Sequencing illumination adjustment system

Abstract

Techniques for monitoring and maintaining illumination uniformity of a line scanning, fluorescence imaging system are described. In one implementation, an imaging system includes: a line generation device including a light source to emit a light beam, and a line forming optic to shape the light beam into a laser line; an objective to project the laser line on a surface of a sample container, causing a sample within the sample container to emit fluorescence emissions; a camera to capture an image corresponding to the fluorescence emissions from the sample; and one or more processors to: calculate, using the image, an illumination quality metric corresponding to the laser line; determine whether the illumination quality metric meets a threshold; and in response to determining that the illumination quality metric does not meet the threshold, adjust the laser line projected on the surface of the sample container.

Description

IP-2823-PCT (47CX-406184-WO)SEQUENCING ILLUMINATION ADJUSTMENT SYSTEMCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Patent Application No. 63 / 684,172, filed August 16, 2024, and titled “SEQUENCING ILLUMINATION ADJUSTMENT SYSTEM,” which is incorporated herein by reference in its entirety.BACKGROUND[0002| In DNA sequencing, illumination plays an important role in the detection process. Lasers activate fluorescent labels on these nucleotide bases, and emitted fluorescence is recorded to determine what base was added in a sequencing cycle to a complimentary sample strand. Different terminator bases (A, T, C, or G) are designed give off distinct colors depending on the base added to a sample strand to enable accurate base identification. Hence, uniform illumination of the sample can be important to enabling high quality sequencing data.

[0003] Optical targets can be used for calibration, alignment, and / or measurement in optical detection systems. The optical target can be utilized to test the accuracy and performance of the optical system. For example, the optical target affords a basis, with respect to which the optical system may quantify optical resolution, depth of focus, optical and mechanical drift, distortion, lens-based aberration, chromatism, and the like. Thus, optical targets can facilitate accurate calibration of alignment and validation of optical detection systems. Optical targets typically include a light emitting material whose emissions are detected by the optical detection system. The emissions are used for the calibration, alignment, and / or measurement.SUMMARY

[0004] Implementations of the disclosure relate to systems and methods for monitoring and maintaining illumination uniformity of a line scanning, fluorescence imaging system.

[0005] In one embodiment, an imaging system comprises: a line generation device comprising: a first light source configured to emit a first light beam at a first wavelength; and one or more line forming optics configured to shape the first light beam emitted by the first light source into a first laser line; an objective configured to project the first laser line on a first surface of a sample container, causing a sample within the sample container to emit first fluorescence emissions; a camera configured to capture a first image corresponding to the first fluorescence emissions from the sample; and one or more processors configured to: calculate, using the first image, a first illumination quality metric corresponding to the first laser line;SMRH:4916-5472-0852 -1-IP-2823-PCT (47CX-406184-WO) determine whether the first illumination quality metric meets a threshold; and in response to determining that the first illumination quality metric does not meet the threshold, adjust the first laser line projected on the first surface of the sample container.

[0006] In some implementations, the sample container comprises a second surface opposite the first surface; the first light source is further configured to emit a second light beam at the first wavelength; the one or more line forming optics are further configured to shape the second light beam emitted by the first light source into a second laser line; the objective is further configured to project the second laser line on the second surface of the sample container, causing the sample to emit second fluorescence emissions; the camera is further configured to capture a second image corresponding to the second fluorescence emissions from the sample; and the one or more processors are further configured to: calculate, using the second image, a second illumination quality metric corresponding to the second laser line; determine whether the second illumination quality metric meets a threshold; and in response to determining that the second illumination quality metric does not meet the threshold, adjust the second laser line projected on the second surface of the sample container.

[0007] In some implementations, the sample container is a flow cell; the first surface is a top interior surface of the flow cell; and the second surface is a bottom interior surface of the flow cell.

[0008] In some implementations, the line generation device further comprises a second light source to emit a second light beam at a second wavelength different from the first wavelength; the one or more line forming optics are further configured to shape the second light beam emitted by the second light source into a second laser line; the objective is further configured to project the second laser line on the first surface of the sample container, causing the sample within the sample container to emit second fluorescence emissions; the camera is further configured to capture a second image corresponding to the second fluorescence emissions from the sample; and the one or more processors are further configured to: calculate, using the second image, a second illumination quality metric corresponding to the second laser line; determine whether the second illumination quality metric meets a threshold; and in response to determining that the second illumination quality metric does not meet the threshold, adjust the second laser line projected on the first surface.

[0009] In some implementations, the first illumination quality metric comprise a uniformity of the first laser line projected on the first surface; and adjusting the first laser lineSMRH:4916-5472-0852 -2-IP-2823-PCT (47CX-406184-WO) projected on the first surface of the sample container comprises adjusting the one or more line forming optics such that the uniformity of the first laser line meets the threshold.

[0010] In some implementations, adjusting the one or more line forming optics such that the uniformity of the first laser line meets the threshold comprises adjusting a beam expander of the first light beam.

[0011] In some implementations, the first illumination quality metric comprise an alignment of the first laser line projected on the first surface; and adjusting the first laser line projected on the first surface of the sample container comprises adjusting an XY position of the first laser line such that the alignment of the first laser line meets the threshold.

[0012] In some implementations, the imaging system further comprises: a network communication interface configured to transmit, over a communication network, a notification to a remote management system indicating whether or not the first illumination quality metric meets the threshold.

[0013] In some implementations, the imaging system further comprises a user interface configured to present to a user of the imaging system, in response to the first illumination quality metric not meeting the threshold, a notification indicating that the first laser line requires an adjustment.

[0014] In some implementations, the imaging system is configured to sequence the sample after adjusting the first laser line projected on the first surface of the sample container.

[0015] In some implementations, the imaging system is configured to sequence the sample before adjusting the first laser line projected on the first surface of the sample container; and the first image is configured to be captured during sequencing of the sample.

[0016] In some implementations, the imaging system is configured to sequence the sample while adjusting the first laser line projected on the first surface of the sample container.

[0017] In one embodiment, a method comprises: projecting, using a first light source of an imaging system, a first laser line on a first surface of a sample container, causing a sample within the sample container to emit first fluorescence emissions; capturing, using a camera of the imaging system, a first image corresponding to the first fluorescence emissions from the sample; calculating, using the first image, a first illumination quality metric corresponding to the first laser line; determining whether the first illumination quality metric meets a firstSMRH:4916-5472-0852 -3-IP-2823-PCT (47CX-406184-WO) threshold; and in response to determining that the first illumination quality metric does not meet the first threshold, adjusting, by the imaging system, the first laser line projected on the first surface.

[0018] In some implementations, the method further comprises sequencing, using the imaging system, the sample after adjusting the first laser line projected on the first surface of the sample container.[0019| In some implementations, the method further comprises sequencing, using the imaging system the sample before adjusting the first laser line projected on the first surface of the sample container, wherein the first image is captured during sequencing of the sample.

[0020] In some implementations, the method further comprises sequencing, using the imaging system, the sample, wherein the first laser line projected on the first surface of the sample container is adjusted during sequencing.

[0021] In some implementations, the method further comprises: projecting, using the first light source, a second laser line on a second surface of the sample container opposite the first surface, causing the sample within the sample container to emit second fluorescence emissions; capturing, using the camera, a second image corresponding to the second fluorescence emissions from the sample; calculating, using the second image, a second illumination quality metric corresponding to the second laser line; determining whether the second illumination quality metric meets a threshold; and in response to determining that the second illumination quality metric does not meet the threshold, adjusting, by the imaging system, the second laser line projected on the second surface.

[0022] In some implementations, the method further comprises: projecting, using a second light source of the imaging system, a second laser line on the first surface, causing the sample within the sample container to emit second fluorescence emissions, the first laser line and the second laser line having different wavelengths; capturing, using the camera, a second image corresponding to the second fluorescence emissions from the sample; calculating, using the second image, a second illumination quality metric corresponding to the second laser line; determining whether the second illumination quality metric meets a threshold; and in response to determining that the second illumination quality metric does not meet the threshold, adjusting, by the imaging system, the second laser line projected on the first surface.

[0023] In one embodiment, a method comprises: projecting, using an imaging system, a laser line beam on a surface of an optical alignment target, causing fluorescent material withinSMRH:4916-5472-0852 -4-IP-2823-PCT (47CX-406184-WO) the optical alignment target to emit fluorescence emissions; capturing, using a camera of the imaging system, a line profile image corresponding to the fluorescence emissions; determining, based on the line profile image, beam edge information of the laser line beam; extracting, based on the line profile image, frequency spectrum information associated with a uniformity of the laser line beam; and calculating, based on the beam edge information and frequency spectrum information, an illumination uniformity of the laser line beam.

[0024] In some implementations, the optical alignment target comprises: a substrate that is translucent or transparent; and a fluorescent layer disposed below a bottom surface of the substrate, the fluorescent layer comprising the fluorescent material; and projecting the laser line beam on the surface of the optical alignment target comprises projecting the laser line beam within the fluorescent layer.

[0025] In some implementations, the optical alignment target further comprises: an opaque material formed over the bottom surface of the substrate in a pattern comprising multiple openings; and each of the openings has a longest dimension that is about 1 pm or less.

[0026] In some implementations, each of the openings is a pinhole.

[0027] In some implementations, the fluorescent material comprises quantum dots.|0028] In some implementations, capturing the line profile image corresponding to the fluorescence emissions comprises capturing, using the camera, a plurality of images of an imaging slit positioned in an optical path between the fluorescent layer and the laser line beam; and constructing the line profile image from the plurality of images.

[0029] In some implementations, capturing the plurality of images of the imaging slit comprises moving the imaging slit across a profile of the laser line beam, wherein each of the plurality of images corresponds to a respective position of the imaging slit relative to the profile of the laser line beam.

[0030] In some implementations, the thickness of the fluorescent layer is less than 10 pm.

[0031] In some implementations, the method further comprises: determining that the illumination uniformity does not meet a threshold; and in response to determining that the illumination uniformity does not meet the threshold: adjusting one or more components of the imaging system used to generate or project the laser line beam; transmitting, over a communication network, a notification to a remote management system indicating that theSMRH:4916-5472-0852 -5-IP-2823-PCT (47CX-406184-WO) illumination uniformity does not meet the threshold; or presenting, to a user of the imaging system, a notification indicating that the illumination uniformity does not meet the threshold.

