous line composed of multiple confocal or semi-confocally directed radiation beams, which still irradiate multiple points along the radiation line 50. These discontinuous beams can be generated by one or more sources that are positioned or scanned to provide excitation radiation 48. As previously described, these beams can be directed confocally or semi-confocally toward the first surface 18 or the second surface 20 of the support structure 16 to irradiate the sites of the biological components 12, 14. As with the continuous semi-confocal line scanning described above, the support structure 16, the guiding optical device 30, the focusing optical device 32, or some combination thereof can be slowly advanced as shown by arrow 52 to irradiate the continuous scan area 54 along the first surface 18 or the second surface 20 of the support structure 16, and thereby irradiate continuous areas of the sites of the biological components 12, 14.

[0055] It should be noted that the system will generally form and direct both the excitation and the returned radiation for imaging. In some embodiments, confocal scanning can be used so that the optical system directs the excitation point or light spot through the biological component by scanning the excitation beam through the objective lens. The detection system images the emission from the excitation point on the detector without "descanning" the reverse beam. The reason for this is that the reverse beam is collected by the objective lens and separated from the excitation beam path before returning through the scanning device. Therefore, depending on the field of view of the original excitation light spot in the objective lens, the reverse beam will appear on the detector 36 at different points. When the excitation point is scanned across the sample, the image of the excitation point at the detector 36 will appear in the shape of a line. For example, if the scanning device cannot accept the reverse beam from the sample for some reason, this structure is useful. Examples are holographic and acousto-optic scanning devices that can scan beams at very high speeds, but use diffraction to generate scans. Therefore, the scanning properties are a function of wavelength. The reverse beam of the emitted radiation is different from the wavelength of the excitation beam. Alternatively or additionally, the emission signals may be collected sequentially after sequential excitation at different wavelengths.

[0056] In certain embodiments, the device or method can be used to measure at least about 0.01 mm 2 / sec. Depending on the particular application, faster rates may also be used, including, for example, at least about 0.02 mm in area scanned or otherwise detected. 2 / sec、0.05mm 2 / sec、0.1mm 2 / sec、1mm 2 / sec、1.5mm 2 / sec、5mm2 / sec、10mm 2 / sec、50mm 2 / sec、100mm 2 / sec or faster. If desired, for example to reduce noise, the detection rate may be about 0.05 mm / sec. 2 / sec、0.1mm 2 / sec、1mm 2 / sec、1.5mm 2 / sec、5mm 2 / sec、10mm 2 / sec、50mm 2 / sec or 100mm 2 The upper limit of / sec.

[0057] In some cases, support structure 16 may be used in a manner in which the biological component is expected to be present on only one surface. However, in some cases, biological material is present on multiple surfaces within support structure 16. For example, Figure 3 A typical support structure 16 is shown in which biological material has been attached to a first surface 18 as well as a second surface 20. In the illustrated embodiment, an attachment layer 56 has been formed on both the first surface 18 and the second surface 20 of the support structure 16. A first excitation radiation source 58 can be used to irradiate one of the many sites of a biological component 12 on the first surface 18 of the support structure 16 and return a first fluorescent emission 60 from the fluorinated nucleotides associated with the irradiated biological component 12. Simultaneously or sequentially, a second excitation radiation source 62 can be used to irradiate one of the many sites of a biological component 14 on the second surface 20 of the support structure 16 and return a second fluorescent emission 64 from the irradiated biological component 14.

[0058] although Figure 3 The embodiment illustrated in FIG. 1 shows excitation from source 58 and source 62 originating from the same side of support structure 16, but it should be understood that the optical system can be configured to impinge on surfaces from opposite sides of support structure 16. Figure 3 For example, the upper surface 18 can be irradiated from the excitation source 58 as shown, and the lower surface 20 can be irradiated from below. Similarly, the emission can be detected from one or more sides of the support structure. In certain embodiments, different sides of the support structure 16 can be excited from the same radiation source by first irradiating one side and then flipping the support structure to bring the other side into position for excitation by the radiation source.

[0059] The distribution of the biological components 12, 14 can follow many different patterns. For example, Figure 4A support structure 16 is shown in which the biological components 12, 14 at sites or features on the first surface 18 and the second surface 20 are evenly distributed in a spatially ordered pattern 66 of biological component sites 68. For example, certain types of microarrays can be used in which the positions of individual biological component sites 68 can be in a regular spatial pattern, such as a hexagonal pattern. The pattern can include sites at predetermined locations. In contrast, in other types of bioimaging arrays, biological components are attached to the surface at sites that occur at random or statistically varying locations, so that imaging the microarray is used to determine the location of each individual biological component feature. Therefore, although it is the product of a synthesis or manufacturing process, the pattern of features does not need to be predetermined.