[0032] Other features and aspects of the disclosed technology will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, the features in accordance with implementations of the disclosed technology. The summary is not intended to limit the scope of any inventions described herein, which are defined by the claims and equivalents.BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The present disclosure, in accordance with one or more implementations, is described in detail with reference to the following figures. The figures are provided for purposes of illustration only and merely depict example implementations. Furthermore, it should be noted that for clarity and ease of illustration, the elements in the figures have not necessarily been drawn to scale.

[0034] Some of the figures included herein illustrate various implementations of the disclosed technology from different viewing angles. Although the accompanying descriptive text may refer to such views as “top,” “bottom” or “side” views, such references are merely descriptive and do not imply or require that the disclosed technology be implemented or used in a particular spatial orientation unless explicitly stated otherwise.

[0035] FIG. 1A shows an imaging system, in accordance with some implementations of the disclosure.

[0036] FIG. IB shows a block diagram illustrating an example two-channel, linescanning modular optical imaging system, in accordance with some implementations of the disclosure.

[0037] FIG. 1C shows a particular example of a line generation module, in accordance with some implementations of the disclosure.

[0038] FIG. ID shows a block diagram illustrating a line generation module alignment system, in accordance with some implementations of the disclosure.

[0039] FIG. IE illustrates a perspective view of the line generation module alignment system of FIG. ID.

[0040] FIG. IF shows a block diagram illustrating a line generation module, an objective lens, and a flow cell, in accordance with some implementations of the disclosure.SMRH:4916-5472-0852 -6-IP-2823-PCT (47CX-406184-WO)

[0041] FIG. 1G shows a block diagram illustrating a line generation module and emission optics module system used to widen the laser line pattern on a flow cell, in accordance with some implementations of the disclosure.

[0042] FIG. 1H shows a sample container having two surfaces, in accordance with some implementations of the disclosure.

[0043] FIG. 2A illustrates changes in line profile uniformity that can occur over about 11 months for a blue light source used in a two-channel line scanning imaging system, in accordance with some implementations of the disclosure.

[0044] FIG. 2B illustrates changes in line profile uniformity that can occur over about 11 months for a green light source used in a two-channel line scanning imaging system, in accordance with some implementations of the disclosure.

[0045] FIG. 3 depicts images of blue and green laser lines on top and bottom surfaces of a flow cell imaged by a multi-channel line scanning imaging system, in accordance with some implementations of the disclosure.

[0046] FIG. 4 is a flow diagram illustrating an example method of monitoring and adjusting illumination uniformity in a line scanning, fluorescent imaging system, in accordance with some implementations of the disclosure.

[0047] FIG. 5 shows a laser line image and plots representative of laser line uniformity of a multi-channel imaging system that sequences a flow cell, before and after laser line adjustment, in accordance with some implementations of the disclosure.

[0048] FIG. 6 shows an example image obtained of an optical alignment target as compared with an image of a flow cell, in accordance with some implementations of the disclosure.

[0049] FIG. 7 depicts a top view of one example of an optical alignment tool that includes one or more optical alignment targets for optical measurements and analysis of an optical instrument, in accordance with some implementations of the disclosure.

[0050] FIG. 8 depicts an example of an optical alignment target, in accordance with some implementations of the disclosure.

[0051] FIG. 9 illustrates one example image of an excitation beam footprint comprising patterned pinholes.SMRH:4916-5472-0852 -7-IP-2823-PCT (47CX-406184-WO)

[0052] FIG. 10 illustrates one example method of sampling an imaging slit to generate a beam footprint image, in accordance with some implementations of the disclosure.

[0053] FIG. 11 is an operational flow diagram illustrating an example method that can be implemented with an imaging system including an optical alignment target to estimate a uniformity of an excitation laser light source, in accordance with some implementations of the disclosure.

[0054] The figures are not exhaustive and do not limit the present disclosure to the precise form disclosed.DETAILED DESCRIPTION

[0055] As used herein to refer to a sample, the term "feature" is intended to mean a point or area in a pattern that can be distinguished from other points or areas according to relative location. An individual feature can include one or more molecules of a particular type. For example, a feature can include a single target nucleic acid molecule having a particular sequence or a feature can include several nucleic acid molecules having the same sequence (and / or complementary sequence, thereof).

[0056] As used herein, the term “swath” is intended to mean a rectangular portion of an object. The swath can be an elongated strip that is scanned by relative movement between the object and a detector in a direction that is parallel to the longest dimension of the strip. Generally, the width of the rectangular portion or strip will be constant along its full length. Multiple swaths of an object can be parallel to each other. Multiple swaths of an object can be adjacent to each other, overlapping with each other, abutting each other, or separated from each other by an interstitial area. A swath can be divided into multiple regions referred to as “tiles”.

[0057] As used herein, the term "fiducial" is intended to mean a distinguishable point of reference in or on an object. The point of reference can be present in an image of the object or in another data set derived from detecting the object. The point of reference can be specified by an x and / or y coordinate in a plane of the object. Alternatively, or additionally, the point of reference can be specified by a z coordinate that is orthogonal to the xy plane, for example, being defined by the relative locations of the object and a detector. One or more coordinates for a point of reference can be specified relative to one or more other features of an object or of an image or other data set derived from the object.SMRH:4916-5472-0852 -8-IP-2823-PCT (47CX-406184-WO)

[0058] As used herein, the term “xy coordinates” is intended to mean information that specifies location, size, shape, and / or orientation in an xy plane. The information can be, for example, numerical coordinates in a Cartesian system. The coordinates can be provided relative to one or both of the x and y axes or can be provided relative to another location in the xy plane. For example, coordinates of a feature of an object can specify the location of the feature relative to location of a fiducial or other feature of the object.

[0059] As used herein, the term "xy plane" is intended to mean a 2-dimensional area defined by straight line axes x and y. When used in reference to a detector and an object observed by the detector, the area can be further specified as being orthogonal to the direction of observation between the detector and object being detected. When used herein to refer to a line scanner, the term “y direction” refers to the direction of scanning.

[0060] As used herein, the term "z coordinate" is intended to mean information that specifies the location of a point, line or area along an axis that is orthogonal to an xy plane. In particular implementations, the z axis is orthogonal to an area of an object that is observed by a detector. For example, the direction of focus for an optical imaging system may be specified along the z axis.

[0061] As used herein, the term "scanning" is intended to mean detecting a 2- dimensional cross-section in an xy plane of an object, the cross-section being rectangular or oblong. For example, in the case of fluorescence imaging an area of an object having rectangular or oblong shape can be specifically excited (at the exclusion of other areas) and / or emission from the area can be specifically acquired (at the exclusion of other areas) at a given time point in the scan.

[0062] As used herein, the term “optical alignment target” is a portion of an optical alignment tool that includes a material that is responsive to excitation light, and whose emissions are used for alignment (e.g., optical alignment in all six degrees of freedom, i.e., X, Y, Z, theta-x, theta-y, theta-z) and / or validation (e.g., calibration, quantification, or characterization of optical properties) of imaging modules of an optical instrument. Such imaging modules can be used in, for example, optical detection of samples, such as those samples detected in nucleic acid sequencing procedures. An optical alignment tool can include a single optical alignment target or multiple optical alignment targets.

[0063] As used herein, the term “quantum dots” (QD) refers to very small semiconductor particles (e.g., from about 2 nm to about 12 nm in diameter) that have opticalSMRH:4916-5472-0852 -9-IP-2823-PCT (47CX-406184-WO) and electronic properties that differ from the properties of larger particles. Quantum dots can be designed to emit light of specific frequencies of interest in response to electricity or light applied thereto. The emission frequencies can be tuned by changing the dot size, shape and / or material. In some examples, nanoscale semiconductor materials tightly confine either electrons or electron holes. By way of example, quantum dots can also be referred to as artificial atoms, a term that emphasizes that a quantum dot is a single object with bound, discrete electronic states, as is the case with naturally occurring atoms or molecules. Quantum dots have optoelectronic properties that change as a function of both size and shape. Larger QDs (radius of 5-6 nm, for example) emit longer wavelengths, resulting in emission colors such as orange or red. Smaller QDs (radius of 2-3 nm, for example) emit shorter wavelengths, resulting in emission colors like blue and green. Specific colors and sizes vary depending on the exact composition of the QD.

[0064] As used herein, the term “translucent” refers to a material that is capable of transmitting some wavelengths of light. The term “transparent” refers to a material that is capable of transmitting all or most wavelengths of light. A transparent material transmits more wavelengths of light than a translucent material. Both translucency and transparency can be quantified using transmittance, i.e., the ratio of light energy falling on a body to that transmitted through the body. The transmittance of a transparent or translucent material will depend upon the thickness of the material, the wavelength of light, and the dosage of the light to which it is exposed. In some of the examples described herein, the transmittance of the transparent or translucent material can range from about 0.25 (25%) to 1 (100%). The transparent or translucent material can be a pure material, a material with some impurities, or a mixture of materials, as long as the resulting transparent or translucent material is capable of the desired transmittance. Additionally, depending upon the transmittance of the transparent or translucent material, the time for light exposure and / or the output power of the light source may be increased or decreased to deliver a suitable dose of light energy through the transparent or translucent material to achieve the desired effect (e.g., optical alignment).

[0065] As discussed above, uniform illumination of a biological sample can be particularly important in fluorescence imaging applications such as sequencing. However, during an imaging system’s life and over the variation in operational conditions, illumination quality can degrade. This can be due to instrument hardware wearing out and / or variations in operating conditions causing the state of the imaging system to become non-ideal, leading to less than optimal illumination quality. This can also be caused by changes in the opticalSMRH:4916-5472-0852 -10-IP-2823-PCT (47CX-406184-WO) imaging system itself induced by the scattering of the excitation energy, degradation of the optics, or heating of the optics induced by transmission of excitation energy. As such, there is a need to design biological sample imaging systems that monitor changes in illumination uniformity over time, and that make suitable optical, hardware, or software adjustments to correct for any degradation in illumination uniformity.|0066] To this end, the disclosure describes techniques for monitoring and adjusting illumination of a line scanning, fluorescence imaging system, enabling illumination adjustment to be carried out remotely and / or automatically in response to degradation in illumination line quality. By virtue of implementing the techniques described herein, consistency and effectiveness of illumination processes can be ensured, preventing damage to the biological sample, extending the effective life of the instrument, and / or greatly improving the data quality performance of a given instrument. The technology described herein can also have the advantage of enabling real time controls to be deployed enabling the adjustment of illumination during the actual imaging process. This can be particularly useful in compensating for transient changes in the optical system due to heating of the optics. These and other benefits that can be realized by implementing the technology described herein are further described below.