[0060] For example, Figure 5 A support structure 16 is shown with sites on the first surface 18 and the second surface 20 positioned in a random spatial distribution 70 of biological component sites 72. However, for both the fixed array 66 and the random distribution 70 of biological sample sites, it is possible to image multiple surfaces 18, 20 of the support structure 16. In addition, it should be noted that in both cases, the biological components at a single site may constitute a population of the same molecules or a random mixture of different molecules. In addition, the density of the biological sample can vary and can be at least 1,000 sites per square millimeter.

[0061] The present technology accommodates such varying physical arrangements of multiple surfaces within the support structure 16, as well as varying arrangements of sites within components on the surfaces. Figure 2 and Figure 3 As discussed, in an embodiment having a support structure 16 with a first surface 18 and a second surface 20, a first excitation radiation source 58 can irradiate sites of a biological component 12 on the first surface 18 and return a first fluorescent emission 60, while a second excitation radiation source 62 can irradiate sites of a biological component 14 on the second surface 20 and return a second fluorescent emission 64, such as Figure 3 As shown in . Therefore, components of the sample volume between the two surfaces do not need to be detected and can be rejected. Selective detection of a surface of the support structure provides preferential detection of that surface compared to the volume of the support structure adjacent to the surface and compared to one or more other surfaces of the support structure.

[0062] In more complex configurations, it may be useful to irradiate more than two surfaces. For example, Figure 6A support structure 16 having N number of plates is shown, including a first plate 42, a second plate 44, ..., an N-2 plate 74, an N-1 plate 76, and an N plate 78. These plates define M number of surfaces, including a first surface 18, a second surface 20, ..., an M-3 surface 80, an M-2 surface 82, an M-1 surface 84, and an M surface 86. In the illustrated embodiment, not only the first surface 18 and the second surface 20 of the support structure 16 can be irradiated, but all M number of surfaces can be irradiated. For example, an excitation radiation source 88 can be used to irradiate biological component sites on the Mth surface 86 of the support structure 16, and return fluorescent emission 90 from the irradiated biological component. For a support structure having multiple surfaces, it may be desirable to excite the upper layer from the top and the lower layer from the bottom to reduce photobleaching. Therefore, components on a layer closer to the first outer side of the support structure can be irradiated from the first side, and components present on a layer closer to the relative outer side can be excited using irradiation from the relative outer side.

[0063] Figure 7 An objective lens 92 is shown through which radiation emitted from the biological components 12, 14 on the first surface 18 and the second surface 20 of the support structure 16 can be detected. The objective lens 92 can be one of the components of the focusing optical device 32 described above. Although not drawn to scale, Figure 7 An exemplary dimension between the objective lens 92 and the support structure 16 is shown. For example, the objective lens 92 can be spaced approximately 500 microns from the upper plate 42 of the support structure 16. The biological sample imaging system 10 can be configured to detect emitted radiation from the biological component 12 on the first surface 18 through the upper plate 42, which can be, for example, made of glass having a thickness of 300 μm to 700 μm. In addition, the biological sample imaging system 10 can also be configured to detect emitted radiation from the biological component 14 on the second surface 20 through the upper plate 42 (which can be, for example, 300 μm-700 μm) and the fluid (e.g., a reagent solution, such as a buffer solution, an imaging solution, or other reagent) within the internal volume 46 of the support structure 46, the thickness of which can vary between 75 μm-100 μm, as shown.

[0064] In certain embodiments, the objective lens 92 may be a plano-convex (PCX) lens with its flat surface facing the sample to avoid turbulence of the liquid flow when the objective lens is used for liquid immersion imaging. In such embodiments, the objective lens 92 may be immersed in the fluid, which may allow it to have a significantly higher numerical aperture than is possible with a dry objective lens, since the numerical aperture will be linearly proportional to the refractive index of the medium surrounding the objective lens. This in turn may increase the throughput of a system based on implementations of the present disclosure, since the throughput will be proportional to the square of the numerical aperture.