[0067] Before describing various implementations of the systems and methods disclosed herein, it is useful to describe an example environment with which the technology disclosed herein can be implemented. One such example environment is that of an imaging system 100 illustrated in FIG. 1A. The example imaging system may include a device for obtaining or producing an image of a sample. It should be noted that although systems and methods may be described herein from time to time in the context of example imaging system 100, these are only examples with which implementations of the illumination uniformity correction methods disclosed herein may be implemented.

[0068] As can be seen in the example of FIG. 1A, subject samples are located on sample container 110 (e.g., a flow cell as described herein), which is positioned on a sample stage 170 under an objective lens 142. Light source 160 and associated optics direct a beam of light, such as laser light, to a chosen sample location on the sample container 110. The sample fluoresces and the resultant light is collected by the objective lens 142 and directed to an image sensor of camera system 140 to detect the florescence. Sample stage 170 is moved relative to objective lens 142 to position the next sample location on sample container 110 at the focal point of the objective lens 142. Movement of sample stage 110 relative to objective lens 142 can be achieved by moving the sample stage itself, the objective lens, some other component of theSMRH:4916-5472-0852 -11-IP-2823-PCT (47CX-406184-WO) imaging system, or any combination of the foregoing. Further implementations may also include moving the entire imaging system over a stationary sample.

[0069] Fluid delivery module 122 directs the flow of reagents (e.g., fluorescently labeled nucleotides, buffers, enzymes, cleavage reagents, etc.) to (and through) sample container 110 and waste valve 120. The system also comprises temperature station actuator 130 and heater / cooler 135 that can optionally regulate the temperature of conditions of the fluids within the sample container 110.

[0070] Sample container 110 can include one or more substrates upon which the samples are provided. For example, in the case of a system to analyze a large number of different nucleic acid sequences, sample container 110 can include one or more substrates on which nucleic acids to be sequenced are bound, attached or associated. In various implementations, the substrate can include any inert substrate or matrix to which nucleic acids can be attached, such as for example glass surfaces, plastic surfaces, latex, dextran, polystyrene surfaces, polypropylene surfaces, polyacrylamide gels, gold surfaces, and silicon wafers. In some applications, the substrate is within a channel or other area at a plurality of locations formed in a matrix or array across the sample container 110.

[0071] In some implementations, the sample container 110 may include a biological sample that is imaged using one or more fluorescent dyes. For example, in a particular implementation the sample container may be implemented as a patterned flow cell including a translucent cover plate, a substrate, and a liquid sandwiched therebetween, and a biological sample may be located at an inside surface of the translucent cover plate or an inside surface of the substrate. The flow cell may include a large number (e.g., thousands, millions, or billions) of wells or regions that are patterned into a defined array (e.g., a hexagonal array, rectangular array, etc.) into the substrate. Each region may form a cluster (e.g., a monoclonal cluster) of a biological sample such as DNA, RNA, or another genomic material which may be sequenced, for example, using sequencing by synthesis. The flow cell may be divided into a number of physically separated lanes (e.g., eight lanes) per flow cell surface, each lane including an array of clusters. The flow cell may include multiple surfaces for imaging. During each cycle of sequencing, each surface of the flow cell (e.g., top surface and bottom surface) may be imaged. During imaging, each lane may be imaged in separate swaths (e.g., three), and any number of images may be collected for each swath. For example, one or more images can be collected for each tile of a swath.SMRH:4916-5472-0852 -12-IP-2823-PCT (47CX-406184-WO)

[0072] Light source 160 (e.g., an excitation laser within an assembly optionally comprising multiple lasers) or other light source can be included to illuminate fluorescent sequencing reactions within the samples via illumination through a fiber optic interface (which can optionally comprise one or more re-imaging lenses, a fiber optic mounting, etc. In some implementations, excitation light source 160 utilizes line scanning to image a sample. For example, the excitation light source 160 may be implemented as part of a line generation module including one or more light sources operating at one or more wavelengths, and a beam shaping optics aligned at a predetermined angle to each light source. The beam shaping optics may be used to provide uniform line illumination at a desired aspect ratio. In a particular implementation, the line generation module is implemented as part of a two-channel imaging system including a first light source operating at a first wavelength, and a second light source operating at a second wavelength. For example, the first wavelength may be a “green” wavelength (e.g., from about 520 to 565 nm), and the second wavelength may be a “blue” wavelength (e.g., from about 405 to 505 nm). Such a line scanning system may be utilized in conjunction with a time delay integration (TDI) sensor.

[0073] Camera system 140 can be included to monitor and track the sequencing of sample container 110. Camera system 140 can be implemented, for example, as a charge- coupled device (CCD) camera (e.g., a TDI CCD camera), which can interact with various filters within filter switching assembly 145, objective lens 142, and focusing laser / focusing laser assembly 150. Camera system 140 is not limited to a CCD camera and other cameras and image sensor technologies can be used. The light emanating from a sample location being imaged can be directed to one or more detectors of camera system 140. An aperture can be included and positioned to allow only light emanating from the focus area to pass to the detector. The aperture can be included to improve image quality by filtering out components of the light that emanate from areas that are outside of the focus area. Emission filters can be included in filter switching assembly 145, which can be selected to record a determined emission wavelength.|0074] Sample container 110 can be ultimately mounted on a sample stage 170 to provide movement and alignment of the sample container 110 relative to the objective lens 142. The sample stage can have one or more actuators to allow it to move in any of three dimensions. For example, in terms of the Cartesian coordinate system, actuators can be provided to allow the stage to move in the X, Y and Z directions relative to the objective lens. This can allow one or more sample locations on sample container 110 to be positioned in optical alignment with objective lens 142.SMRH:4916-5472-0852 -13-IP-2823-PCT (47CX-406184-WO)

[0075] A focus (z-axis) component 175 is shown in this example as being included to control positioning of the optical components relative to the sample container 110 in the focus direction (typically referred to as the z axis, or z direction). Focus component 175 can include one or more actuators physically coupled to the optical stage or the sample stage, or both, to move sample container 110 on sample stage 170 relative to the optical components (e.g., the objective lens 142) to provide proper focusing for the imaging operation. For example, the actuator may be physically coupled to the respective stage such as, for example, by mechanical, magnetic, fluidic or other attachment or contact directly or indirectly to or with the stage. The one or more actuators can be configured to move the stage in the z-direction while maintaining the sample stage in the same plane (e.g., maintaining a level or horizontal attitude, perpendicular to the optical axis). The one or more actuators can also be configured to tilt the stage. This can be done, for example, so that sample container 110 can be leveled dynamically to account for any slope in its surfaces. Focusing of the system generally refers to aligning the focal plane of the objective lens with the sample to be imaged at the chosen sample location. Because the sample container is not perfectly flat as fixtured in the instrument, focus component 175 may be set up to follow this profile while moving along in the scanning direction (herein referred to as the y-axis).[0076| In some implementations, the imaging system 100 can also include an optical alignment target (not shown) that can be imaged periodically using the same camera system 140 used to image sample container 110 or using a different camera system. Based on images of the optical alignment target, control software can be used to calculate the imaging system’s optical alignment and / or health, including, for example, measuring illumination uniformity.

[0077] Output data from the sensors of camera system 140 may be communicated to an image analysis module 225 of imaging system 100 that may be implemented as a software application or instructions that analyze the image data or characteristics of the excitation light source / laser beam (e.g., uniformity, shape, intensity, power, brightness, position). Image analysis module, in various implementations, executes computer readable instructions for analyzing the image data, and / or reporting or displaying the characteristics of the beam to a graphical user interface (GUI) or remote management system 199. These operations may be performed in real-time during imaging cycles to minimize downstream analysis time and provide real time feedback and troubleshooting during an imaging run. In implementations, image analysis module 225 may be a computing device that is communicatively coupled to and controls imaging system 100.SMRH:4916-5472-0852 -14-IP-2823-PCT (47CX-406184-WO)

[0078] The data collected by image analysis module 225 can be communicated to a remote management system 199 over communication network 195 and / or used to correct for illumination uniformity issues with the imaging system 100. For example, the remote management system 199 can receive imaging data indicating that illumination uniformity is degrading or not adjusted for a particular line scanning light source. To correct for illumination uniformity issues, remote management system 199 can communicate configuration data to imaging system 100 via communication network 195. The configuration data can be used to adjust light source 160, optical components in the light path of light source 160, or other components of imaging system 100. The communication network 195 can include the Internet. Communications can be implemented using any suitable communication medium including cable, cellular, and / or satellite. Some example implementations of a remote management system that can be used with the technology described herein are further described in U.S. Patent No. 10,871,497, which is incorporated herein by reference.

[0079] A controller 190 can be provided to control the operation of the scanning system. The controller can be implemented to control aspects of system operation such as, for example, illumination of the sample, focusing, stage movement, and imaging operations. In various implementations, the controller can be implemented using hardware, algorithms (e.g., machine executable instructions), or a combination of the foregoing. For example, in some implementations the controller can include one or more CPUs or processors with associated memory. As another example, the controller can comprise hardware or other circuitry to control the operation, such as a computer processor and a non-transitory computer readable medium with machine-readable instructions stored thereon. For example, this circuitry can include one or more of the following: field programmable gate array (FPGA), application specific integrated circuit (ASIC), programmable logic device (PLD), complex programmable logic device (CPLD), a programmable logic array (PLA), programmable array logic (PAL) or other similar processing device or circuitry. As yet another example, the controller can comprise a combination of this circuitry with one or more processors. In some implementations, the controller 190 can perform these operations in conjunction with the data collected by image analysis module 225. In some implementations, image analysis module 225 and controller 190 can be components of the same computing system.