[0065] While liquid immersion can allow for an increase in numerical aperture, it can also increase aberrations such as wavefront error (e.g., spherical aberration) when performing multi-surface imaging. This can be seen in Figure 8 Seen in Figure 8 The ray tracing from the light source to the first lens of the objective lens for imaging the top surface 801 and the bottom surface 802 in a dual surface imaging configuration is shown. As shown in the figure, the segment M on the marginal ray c M i The length will vary with the angle θ between the top surface image and the bottom surface image. i Therefore, since the maximum wavefront error can be evaluated by the optical path difference between the axial ray and the marginal ray, the refractive index of the medium separating the objective from the top surface (n i ) is larger, the angle θ i The larger , and therefore the greater the error in sequencing using the depicted optical layout.

[0066] Although liquid immersion increases wavefront error, this error is predictable and can therefore be compensated for by the design of the objective 92. For example, by designing the objective to compensate for the wavefront error expected for imaging the top surface, a diffraction limited quality image of the top surface can be obtained. Figure 8 As shown in , because the optical path taken by light is different when imaging on different surfaces of the support structure 16, optimization for imaging on the first surface 18 will result in degradation of the image from the second surface 20. This is shown in Fig. 9 This is shown graphically in the figure, which shows Figure 7 1 and 2. Example images expected for the first surface 18 and second surface 20 of the support structure 16 in the configuration shown corresponding to the thickness of the upper plate 42 (e.g., 300 microns), where the imaging system is optimized for the first surface 18. As shown, the imaging system is able to provide high image quality on the first surface 18 because Fig. 9 In the example of , it is designed for this. However, in a system optimized for imaging from the first surface, the image taken for the second surface 20 may contain aberrations. Similarly, if the system is optimized for imaging from the second surface 20, the image taken from the first surface 18 may contain aberrations.

[0067] The errors that a system optimized for imaging different surfaces may encounter when imaging from one surface are expressed mathematically in Equations 1-3 below. In these equations, W 顶部 is the wavefront error of the upper surface, W 底部 is the wavefront error of the bottom surface, n c is the refractive index of the cover glass, n i is the refractive index of the immersion fluid, n gis the refractive index of the first lens of the objective, n b is the refractive index of the reagent solution, θ c is the angle between the marginal ray and the axial ray passing through the coverslip, θ i is the angle between the marginal ray and the axial ray passing through the immersion fluid, θ g is the angle between the marginal ray and the axial ray passing through the first lens of the objective, θ b is the angle between the marginal ray and the axial ray passing through the reagent solution, t c is the thickness of the cover glass, t i is the thickness of the immersion fluid used for top surface imaging, t i |' is the thickness of the immersion fluid used for bottom surface imaging, t g is the thickness of the first lens of the objective, t b is the thickness of the reagent solution, and Δ is the wavefront error difference between the top and bottom surfaces (eg, the aberration associated with bottom surface imaging in a system optimized for top surface imaging).

[0068]

[0069] A variety of methods can be used to minimize the effects of this spherical aberration. For example, a compensator such as described in U.S. Pat. No. RE48,561, which is incorporated herein by reference in its entirety, can be used. However, for some systems, the aberration may be significant enough that the expense and / or complexity of a compensator-based system may be a barrier. Therefore, in some embodiments, in addition to or as an alternative to incorporating a compensator into the imaging system, the aberration may be addressed by coordinating the choice of immersion fluid with the movement of objective lens 92 between imaging different surfaces (e.g., the top and bottom surfaces) so that, given n associated with the system, the aberration is minimized. b ,t b and θ b For example, if the immersion fluid is selected to have the same refractive index as the reagent solution in the interior volume 46 of the support structure 16 (e.g., n b =1.36). This can be done, for example, in some systems that use the same fluid as the buffer fluid and the immersion fluid and keep the temperature of these fluids constant, in systems that use water with salt water or other organic compounds dissolved in it to obtain the appropriate refractive index, or in other types of systems (for example, systems that use matching liquids). In this case, n b will be equal to n i , and θ b will be equal to θ i , thereby allowing Equation 5 to be simplified to Equation 4 below.

[0070]

[0071] Using this formula, we can i Set to be equal to t i -t b to achieve a value of Δ of 0 (i.e., to obtain the same image quality for the bottom surface as for the top surface). Therefore, by using an immersion fluid with a refractive index equal to that of the reagent solution, the objective lens is separated from the cover glass by a distance t i The first imaging run of the top surface is performed with the objective lens and coverslip separated by an amount equal to t i Subtract t b By performing a second imaging run of the bottom surface at a distance of 1.50 mm / s, the imaging system can obtain diffraction limited image quality of both the top and bottom surfaces of the flow cell.