[0080] FIG. 1H illustrates an example sample container 16 that can be used with some of the implementations described herein. In this example, the sample container 16 includes an upper plate 42 and a lower plate 44 with an internal volume between the upper and lower platesSMRH:4916-5472-0852 -15-IP-2823-PCT (47CX-406184-WO)42, 44. The upper and lower plates 42, 44 can be made of any of a variety of materials but may preferably be made of a substrate material that is substantially transparent at the wavelengths of the excitation radiation, allowing for the passage of excitation radiation and returned fluorescent emissions without significant loss of signal quality. Biological material is attached to a first surface 18 as well as to a second surface 20. An attachment layer 56 has formed on both the first surface 18 and the second surface 20 of the sample container 16. A first excitation radiation 58 source can be used to irradiate one of many sites of biological component 12 on the first surface 18 of the sample container 16 and return a first fluorescent emission 60 from the irradiated biological component 12. Simultaneously or sequentially, a second source of excitation radiation 62 (or the same source) can be used to irradiate one of many sites of biological component 14 on the second surface 20 of the sample container 16 and return a second fluorescent emission 64 from the irradiated biological component 14. Particular example implementations of performing imaging and / or focusing operations on a multi-surface sample container are further described in U.S. Patent No. US 8,039,817, which is incorporated herein by reference in its entirety.

[0081] FIG. IB is block diagram illustrating an example two-channel, line-scanning modular optical imaging system 200 that may be implemented in particular embodiment of imaging system 100. It should be noted that although systems and methods may be described herein from time to time in the context of example imaging system 200, these are only examples with which implementations of the technology disclosed herein may be implemented.

[0082] In some implementations, system 200 may be used for the sequencing of nucleic acids. Applicable techniques include those where nucleic acids are attached at fixed locations in an array (e.g., the wells of a flow cell) and the array is imaged repeatedly. In such implementations, system 200 may obtain images in two different color channels, which may be used to distinguish a particular nucleotide base type from another. More particularly, system 200 may implement a process referred to as “base calling,” which generally refers to a process of a determining a base call (e.g., adenine (A), cytosine (C), guanine (G), or thymine (T)) for a given spot location of an image at an imaging cycle. During two-channel base calling, image data extracted from two images may be used to determine the presence of one of four base types by encoding base identity as a combination of the intensities of the two images. For a given spot or location in each of the two images, base identity may be determined based on whether the combination of signal identities is [on, on], [on, off], [off, on], or [off, off].SMRH:4916-5472-0852 -16-IP-2823-PCT (47CX-406184-WO)[00831 Referring again to imaging system 200, the system includes a line generation module (LGM) 210 with two light sources, 211 and 212, disposed therein. Light sources 211 and 212 may be coherent light sources such as laser diodes which output laser beams. Light source 211 may emit light in a first wavelength (e.g., a blue color wavelength), and light source 212 may emit light in a second wavelength (e.g., a green color wavelength). The light beams output from laser sources 211 and 212 may be directed through a beam shaping lens or lenses 213. In some implementations, a single light shaping lens may be used to shape the light beams output from both light sources. In other implementations, a separate beam shaping lens may be used for each light beam. In some examples, the beam shaping lens is a Powell lens, such that the light beams are shaped into line patterns. The beam shaping lenses of LGM 210 or other optical components imaging system be configured to shape the light emitted by light sources 211 and 212 into a line patterns (e.g., by using one or more Powel lenses, or other beam shaping lenses, diffractive or scattering components). LGM 210 may further include mirror 214 and semi-reflective mirror 215 configured to direct the light beams through a single interface port to an emission optics module (EOM) 230. The light beams may pass through a shutter element 216. FIG. 1C illustrates a particular example of a LGM 210 that can be implemented in some implementations of the disclosure.[0084| EOM 230 may include objective 235 and a z-stage 236 which moves objective 235 longitudinally closer to or further away from a target 250. For example, target 250 may include a liquid layer 252 and a translucent cover plate 251, and a biological sample may be located at an inside surface of the translucent cover plate as well an inside surface of the substrate layer located below the liquid layer. The z-stage may then move the objective as to focus the light beams onto either inside surface of the flow cell (e.g., focused on the biological sample). The biological sample may be DNA, RNA, proteins, or other biological materials responsive to optical sequencing as known in the art.

[0085] EOM 230 may include semi-reflective mirror 233 to reflect a focus tracking light beam emitted from a focus tracking module (FTM) 240 onto target 250, and then to reflect light returned from target 250 back into FTM 240. FTM 240 may include a focus tracking optical sensor to detect characteristics of the returned focus tracking light beam and generate a feedback signal to focus objective 235 on target 250.

[0086] EOM 230 may also include semi-reflective mirror 234 to direct light through objective 235, while allowing light returned from target 250 to pass through. In some implementations, EOM 230 may include a tube lens 232. Light transmitted through tube lensSMRH:4916-5472-0852 -17-IP-2823-PCT (47CX-406184-WO)232 may pass through filter element 231 and into camera module (CAM) 220. CAM 220 may include one or more optical sensors 221 to detect light emitted from the biological sample in response to the incident light beams (e.g., fluorescence in response to blue and green light received from light sources 211 and 212).

[0087] Output data from the sensors of CAM 220 may be communicated to an image analysis module 225 as described above. For example, it can analyze the image data or characteristics of the excitation light sources / laser beams 211 and 212. It also can communicate image data to remote management system 199. In implementations further described below, image analysis module 225 can execute computer readable instructions for analyzing and correcting illumination uniformity on target 250 of either light beam generated by LGM 210 and output by EOM 230.

[0088] To generate a uniform line illumination through an objective lens an LGM alignment system can be used in conjunction with LGM 210 to align the internal components of the LGM when the LGM is being assembled, maintained, or operated. The LGM can use one or more Powell lenses to spread and / or shape the laser beams from single or near- single mode laser light sources. Other beam shaping optics may be used to control uniformity and increase tolerance such as an active beam expander, an attenuator, one relay lenses, cylindrical lenses, actuated mirrors, diffractive elements, and scattering components. Laser beams may intersect at the back focal point of objective lens to provide better tolerance on flow cell surfaces (e.g., as illustrated in FIG. IF). A Powell lens may be located near the objective lens, or near a relay lens. The angle of the laser beam entering the imaging optics may be adjusted to match the field view of imaging optics. The direction, size, and / or polarization of the laser beams may be adjusted by using lenses, mirrors, and / or polarizers. Optical lenses (e.g., cylindrical, spherical, or aspheric) may be used to actively adjust the illumination focus on dual surfaces of the flow cell target. The light modules on LGM 210 may be replaceable individually for field service.

[0089] To maintain uniform illumination and avoid photo-bleaching on adjacent area or photo- saturation of fluorophores, illumination line profiles can be adjusted. For example, in some implementations, by widening the laser line patterns at the flow cell and / or sensor, higher scan speeds and laser powers may be employed. Methods for adjusting illumination line width using optics may include adding a defocus lens, prism array, or diffuser after or before the Powell lens. In some implementations, these methods may also include reducing or increasing the laser illumination beam size and / or reducing objective lens infinite conjugation design.SMRH:4916-5472-0852 -18-IP-2823-PCT (47CX-406184-WO)FIG. 1G illustrates a block diagram of an LGM and EOM system used to widen the laser line pattern on a flow cell to avoid photo- saturation and photo-bleaching. The laser beam line width incident on the flow cell may be increased to adjust illumination uniformity, reduce excitation power density, and / or avoid photo-saturation. Line width may be increased, for example, by incorporating a defocus lens, prism, array, or diffuser either in front of or behind the Powell lens. In some implementations, the line width may be increased by defocusing the objective lens, as illustrated in FIG. 1G (e.g., moving the objective lens in the Z-axis) to focus the line pattern beyond the surfaces of the flow cell. In some examples, defocusing the line pattern to a distance of between about 50 microns and about 150 microns from a distal surface of the flow cell may generate a line width larger than 10 microns, and effectively reduce photo-saturation and photo-bleaching effects.

[0090] FIG. ID illustrates a block diagram of a LGM alignment system. FIG. IE illustrates a perspective view of an LGM alignment system. As illustrated, in some implementations, a green laser module may generate a first laser beam that reflects off two PZT mirrors. Similarly, a blue laser module may generate a second laser beam that also reflects off of two PZT mirrors and is combined with the first laser beam. Both laser beams may then pass through a Powell lens to generate a line pattern, and then through a shutter, EOM optics, and an objective lens. In some implementations, the laser beams may be defocused using a defocus lens prior to passing through the objective as to increase the line width of the laser beams. Alternatively, the laser beams may be defocused by articulating the objective in the Z- axis. By focusing the laser beams at a focal point beyond the surfaces of the flow cell, the laser lines may be widened. In some implementations, the line patterns may be increased in width from less than 5 microns to more than 13 microns.

[0091] The LGM alignment system may include control surfaces to adjust or manipulate relative positioning of mirrors, lenses, lasers, and / or other components or optics in the LGM 210. For example, one or more of the optical components may be adjusted or manipulated automatically. Automatic control devices may include a motorized translation stage, an actuation device, one or more piezo stages, and / or one or more automatic switch and flip mirrors and lenses. A software interface may be used to control all the devices, test system, calibration, and test procedure. The alignment system can include a beam profiler, imaging lens (replacing EOM objective lens), attenuator, and / or alignment targets. In other implementations, adjustments may be made using manual manipulation of control knobs, screws, or other components.SMRH:4916-5472-0852 -19-IP-2823-PCT (47CX-406184-WO)

[0092] The internal components of the LGM may be aligned during a maintenance activity. The internal components of the LGM may also be assembled and aligned prior to installation in the modular optical analytic system. In some implementations, further described below, alignment of the LGM optical components may be accomplished using actuated devices for automatic tracking and adjustment during sequencing or between sequencing cycles / runs. For example, the actuated devices can be a piezo stage, a motorized actuator, or similar devices. The actuated devices may also compensate for drift caused by temperature changes, as well as decay of optical components including lasers, lens, and mounts.[0093| As noted above, during an imaging system’s life and over the variation in operational conditions, illumination uniformity of laser lines can change. For example, laser lines may become misaligned, lose intensity along certain portions (e.g., less intensity along edges), or overall lose intensity. These changes in illumination uniformity can depend on the wavelength of the light source that generates the laser line used to image a sample. For example, FIG. 2A-2B illustrates changes in line profile uniformity that can occur over about 11 months for blue and green light sources used in a two-channel line scanning imaging system, in accordance with some implementations of the disclosure. As depicted, while both light sources experience a change in illumination uniformity over time (measured as normalized intensity as a function of field position in pixels), the change in illumination uniformity in this example system is substantially more significant in the blue light source, showing a decline of over 30%. The foregoing examples also illustrate that illumination at the field edges may experience a greater drop off in intensity.