[0072] In addition to the compensator-based approach described in RE48,561 or matching n b and n i And set t i 'In addition to reducing Δ to 0, other methods are also possible. For example, in some cases, instead of making n b and n i The difference between them is equal to 0, but the system implemented based on the present disclosure uses b n within a certain tolerance of i The immersion fluid is of a value such that adequate image quality can still be obtained given the application in which the system is assumed to be employed. To illustrate how this type of approach can be implemented in practice, consider a system with a numerical aperture of 1.2, which is able to achieve diffraction limited performance as long as the combination of primary and higher order spherical aberrations is less than or equal to 0.07 waves. In this case, the higher and lower order spherical aberrations resulting from the mismatch between the refractive indices of the buffer fluid and the immersion fluid can be calculated by applying Zernike analysis using equations 5 and 6 below.

[0073]

[0074]

[0075] In equations 5 and 6, ρ is the value of sin(ψ) in the unit circle, and ψ is equal to θ b -θ i The angle, c qm is the Zernike radial variable The Zernike expansion coefficient and δ m0is a normalized variable equal to 1 if m is 0 and 0 otherwise. Application of this analysis to an exemplary 1.2NA system provides the primary and higher order spherical aberration information listed in Table 1 below for various exemplary theoretical immersion fluids having a specified refractive index difference (i.e., Δn) with the buffer used in the system.

[0076]

[0077] Table 1

[0078] As can be seen from Table 1, in this embodiment, diffraction limited performance can be achieved as long as the refractive index difference between the buffer and the immersion fluid is no greater than 0.0003, which means that immersion fluids G, H, I or J can be used despite the fact that only immersion fluid J has the same refractive index as the buffer fluid used in the system.

[0079] As another type of variation, while the above examples discuss systems in which the objective is configured to compensate for expected aberrations during imaging of the top surface and then the manipulable parameters are selected to allow the bottom surface to match, the objective can be configured to compensate for expected aberrations during imaging of the bottom surface, where the manipulable parameters are selected to cause the top surface imaging to match the aberrations. The above method can also be applied to more than two surfaces, such as by selecting an immersion fluid and manipulating the position of the objective between imaging runs so that the difference between the axial ray and the marginal ray is the same for each surface. Other variations (e.g., setting the manipulable parameters of the system to sufficiently reduce aberrations to allow the use of a less complex compensator, rather than setting them to completely eliminate the need for a compensator) are also possible and will be immediately apparent to those skilled in the art in light of this disclosure. Therefore, the above description of methods for addressing aberration differences between surfaces and exemplary variations thereof should be understood to be illustrative only and should not be considered limiting.

[0080] In certain embodiments, the system based on the present disclosure can utilize sequencing by synthesis (SBS). In SBS, one or more fluorescently labeled modified nucleotides are used to determine the nucleotide sequence of nucleic acids present on the surface of a support structure (such as a flow cell). Exemplary SBS systems and methods that can be utilized together with the apparatus and methods described herein are described in U.S. Patent No. 7057026; U.S. Patent Application Publication Nos. 2005 / 0100900, 2006 / 0188901, 2006 / 0240439, 2006 / 0281109, and 2007 / 0166705; and PCT Publication Nos. WO 05 / 065814, WO 06 / 064199, and WO07 / 010251; Each of these patents is incorporated herein by reference.

[0081] In the specific use of the apparatus and methods herein, a flow cell containing arranged nucleic acids is processed by several repeated cycles of the entire sequencing process. Nucleic acids are prepared so that they contain oligonucleotide primers adjacent to unknown target sequences. In order to start the first SBS sequencing cycle, one or more differently labeled nucleotides and DNA polymerase are flowed into the flow cell. Single nucleotides can be added once, or the nucleotides used in the sequencing process can be specially designed to have reversible termination properties, so that each cycle of the sequencing reaction occurs simultaneously in the presence of all four labeled nucleotides (A, C, T, G). After adding nucleotides, features on the surface can be imaged to determine the characteristics (identity) of the incorporated nucleotides (based on the marks on the nucleotides). Then, reagents can be added to the flow cell to remove the closed 3' ends (if appropriate) and remove the marks from each incorporated base. Such cycles are then repeated, and the sequence of each cluster is read through multiple chemical cycles. In a further specific implementation, fluorinated nucleotides or other detectable components can only temporarily associate with the unknown target sequence for imaging in a step separated from advancing to the next nucleotide.

[0082] Other sequencing methods utilizing cyclic reactions, such as pyrophosphate sequencing and sequencing by ligation, can also be used, wherein each cycle includes a step of delivering one or more reagents to nucleic acids on a surface and imaging the surface-bound nucleic acids. Useful pyrophosphate sequencing reactions are described in, for example, U.S. Patent No. 7,244,559 and U.S. Patent Application Publication No. 2005 / 0191698, each of which is incorporated herein by reference. Sequencing by ligation is described in, for example, Shendure et al. Science 309:1728-1732 (2005); and U.S. Patent Nos. 5,599,675 and 5,750,341, each of which is incorporated herein by reference.

[0083] The methods and devices described herein can also be used to detect features present on surfaces used in genotyping assays, expression analysis, and other assays known in the art, such as those described in U.S. Patent Application Publications 2003 / 0108900, US2003 / 0215821, and US2005 / 0181394, each of which is incorporated herein by reference.

[0084] Although various specific implementations of the present disclosure have been described above, it should be understood that they are presented only by way of example and not limitation. Similarly, each figure may depict an exemplary architecture or other configuration of the present disclosure, which is done to help understand the features and functions that may be included in the present disclosure. The present disclosure is not limited to the exemplary architecture or configuration shown, but various alternative architectures and configurations may be used to achieve the desired features. In fact, it is obvious to those skilled in the art how to implement alternative functions, logical or physical partitions and configurations to achieve the desired features of the present disclosure. In addition, many different component module names other than those described herein may be applied to various partitions. In addition, with respect to flow charts, operational descriptions, and method claims, unless the context otherwise dictates, the order of the actions presented herein should not enforce that various specific implementations perform the functions in the same order.

Claims

1. A multi-surface biological sample imaging machine, characterized in that: The machine comprises: a lens having a field of view and immersed in a first fluid having a first refractive index; a flow cell comprising a first surface and a second surface separated by a second fluid having a second refractive index; and A controller, the controller being used to: moving the lens from a first position having a first distance from the flow cell to a second position having a second distance from the flow cell; capturing light emitted from nucleic acid disposed on the first surface of the flow cell using the lens when the lens is separated from the flow cell by the first distance; and capturing light emitted from nucleic acid disposed on the second surface of the flow cell using the lens when the lens is separated from the flow cell by the second distance; Wherein the optical path difference between the marginal light and the axial light in the field of view of the lens when the lens is separated from the circulation pool by the first distance is substantially equal to the optical path difference between the marginal light and the axial light in the field of view of the lens when the lens is separated from the circulation pool by the second distance.

2. The multi-surface biological sample imaging machine according to claim 1, characterized in that: The first refractive index and the second refractive index are substantially equal.

3. The multi-surface biological sample imaging machine according to claim 2, characterized in that: The lens comprises an optic adapted to capture light emitted from nucleic acid sequences disposed on the first surface of the flow cell and to allow the biological sample to be imaged on a detector with a diffraction limited imaging quality; as well as The first refractive index and the second refractive index are substantially equal, meaning that spherical aberration caused by any difference between the first refractive index and the second refractive index is sufficiently low to not prevent diffraction limited imaging of the biological sample, the diffraction limited imaging being based on: light emitted from nucleic acid sequences disposed on the first surface of the flow cell; as well as Light emitted from nucleic acid sequences disposed on the second surface of the flow cell.

4. The multi-surface biological sample imaging machine according to claim 2, characterized in that: The first fluid and the second fluid are the same fluid.

5. The multi-surface biological sample imaging machine according to claim 2, characterized in that: The first fluid and the second fluid are different fluids.

6. The multi-surface biological sample imaging machine according to claim 1, characterized in that: The first surface of the flow cell is a top surface of the flow cell, and the second surface of the flow cell is a bottom surface of the flow cell.

7. The multi-surface biological sample imaging machine according to claim 1, characterized in that: The lens comprises an optic adapted to capture light emitted from nucleic acid sequences disposed on the first surface of the flow cell and to allow the biological sample to be imaged on a detector with a diffraction limited imaging quality; as well as The optical path difference between marginal rays and axial rays in the field of view of the lens when the lens is separated from the flow cell by the first distance is substantially equal to the optical path difference between marginal rays and axial rays in the field of view of the lens when the lens is separated from the flow cell by the second distance, meaning that spherical aberration is sufficiently low so as not to prevent diffraction limited imaging of the biological sample, the diffraction limited imaging being based on: light emitted from nucleic acid sequences disposed on the first surface of the flow cell; as well as Light emitted from nucleic acid sequences disposed on the second surface of the flow cell.

Images