[0094] In addition to the wavelength of the light source, illumination uniformity can depend on the surface of a sample container that is being imaged by the imaging system. For example, FIG. 3 depicts images of blue and green laser lines on top and bottom surfaces of a flow cell imaged by a multi-channel line scanning imaging system. In this example, while the measured top blue line profile uniformity is over 80%, the measured bottom blue line profile uniformity is under 25%. Similarly, while the measured top green line profile uniformity is over 80%, the measured bottom green line profile uniformity is under 50%.

[0095] As the foregoing examples illustrate, in a line-scanning imaging system, illumination uniformity may need to be independently adjusted for a given channel (i.e., for a particular wavelength) and / or a given illumination surface / depth. In addition, when adjusting illumination uniformity, the lower illumination at field edges may need to be accounted for. This can be particularly beneficial in sequencing systems that use base calling operations basedSMRH:4916-5472-0852 -20-IP-2823-PCT (47CX-406184-WO) on intensity of clusters within images. Intensity non-uniformities across a field of view can impact base calling and limit the number / percentage of clusters for which high quality data can be captured. While the imaging system would ideally uniformly illuminate clusters within a flow cell to minimize errors, this may not occur in practice. While a gain and offset of the pixels in the imaging camera can be calibrated during manufacturing, the potential exists that the calibration of camera pixels can change over time and / or operational conditions (e.g., temperature) as discussed above.

[0096] FIG. 4 is a flow diagram illustrating an example method of monitoring and adjusting illumination uniformity in a line scanning, fluorescent imaging system, in accordance with some implementations of the disclosure. The example operations of FIG. 4 can be implemented using any of the aforementioned imaging systems, including, for example, imaging system 100 or imaging system 200.

[0097] Operation 410 includes projecting a laser line on a surface of a sample container, causing a sample within the sample container to emit fluorescence emissions. For example, the imaging system can include a first laser light source that emits a first light beam at a first wavelength, and one or more line forming optics to shape the first light beam emitted by the first light source into a line. The imaging system can also include an objective lens for focusing the light beam either within or external to the sample container. Focusing can be at or outside (e.g., to prevent photobleaching) the imaged surface.

[0098] Operation 420 includes capturing, using a camera of the imaging system, an image corresponding to the fluorescence emissions from the sample. For example, a TDI sensor can be used to detect fluorescence emissions from the sample and generate a line profile image as discussed and illustrated above. In some implementations, an area-mode or frame-mode image of the laser line can be captured. In a particular embodiment where the sample container is a flow cell, the TDI image can be obtained at a tile with the objective focused at a predetermined depth inside or outside the flow cell.

[0099] Operation 430 includes calculating, using the captured image, one or more illumination quality metrics corresponding to the projected laser line. One or more processors of the imaging system can be used to calculate the illumination quality metrics. For example, an image analysis module 225 can be used to calculate the illumination quality metrics based on an intensity and / or distribution of pixels in the captured image. The calculated illumination quality metrics can indicate a uniformity and / or alignment of the projected laser line. For example, the illumination quality metrics can indicate a uniformity across the entire imaging SMRH:4916-5472-0852 _2 | >IP-2823-PCT (47CX-406184-WO) field, uniformity across a center of the imaging field, a position of the line, a width of the line, a rotation of the line, and / or other aspects of the laser line. The illumination quality metrics can be calculated for a given point in time (e.g., uniformity at a given time point) and / or as a change in time (e.g., change in uniformity over time).

[0100] Operation 440 includes determining whether the one or more illumination quality metrics meet one or more respective thresholds. For example, the illumination quality metrics can be compared to preconfigured thresholds to determine whether the laser is rotated out of position, rotating out of position above some threshold over time, translated out of position, translating out of position above some threshold over time, insufficiently uniform across the field, and / or or losing uniformity above some threshold across the field over time. In some implementations, the imaging system can store one or more flags or other identifiers indicating whether the one or more illumination quality thresholds are being met. In some implementations, the imaging system can notify the user of the imaging system via a GUI or other means whether the one or more illumination quality thresholds are being met.

[0101] Optional operation 450 includes transmitting from the imaging system to a remote management system, over a communication network, one or more notifications indicating whether the one or more illumination quality thresholds are met. For example, an image analysis module 225 can transmit the notifications to a remote management system 199 via communication network 195. If the notifications indicate that the illumination quality metrics do not meet one or more thresholds, the remote management system 199 can flag the imaging system for future service by a field service engineer. To that end, the remote management system 199 can store an identifier of the imaging system and associate it with a service flag. In some implementations, the one or more illumination quality metrics can be transmitted to the remote management system via the communication network, and the remote management system can determine whether the one or more metrics meet the one or more respective thresholds.

[0102] If the one or more illumination quality thresholds are not met, the imaging system 100 can be configured to make one or more automatic adjustments and thereafter iterate the method of FIG. 4. To that end, operation 460 includes adjusting the projected laser line based one or more illumination quality thresholds that are not met. For example, if an illumination uniformity threshold is not met, the imaging system can be configured to adjust a laser beam expander until the threshold is met. In some implementations, if a laser beam position does not meet a threshold, adjusting the projected laser line includes adjusting the laserSMRH:4916-5472-0852 -22-IP-2823-PCT (47CX-406184-WO) line XY position. In other implementations, other optical components of the imaging system, can be adjusted. For example, one or more components of a LGM or EOM as described above can be adjusted using one or more actuators or translation stages. In some implementations, the adjustments can be made using a controller 190 as described above.

[0103] It should be appreciated that the foregoing operations of the method of FIG. 4 can be performed over multiple locations on a given sample surface. For example, in implementations where a flow cell is imaged, illumination can be adjusted for each tile, lane, or swath. The imaging system adjustments needed (if any) for a given tile, lane, or swath, can be stored in a memory (e.g., as a table) for future reference during a sequencing run.

[0104] It should also be appreciated that the foregoing operations of the method of FIG. 4 can be independently performed for each light source of a multi-channel, fluorescence imaging system. In addition, the foregoing operations of the method of FIG. 4 can be independently performed for each surface of the sample that is imaged separately (e.g., top and bottom surfaces of a flow cell). As such, illumination uniformity monitoring and adjustment can be tailored for a given light source and sample surface.

[0105] Although implementations of FIG. 4 have been described in reference to fluorescence emission from a sample, in other implementations, the method of FIG. 4 is utilized in reference to fluorescence emission from an optical alignment target. In certain implementations of FIG. 4, the method of FIG. 4 can be performed in conjunction with focus model generation. For example, US 8,422,031 B2 discloses one example of focus model generation.

[0106] Certain embodiments can be implemented with an illumination and imaging system utilizing an area illumination and area imaging. For example, US 9,193,996, discloses one example of an area illumination and area imaging system, which is incorporated herein by reference in its entirety.|0107] In implementations where the imaging system is a sequencing system, illumination quality monitoring and adjustment can be performed before a sequencing run, during a sequencing run, and / or after every sequencing run. For example, in some implementations, images can be captured before a sequencing run as part of a calibration method that includes a pre-run illumination check. The captured images can be processed through algorithms as described above to determine changes needed in the system to adjust illumination for the subsequent run. In some implementations, automated image acquisitionSMRH:4916-5472-0852 -23-IP-2823-PCT (47CX-406184-WO) can occur after a sequencing run to quantify the quality of illumination, and the images can be processed through algorithms as described above to determine changes needed in the system to adjust illumination for the subsequent run. In some implementations, integrated illumination adjustment can be completed at the end of each image scan, enabling adjustment of the next imaging scan, which could be particularly useful in high numerical optical systems where heating of the optics can become important in image quality.

[0108] In some implementations, real time controls of the illumination can be based on real time analysis of images acquired during sequencing, such as those processed through real time base calling operations. In certain embodiments, real time control comprises servo-control of the illumination system.

[0109] Although the foregoing examples have been described in the context of illumination monitoring and adjustment of a line-scanning, fluorescent imaging system based on a fluorescent emissions from a sample that is imaged, the illumination uniformity monitoring and adjustment techniques described herein could also be implemented by imaging an optical alignment target that is built in or removable from the sample container. The optical target could enable fluorescence emissions that are constant over time without photodegradation at a given optical power, enabling its use for power metering and power calibration of instrument illumination sources while in the field. Particular implementations of an optical target are further described in U.S. Patent No. 10,830,700, which is incorporated herein by reference in its entirety.

[0110] FIG. 5 shows a laser line image and plots representative of laser line uniformity of a multi-channel imaging system that sequences a flow cell, before and after laser line adjustment, in accordance with some of the implementations described herein. As depicted, laser line uniformity substantially improved over the two color channels, and the two surfaces of the flow cell. In particular, due to significant improves in data quality and cross field variability, it was observed that a higher mean percentage of clusters could be imaged during sequencing.10111] FIG. 6 shows an example image obtained of an optical alignment target as compared with an image of a flow cell, in accordance with some implementations of the disclosure.

[0112] As noted above, the illumination uniformity monitoring and adjustment techniques described herein can be implemented by imaging an optical alignment target havingSMRH:4916-5472-0852 -24-IP-2823-PCT (47CX-406184-WO) a fluorescing material contained therein. The optical alignment target can have a similar shape and / or size as the sample container (e.g., flow cell) or its components. In some implementations, the optical alignment target can be built in, removable from, and / or adjacent to the sample container. In some implementations, further described in U.S. Patent No. 9,540,690 the optical alignment target can be incorporated in a cartridge that is placed in the imaging system in a manner similar to how a cartridge containing a sample container (e.g., flow cell) would be placed in the imaging system. The optical alignment target can be used to measure the performance of the optical system over time, including illumination uniformity.

[0113] By way of illustration, FIG. 7 depicts a top view of one example of an optical alignment tool 700 that includes one or more optical alignment targets 800 for optical measurements and analysis of an optical instrument, in accordance with some implementations of the disclosure. Two optical alignment targets are shown in FIG. 7. In this example, optical alignment tool 700 is designed for an optical instrument (e.g., a sequencer), and the dimensions are similar to the analytical apparatus (e.g., a flow cell) that is to be optically addressed using the optical instrument. This example of optical alignment target 800 can be readily positioned on the stage of the sequencer for alignment and validation procedures. The optical alignment target(s) 800 can have any number of features of any shape and / or size. Such features include, but are not limited to: i) features that are the same as or similar to the optically addressable component(s) of the analytical apparatus (e.g., flow cell), and / or ii) features that can be used for alignment (e.g., optical alignment in all six degrees of freedom, i.e., X, Y, Z, theta-x, theta- y, theta-z), and / or (iii) features that can be used for validation (e.g., calibration, quantification, or characterization of optical properties such as illumination uniformity) of imaging modules of the optical instrument, and / or (iv) other appropriate features. In certain embodiments, each optical alignment target 800 comprises one or more areas or tiles comprising a feature 802 to measure excitation laser line quality.

[0114] FIG. 8 depicts an example of an optical alignment target 800, in accordance with some implementations of the disclosure. The optical alignment target 800 includes a translucent or transparent substrate 818 having a bottom surface 820; an opaque material 822 formed over the bottom surface 820 in a pattern, and a fluorescent layer 814 disposed below the opaque material 822 and containing fluorescent composition 816.

[0115] The substrate 818 can be made of any material that is capable of transmitting the light that is used to excite the fluorescent material of fluorescent composition 816 and that is emitted from the fluorescent material. In one specific example, the substrate 818 is capableSMRH:4916-5472-0852 -25-IP-2823-PCT (47CX-406184-WO) of transmitting blue and green excitation light (e.g., from 450 nm to 570 nm), and blue and green emission light that is slightly red-shifted from the excitation light. As examples, suitable materials can include siloxanes, glass, modified or functionalized glass, some polymeric and / or resin materials, inorganic oxides, fused silica, silica-based materials, silicon nitride (SisN4), or combinations thereof. In some examples, the substrate material has a UV transmittance that ranges from about 0.5 to about 1, e.g., from about 0.75 to about 1, from about 0.9 to about 0.99. The thickness of the substrate 818 can vary depending upon the type of instrument the optical alignment tool 700 is used with or incorporated into. In an example, thickness of the substrate 818 may range from about 650 pm to about 750 pm. In another example, the thickness of the substrate 18 is about 700 pm + / - 10 pm.

[0116] As depicted, the pattern of opaque material 822 includes opaque portions (OP) and gap portions (GP) devoid of the opaque material 822. The pattern can include pinholes, slits, stripes, dots, cross shapes, or the like. In some implementations, the opaque material 822 can be a substantially continuous coating that includes GP of a predetermined size formed at a predetermined distance apart from each other. Examples of a suitable opaque material 22 include chromium (Cr), silver (Ag), and titanium (Ti). In one example, the thickness of the opaque material 22 is about 50 nm. The opaque material 22 can be formed using any suitable method, including selective application techniques such as printing, masking and depositing, or the like.

[0117] The OP and GP can respectively block and allow transmission of i) the excitation light directed toward the optical alignment target 800 and ii) the emissions from the fluorescent material of fluorescent composition 816. In alternative implementations, further described below, the opaque material can be omitted from optical alignment target 800.

[0118] Depending on the implementation of optical alignment target 800, the fluorescent composition 816 of fluorescent layer 814 disposed below the opaque material 822 can be a solid host material containing fluorescent materials or a fluid suspension containing a liquid carrier and fluorescent materials that are solid or liquid.|0119] In implementations where fluorescent composition 816 is solid, it can be implemented as a solid host material with fluorescing material contained therein, e.g., via doping. In such implementations, the fluorescent layer 814 can be a solid layer containing fluorescent composition 816. The solid host material can be entirely or at least partially transparent where a degree of transparency in the solid host material may be based, in part, on a desired intensity of fluorescing emissions that are emitted. By way of example, the solid host SMRH:4916-5472-0852 _17_IP-2823-PCT (47CX-406184-WO) material can be a glass substrate or another solid host material having desired mechanical and optical properties. For example, the solid host material can include at least one of glass, amorphous polymers, crystalline materials, semi-crystalline polymers, metallic glass, ceramic and the like. As one example, the solid host material can be indium-fluoride glass. The solid host material can represent heavy metal fluoride glasses (e.g., ZBLAN). ZBLAN glass can utilize various combinations with fluoride, such as ZrF4, BaF2, LaFa. AIF3, and NaF. Optionally, the solid host material can be CaF2. The solid host materials can be selected to exhibit a low maximum phonon energy level.[0120| The fluorescing material embedded in the solid host material can be a rare-earth element such as rare earth ions: Tm3+(455 nm), Ho3+(550 nm), Tb3+(540 nm), Eu3+(611 nm), Sm3+(550 nm), Pr3+(488, 590 nm), Dy3+(480 nm & 575 nm), or Er3+(550 nm & 660 nm); an element from the Actinide series: U; transition metal ions: Ti3+, Cr2+ / 3+, etc. In other implementations, the fluorescing material in the solid host material can be quantum dots. For example, a solid body of at least one of an epoxy or polymer can enclose quantum dots that emit fluorescence in one or more predetermined emission bands of interest when irradiated by the excitation light. The fluorescing material can be distributed in an even and homogeneous fixed manner throughout the solid host material. The fluorescing material can emit in one or more emission channels of interest. The fluorescence material can be provided in various concentrations within the solid host material, where the concentration of the fluorescing material is managed based, in part, on a desired intensity of fluorescence emission to be obtained in response to an expected excitation light intensity. Particular examples of solid fluorescent compositions 816 that can be implemented in an optical alignment target 800 are further described in U.S. Patent No. 10,261,018.

[0121] In implementations where fluorescent composition 816 is implemented as a fluid suspension containing a liquid carrier and fluorescent materials, the fluorescent layer 814 can be a channel configured to enclose the liquid suspension therein. In such implementations, the carrier liquid of the fluid suspension can be water, ethanol, or ethylene glycol. The fluorescent materials can be quantum dots or cerium powder. As examples, the quantum dots can be silicon, cadmium selenide, cadmium sulfide, cadmium telluride, graphene, perovskite, indium phosphide, and lead sulfide. When fluorescent material is the cerium powder, the cerium powder can include particles having an average particle size (among the population of particles) from 1 nm to less than 100 nm. Within the fluid suspension, a concentration of the fluorescent material in the carrier liquid can range from about 0.1% to about 10% in numberSMRH:4916-5472-0852 -27-IP-2823-PCT (47CX-406184-WO) density. The quantum dots or cerium powder can be distributed substantially evenly throughout the carrier liquid, such that when the fluid suspension is irradiated by an excitation light, the quantum dots or cerium powder emit(s) fluorescence in one or more predetermined emission bands of interest.

[0122] In yet other implementations, the fluorescent composition 816 is a cured fluorescent material formed as a solid within a channel (i.e., fluorescent layer 814). In such implementations, the cured fluorescent material can fill the GP of the pattern. In such implementations, a curable fluid suspension can be introduced into the channel corresponding to fluorescent layer 814 and cured using a suitable curing technique such as ultraviolet (UV) light curing. For example, a UV light curable fluid suspension can include a UV curable material that is i) transparent to an excitation wavelength and an emission wavelength of the fluorescent material and ii) selected from the group consisting of a liquid photopolymer and an epoxy; and the fluorescent material selected from the group consisting of organic dyes, quantum dots, and cerium powder.

[0123] As shown in FIG. 8, the substrate 818 can, optionally, have one or more fluid ports 830, 830’ defined therein that extend from an exterior of the substrate 818 through the bottom surface 820 to the fluorescent layer 814. The fluid ports 830, 830’ can be included to introduce a fluid suspension in implementation where a fluorescent composition 816 composed of a fluid suspension is used. In implementations, where the fluorescent composition 816 is a cured solid composition, the fluid ports 830, 830’ may or may not be including depending on whether they are needed for introducing curable fluid. In implementations where the fluorescent composition 816 is a solid host material with fluorescing material contained therein, the fluid ports 830, 830’ can be omitted from optical alignment target 800. In some implementations, a single fluid port 830 is used for fluid ingress and egress. In other implementations, one fluid port 830 is dedicated for introducing fluid(s) into the channel and a second fluid portion 830’ is dedicated for removing fluid(s) from the channel.

[0124] In the example shown in FIG. 8, the optical alignment target 800 can further include a second substrate 828 secured to the translucent or transparent substrate 818 such that the enclosed fluorescent layer 814 is defined between the two substrates 818, 828. Any of the example materials set forth herein for the substrate 818 can be used for the substrate 828. In particular, if it is desirable for the second substrate 828 to have the same optical properties as the substrate 818, the same material may be used for both substrates 818, 828. Alternatively, if it is desirable for the substrate 828 to block transmission of the excitation or emission light,SMRH:4916-5472-0852 -28-IP-2823-PCT (47CX-406184-WO) a metal (e.g., aluminum) may be a suitable material for the substrate 828. The substrate 828, in some implementations, can be used for mechanical positioning of the portion of the target being imaged, and thus any material that has well controlled flatness and thickness can be used. Examples of such materials include glass, ceramic, plastics, or metal. The substrates 818 and 828 can be secured together at bonding regions 832 using an adhesive, a thin metal layer, or a polymeric interposer. The bonding regions 832 can be designated areas of each of the substrates 818, 828 for direct or indirect attachment to one another. As depicted in the example of FIG. 8, the bonding regions 832 form an interface where the surface 820 of the substrate 818 and a surface of the substrate 828 come into direct contact.

[0125] As alluded to above, imaging systems such as DNA sequencers rely on fluorescence signaling from the sample that is excited using one or more excitation beams such as a laser line illumination beam. Maintaining a consistent and uniform illumination of the sample during imaging can improve imaging data fidelity. As such, it can be important to monitor the uniformity of the laser line illumination over time. To this end, optical alignment targets such as optical alignment target 800 can be designed to facilitate imaging of the excitation beam footprint and obtain high fidelity information about the characteristics (e.g., illumination uniformity) of the excitation beam.|0126] In accordance with some implementations of the disclosure, the optical alignment target 800 can be structured to limit the excitation depth at which a laser line illuminator excites the fluorescent material of the fluorescent layer 814 contained therein. Having a greater depth of excitation can degrade the fidelity of the illumination measurements due to factors such as background fluorescence, increased scattering or absorption of the light, and / or increased photobleaching.

[0127] In one embodiment, the pattern of opaque material 822 can be designed to limit the excitation depth. To this end, in some implementations the GP or openings corresponding to opaque material 822 can be limited in size to limit the excitation depth of the optical alignment target 800. For example, in one particular embodiment, the openings of patterned opaque material 822 can have a longest dimension that is about 1 pm or less. The longest dimension of a regularly shaped opening (e.g., circular, rectangular, etc.) can refer to a diameter of a circular opening, a length of a side of a square opening or longest side of a rectangular opening, a major axis of an ellipse. The longest dimension of an irregularly shaped opening (e.g., irregular polygon) can be measured along one or more axes of the opening (e.g., x or y axis).SMRH:4916-5472-0852 -29-IP-2823-PCT (47CX-406184-WO)

[0128] In some implementations, the opaque material 822 can comprise small patterned circular openings (e.g., “pinholes”). FIG. 9 illustrates one example image 900 of an excitation beam footprint comprising patterned pinholes 910. The pinholes can be very small in diameter to limit the depth of excitation of the test target. For example, in one particular embodiment the pinholes can have diameters from about 0.5 pm to about 1 pm. In some implementations, to avoid fluorescence signal saturation, the spacing between openings (e.g., pinholes or other openings) can be minimized to provide a proper sampling rate. In some implementations, a patterned opaque material 822 having small openings can be used with any thickness of fluorescent layer 814.

[0129] In one embodiment, an imaging slit can be used to sample the excitation beam during imaging to reduce blur from out-of-focus light and improve the fidelity of measurements. The imaging slit can be positioned in an optical path between the excitation beam and the optical alignment target 800 during imaging. In certain embodiments, the imaging slit can be formed on the optical alignment target 800, such as a slit or patterned opening in an opaque layer over the fluorescent material. The imaging slit can be used to sample the beam while moving the imaging slit across the beam during imaging. Depending on the excitation beam’s dimensions and imaging slit’s dimensions, the foregoing technique can be applied to moving a vertical imaging slit in the horizontal direction and collecting multiple images that are combined, or moving a horizontal imaging slit in the vertical direction and collecting multiple images that are combined. In one particular embodiment, an imaging slit can have a width from about 100 nm to about 1000 nm.

[0130] FIG. 10 illustrates one example method of sampling an imaging slit to generate a beam footprint image 1050, in accordance with some implementations of the disclosure. In this example, a single, horizontal imaging slit is imaged in multiple positions across the beam’s profile. A first slit image 1010-1 is generated with the imaging slit in a first position. The imaging slit is subsequently moved to a next position (e.g., by a distance about the same as the slit’s width) to capture a second slit image 1010-2, and this process is repeated until a final slit image 1010-n is captured. The captured slit images 1010-1 to 1010-n can then be combined (e.g., using image construction algorithms) to generate the beam footprint image.

[0131] In another example method of sampling an imaging slit to generate a beam footprint image, the imaging camera can be set to a long exposure mode, and while camera starts imaging, the imaging slit can be moved down to pass through the whole beam profile. InSMRH:4916-5472-0852 -30-IP-2823-PCT (47CX-406184-WO) this example method, the laser intensity, slit moving speed and camera integration time can be tuned to obtain an image of the beam with proper intensity.

[0132] In one embodiment, the fluorescent layer 814 of the optical alignment target 800 can be designed to be as thin as possible. For example, it can be implemented as a very thin fluorescent film that is attached on an underside of substrate 818. In some implementations, the thin fluorescent layer has a thickness of less than about 10 pm. In some implementations, the thin fluorescent layer has a thickness of less than about 1 pm. In some implementations, the thin fluorescent layer has a thickness of less than about 100 nm. In some implementations, the thin fluorescent layer has a thickness ranging from about 10 nm to 100 nm. In some implementations, the thin fluorescent layer has a thickness ranging from about 10 nm to 10 pm.

[0133] By physically limiting the excitation depth, such an embodiment can avoid the degradation of the fidelity of the illumination measurements that can be observed in optical alignment targets having thick fluorescent layers. Any suitable fluorescent layer 814 as described above with reference to FIG. 8 that is suitably thin can be implemented in such implementations. For example, in some implementations, the fluorescent layer can comprise a fluorescent composition 816 that is a solid composition comprising fluorescent materials or a fluid suspension containing a liquid carrier and fluorescent materials. The fluorescent materials can be quantum dots or other fluorescent materials described above. In some implementations, an optical alignment target 800 having a thin fluorescent layer 814 can exclude patterned opaque material 822. In some implementations, a thin fluorescent layer 814 can be used in combination with a patterned opaque material 822 having small openings (e.g., small pinholes) and / or an imaging slit for sampling the excitation beam.

[0134] FIG. 11 is an operational flow diagram illustrating an example method that can be implemented with an imaging system including an optical alignment target 800 to estimate a uniformity of an excitation laser light source, in accordance with some implementations of the disclosure.

[0135] Operation 1110 includes projecting a laser line beam of the imaging system on a surface of an optical alignment target 800, causing fluorescent material within the optical alignment target to emit fluorescence emissions. For example, the laser line beam can be projected within the body of a fluorescent layer of the optical alignment target. In some implementations, the optical alignment target 800 can be structured as described above with reference to FIGs. 8-10. For example, the optical alignment target can include a thin fluorescent SMRH:4916-5472-0852 1 >IP-2823-PCT (47CX-406184-WO) layer 814. The optical alignment target 800 could also or alternatively include a patterned opaque material 822 having small openings such as small pinholes.

[0136] Operation 1120 includes capturing, using a camera of the imaging system, a line profile image corresponding to the fluorescence emissions. The line profile image can provide a visual representation of the intensity of the projected laser line. In some implementations, slit imaging techniques as described above can be implemented to capture the line profile image. In such implementations, an imaging slit can be positioned in an optical path between the laser line beam and a fluorescent layer of the optical alignment target.

[0137] Operation 1130 includes determining, based on the line profile image, beam edge information of the laser line beam. In some implementations, a width of the laser line beam along a first axis of the plane (e.g., X-axis) and a width of the laser line along a second axis of the plane substantially orthogonal to the first axis (e.g., Y-axis) can be determined. In some implementations, calculating the width of the laser line along each axis comprises smoothing the original line profile image to remove noise or other artifacts. In some implementations, full width at half maximum (FWHM) techniques can be applied to calculate edges of the laser line (i.e., the width along each axis) based on an intensity profile of the laser line in the line profile image. For example, beam edge points can be identified based on where the intensity profile of the beam matches one or more threshold values.

[0138] Operation 1140 includes extracting, based on the line profile image, frequency spectrum information associated with a uniformity of the laser line beam. The line profile image, containing pixels of varying brightness, can encode a spatial frequency spectrum that can be converted to the frequency domain. In some implementations, Fourier Transform techniques can be applied to transform the line profile image (or a processed version of the line profile image) from the spatial domain to the frequency domain. In some implementations, a low pass filter can be applied to the frequency spectrum information to obtain filtered frequency spectrum information of the laser line beam uniformity.

[0139] Operation 1150 includes calculating, based on the beam edge information and frequency spectrum information, a uniformity of the laser line beam. In some implementations, the uniformity of the laser line beam can be calculated as laser speckle contrast values.

[0140] Although the foregoing method of FIG. 11 has been described in the context of an optical alignment target, it is envisioned that this method could also be implemented with a sample holder (e.g., flow cell) having features similar to the optical alignment targets describedSMRH:4916-5472-0852 -32-IP-2823-PCT (47CX-406184-WO) above with reference to FIGs. 8-9. For example, the sample container could have a patterned layer of opaque material with a fluorescent layer below. The sample container could incorporate a thin fluorescent layer. In addition, slit imaging techniques could be applied with the sample container.

[0141] In this document, the terms “machine readable medium,” "computer readable medium,” and similar terms are used to generally refer to non-transitory mediums, volatile or non-volatile, that store data and / or instructions that cause a machine to operate in a specific fashion. Common forms of machine readable media include, for example, a hard disk, solid state drive, magnetic tape, or any other magnetic data storage medium, an optical disc or any other optical data storage medium, any physical medium with patterns of holes, a RAM, a PROM, EPROM, a FLASH-EPROM, NVRAM, any other memory chip or cartridge, and networked versions of the same.

[0142] These and other various forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processing device for execution. Such instructions embodied on the medium, are generally referred to as “instructions” or “code.” Instructions may be grouped in the form of computer programs or other groupings. When executed, such instructions may enable a processing device to perform features or functions of the present application as discussed herein.

[0143] In this document, a “processing device” may be implemented as a single processor that performs processing operations or a combination of specialized and / or general- purpose processors that perform processing operations. A processing device may include a CPU, GPU, NPU, APU, DSP, FPGA, ASIC, SOC, and / or other processing circuitry.

[0144] The terms “substantially” and “about” used throughout this disclosure, including the claims, are used to describe and account for small fluctuations, such as due to variations in processing. For example, they can refer to less than or equal to ±5%, such as less than or equal to ±2%, such as less than or equal to ±1%, such as less than or equal to ±0.5%, such as less than or equal to ±0.2%, such as less than or equal to ±0.1%, such as less than or equal to ±0.05%.

[0145] To the extent applicable, the terms “first,” “second,” “third,” etc. herein are merely employed to show the respective objects described by these terms as separate entities and are not meant to connote a sense of chronological order, unless stated explicitly otherwise herein.SMRH:4916-5472-0852 -33-IP-2823-PCT (47CX-406184-WO)

[0146] Terms and phrases used in this document, and variations thereof, unless otherwise expressly stated, should be construed as open ended as opposed to limiting. As examples of the foregoing: the term “including” should be read as meaning “including, without limitation” or the like; the term “example” is used to provide exemplary instances of the item in discussion, not an exhaustive or limiting list thereof; the terms “a” or “an” should be read as meaning “at least one,” “one or more” or the like; and adjectives such as “conventional,” “traditional,” “normal,” “standard,” “known” and terms of similar meaning should not be construed as limiting the item described to a given time period or to an item available as of a given time, but instead should be read to encompass conventional, traditional, normal, or standard technologies that may be available or known now or at any time in the future. Likewise, where this document refers to technologies that would be apparent or known to one of ordinary skill in the art, such technologies encompass those apparent or known to the skilled artisan now or at any time in the future.

[0147] The presence of broadening words and phrases such as “one or more,” “at least,” “but not limited to” or other like phrases in some instances shall not be read to mean that the narrower case is intended or required in instances where such broadening phrases may be absent.|0148] Additionally, the various embodiments set forth herein are described in terms of exemplary block diagrams, flow charts and other illustrations. As will become apparent to one of ordinary skill in the art after reading this document, the illustrated embodiments and their various alternatives can be implemented without confinement to the illustrated examples. For example, block diagrams and their accompanying description should not be construed as mandating a particular architecture or configuration.

[0149] While various embodiments of the present disclosure have been described above, it should be understood that they have been presented by way of example only, and not of limitation. Likewise, the various diagrams may depict an example architectural or other configuration for the disclosure, which is done to aid in understanding the features and functionality that can be included in the disclosure. The disclosure is not restricted to the illustrated example architectures or configurations, but the desired features can be implemented using a variety of alternative architectures and configurations. Indeed, it will be apparent to one of skill in the art how alternative functional, logical or physical partitioning and configurations can be implemented to implement the desired features of the present disclosure. Also, a multitude of different constituent module names other than those depicted herein canSMRH:4916-5472-0852 -34-IP-2823-PCT (47CX-406184-WO) be applied to the various partitions. Additionally, with regard to flow diagrams, operational descriptions and method claims, the order in which the steps are presented herein shall not mandate that various embodiments be implemented to perform the recited functionality in the same order unless the context dictates otherwise.

[0150] Although the disclosure is described above in terms of various exemplary embodiments and implementations, it should be understood that the various features, aspects and functionality described in one or more of the individual embodiments are not limited in their applicability to the particular embodiment with which they are described, but instead can be applied, alone or in various combinations, to one or more of the other embodiments of the disclosure, whether or not such embodiments are described and whether or not such features are presented as being a part of a described embodiment. Thus, the breadth and scope of the present disclosure should not be limited by any of the above-described exemplary embodiments.

[0151] It should be appreciated that all combinations of the foregoing concepts (provided such concepts are not mutually inconsistent) are contemplated as being part of the inventive subject matter disclosed herein. In particular, all combinations of claimed subject matter appearing in this disclosure are contemplated as being part of the inventive subject matter disclosed herein.SMRH:4916-5472-0852 -35-

Claims

IP-2823-PCT (47CX-406184-WO)ClaimsWhat is claimed is:

1. An imaging system, comprising: a line generation device comprising: a first light source configured to emit a first light beam at a first wavelength; and one or more line forming optics configured to shape the first light beam emitted by the first light source into a first laser line; an objective configured to project the first laser line on a first surface of a sample container, causing a sample within the sample container to emit first fluorescence emissions; a camera configured to capture a first image corresponding to the first fluorescence emissions from the sample; and one or more processors configured to: calculate, using the first image, a first illumination quality metric corresponding to the first laser line; determine whether the first illumination quality metric meets a threshold; and in response to determining that the first illumination quality metric does not meet the threshold, adjust the first laser line projected on the first surface of the sample container.

2. The imaging system of claim 1, wherein: the sample container comprises a second surface opposite the first surface; the first light source is further configured to emit a second light beam at the first wavelength; the one or more line forming optics are further configured to shape the second light beam emitted by the first light source into a second laser line; the objective is further configured to project the second laser line on the second surface of the sample container, causing the sample to emit second fluorescence emissions; the camera is further configured to capture a second image corresponding to the second fluorescence emissions from the sample; and the one or more processors are further configured to:SMRH:4916-5472-0852 -36-IP-2823-PCT (47CX-406184-WO) calculate, using the second image, a second illumination quality metric corresponding to the second laser line; determine whether the second illumination quality metric meets a threshold; and in response to determining that the second illumination quality metric does not meet the threshold, adjust the second laser line projected on the second surface of the sample container.

3. The imaging system of claim 2, wherein: the sample container is a flow cell; the first surface is a top interior surface of the flow cell; and the second surface is a bottom interior surface of the flow cell.

4. The imaging system of claim 1, wherein: the line generation device further comprises a second light source to emit a second light beam at a second wavelength different from the first wavelength; the one or more line forming optics are further configured to shape the second light beam emitted by the second light source into a second laser line; the objective is further configured to project the second laser line on the first surface of the sample container, causing the sample within the sample container to emit second fluorescence emissions; the camera is further configured to capture a second image corresponding to the second fluorescence emissions from the sample; and the one or more processors are further configured to: calculate, using the second image, a second illumination quality metric corresponding to the second laser line; determine whether the second illumination quality metric meets a threshold; and in response to determining that the second illumination quality metric does not meet the threshold, adjust the second laser line projected on the first surface.

5. The imaging system of claim 1, wherein: the first illumination quality metric comprise a uniformity of the first laser line projected on the first surface; andSMRH:4916-5472-0852 -37-IP-2823-PCT (47CX-406184-WO) adjusting the first laser line projected on the first surface of the sample container comprises adjusting the one or more line forming optics such that the uniformity of the first laser line meets the threshold.

6. The imaging system of claim 5, wherein adjusting the one or more line forming optics such that the uniformity of the first laser line meets the threshold comprises adjusting a beam expander of the first light beam.

7. The imaging system of claim 1, wherein: the first illumination quality metric comprise an alignment of the first laser line projected on the first surface; and adjusting the first laser line projected on the first surface of the sample container comprises adjusting an XY position of the first laser line such that the alignment of the first laser line meets the threshold.

8. The imaging system of claim 1, further comprising: a network communication interface configured to transmit, over a communication network, a notification to a remote management system indicating whether or not the first illumination quality metric meets the threshold.

9. The imaging system of claim 1, further comprising: a user interface configured to present to a user of the imaging system, in response to the first illumination quality metric not meeting the threshold, a notification indicating that the first laser line requires an adjustment.

10. The imaging system of claim 1, wherein the imaging system is configured to sequence the sample after adjusting the first laser line projected on the first surface of the sample container.

11. The imaging system of claim 1, wherein: the imaging system is configured to sequence the sample before adjusting the first laser line projected on the first surface of the sample container; and the first image is configured to be captured during sequencing of the sample.SMRH:4916-5472-0852 -38-IP-2823-PCT (47CX-406184-WO)12. The imaging system of claim 1, wherein the imaging system is configured to sequence the sample while adjusting the first laser line projected on the first surface of the sample container.

13. A method, comprising: projecting, using a first light source of an imaging system, a first laser line on a first surface of a sample container, causing a sample within the sample container to emit first fluorescence emissions; capturing, using a camera of the imaging system, a first image corresponding to the first fluorescence emissions from the sample; calculating, using the first image, a first illumination quality metric corresponding to the first laser line; determining whether the first illumination quality metric meets a first threshold; and in response to determining that the first illumination quality metric does not meet the first threshold, adjusting, by the imaging system, the first laser line projected on the first surface.

14. The method of claim 13, further comprising: sequencing, using the imaging system, the sample after adjusting the first laser line projected on the first surface of the sample container.

15. The method of claim 13, further comprising: sequencing, using the imaging system the sample before adjusting the first laser line projected on the first surface of the sample container, wherein the first image is captured during sequencing of the sample.

16. The method of claim 13, further comprising: sequencing, using the imaging system, the sample, wherein the first laser line projected on the first surface of the sample container is adjusted during sequencing.

17. The method of claim 13, further comprising: projecting, using the first light source, a second laser line on a second surface of the sample container opposite the first surface, causing the sample within the sample container to emit second fluorescence emissions; capturing, using the camera, a second image corresponding to the second fluorescence emissions from the sample;SMRH:4916-5472-0852 -39-IP-2823-PCT (47CX-406184-WO) calculating, using the second image, a second illumination quality metric corresponding to the second laser line; determining whether the second illumination quality metric meets a threshold; and in response to determining that the second illumination quality metric does not meet the threshold, adjusting, by the imaging system, the second laser line projected on the second surface.

18. The method of claim 13, further comprising: projecting, using a second light source of the imaging system, a second laser line on the first surface, causing the sample within the sample container to emit second fluorescence emissions, the first laser line and the second laser line having different wavelengths; capturing, using the camera, a second image corresponding to the second fluorescence emissions from the sample; calculating, using the second image, a second illumination quality metric corresponding to the second laser line; determining whether the second illumination quality metric meets a threshold; and in response to determining that the second illumination quality metric does not meet the threshold, adjusting, by the imaging system, the second laser line projected on the first surface.

19. A method, comprising: projecting, using an imaging system, a laser line beam on a surface of an optical alignment target, causing fluorescent material within the optical alignment target to emit fluorescence emissions; capturing, using a camera of the imaging system, a line profile image corresponding to the fluorescence emissions; determining, based on the line profile image, beam edge information of the laser line beam; extracting, based on the line profile image, frequency spectrum information associated with a uniformity of the laser line beam; and calculating, based on the beam edge information and frequency spectrum information, an illumination uniformity of the laser line beam.SMRH:4916-5472-0852 -40-IP-2823-PCT (47CX-406184-WO)20. The method of claim 19, wherein: the optical alignment target comprises: a substrate that is translucent or transparent; and a fluorescent layer disposed below a bottom surface of the substrate, the fluorescent layer comprising the fluorescent material; and projecting the laser line beam on the surface of the optical alignment target comprises projecting the laser line beam within the fluorescent layer.

21. The method of claim 20, wherein: the optical alignment target further comprises: an opaque material formed over the bottom surface of the substrate in a pattern comprising multiple openings; and each of the openings has a longest dimension that is about 1 pm or less.

22. The method of claim 21, wherein each of the openings is a pinhole.

23. The method of claim 20, wherein the fluorescent material comprises quantum dots.

24. The method of claim 20, wherein capturing the line profile image corresponding to the fluorescence emissions comprises: capturing, using the camera, a plurality of images of an imaging slit positioned in an optical path between the fluorescent layer and the laser line beam; and constructing the line profile image from the plurality of images.

25. The method of claim 24, wherein capturing the plurality of images of the imaging slit comprises moving the imaging slit across a profile of the laser line beam, wherein each of the plurality of images corresponds to a respective position of the imaging slit relative to the profile of the laser line beam.

26. The method of any one of claims 20-25, wherein a thickness of the fluorescent layer is less than 10 pm.

27. The method of claim 19, further comprising: determining that the illumination uniformity does not meet a threshold; and in response to determining that the illumination uniformity does not meet the threshold:SMRH:4916-5472-0852 -41-IP-2823-PCT (47CX-406184-WO) adjusting one or more components of the imaging system used to generate or project the laser line beam; transmitting, over a communication network, a notification to a remote management system indicating that the illumination uniformity does not meet the threshold; or presenting, to a user of the imaging system, a notification indicating that the illumination uniformity does not meet the threshold.SMRH:4916-5472-0852 -42-

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