Apparatus and method for acquiring images of a sample in transit

Abstract

The method is used to generate analytical images of a moving sample based on one or more exposures. An illumination source illuminates the camera's field of view for one or more pulses while the sample moves through the field of view. The distance traveled by the sample during each of these one or more pulses can be less than the size of one pixel in the image captured by the camera.

Description

Technical Field

[0001] (Cross - Reference to Related Applications) This application claims the benefit of U.S. Provisional Patent Application No. 63 / 110,720, filed Nov. 6, 2020, entitled “Apparatus and Method of Obtaining an Image of a Sample in Motion,” which is hereby incorporated by reference in its entirety.

Background Art

[0002] The subject matter considered in this section should not be assumed to be prior art merely as a result of mention in this section. Similarly, problems mentioned in this section, or problems associated with the subject matter provided as background, should not be assumed to have been previously recognized in the prior art. The subject matter of this section merely represents different approaches and, in itself, may also correspond to embodiments of the claimed technology.

[0003] For an object to be imaged, photons must be collected while the object is within the field of view of the imaging device. This, in turn, requires that the object be illuminated. When the object to be imaged is within the field of view for only a limited time, the imaging system needs to ensure that the energy applied through illumination while the object is within the field of view is sufficient to collect the required photons. High - precision motion stages, time delay integration (TDI) cameras, and diode pumped solid state (DPSS) lasers are among the components that have been used to achieve this purpose.

Summary of the Invention

Problems to be Solved by the Invention

[0004] The examples disclosed herein concern techniques for illuminating objects, with particular focus on techniques for illuminating samples of genetic material to be sequenced. [Means for solving the problem]

[0005] One embodiment relates to a machine comprising a camera for capturing an image containing pixels, each pixel having a pixel size corresponding to its distance on a stage in the direction of movement of a sample container. The machine further comprises a stage for moving the sample container relative to the camera's field of view, which overlaps with the stage, and the sample container comprises an array of features having a pitch length in the direction of movement of the sample container. The machine further comprises an illumination source for illuminating the camera's field of view. The machine further comprises a controller for acquiring an analytical image by performing an action which includes acquiring one or more exposures of a feature from the array of features while that feature is in the camera's field of view and is moving relative to the camera's field of view. Acquiring one or more exposures of a feature may be performed by performing an action for each of the one or more exposures. The action may include illuminating the camera's sensor for a first duration and illuminating the camera's field of view with the illumination source for a period having a second duration which elapses while the camera's sensor is illuminated. In such a machine, the displacement of the feature in the camera's field of view from the start to the end of the period having the second duration is less than or equal to the pitch length in the direction of movement of the sample container.

[0006] In some embodiments, in the machine described above, the displacement of features in the direction of movement of the sample container within the camera's field of view from the start to the end of a period having a second duration may be less than or equal to the pixel size.

[0007] In some embodiments of the machine, as described in either of the two preceding paragraphs of the summary of the present invention, obtaining exposure of one or more features includes obtaining multiple exposures of the feature. The actions performed by the controller include overlaying the multiple exposures of the feature, based on translating one or more of the multiple exposures of the feature.

[0008] In some embodiments of the machine, as described in the preceding paragraphs of the summary of the present invention, the actions performed by the controller include, for each exposure, obtaining a corresponding value of the position of the sample container when the camera's field of view is illuminated by the illumination source. In some such embodiments, the controller translates one or more of a plurality of exposures of the feature based on the difference between the corresponding values ​​of the exposures of the position of the sample container.

[0009] In some embodiments of the machine, as described in the preceding paragraphs of the summary of the present invention, the machine comprises an encoder for providing a value for the position of the sample container. In some such embodiments, for each exposure, the controller obtains from the encoder a corresponding value for the position of the sample container when the camera's field of view is illuminated by the light source.

[0010] In some embodiments of the machine as described in the preceding paragraphs of the summary of the present invention, the encoder has a resolution that distinguishes distances smaller than the distance on the stage corresponding to the pixel size, and overlaying multiple exposures of a feature includes colregiping each of the multiple exposures with the resolution of the encoder.

[0011] In some embodiments of the machine as described in the preceding paragraphs of the summary of the present invention, colregiping each of a plurality of exposures at the encoder resolution includes obtaining a frequency-space representation by performing a fast Fourier transform of the exposure for at least one of the one or more exposures. Collegging each of a plurality of exposures at the encoder resolution further includes translating the frequency-space representation for at least one of the one or more exposures by a distance that is not an integer multiple of the distance on the stage corresponding to the pixel size. Collegging each of a plurality of exposures at the encoder resolution further includes performing an inverse fast Fourier transform of the translated frequency-space representation for at least one of the one or more exposures.

[0012] In some embodiments of the machine, such as those described in either of the two preceding paragraphs of the summary of the present invention, colresisting each of a plurality of exposures at the resolution of the encoder includes, for each plurality of exposures, upsampling that exposure to the resolution of the encoder based on interpolating data between pixels, and translating one or more of the exposures after upsampling.

[0013] In some embodiments of the machine, as described in any of the preceding paragraphs of the summary of the present invention, the sample container may include a plurality of reference points, and the controller may translate one or more of the plurality of exposures of the feature based on the difference in the location of the reference points between exposures.

[0014] In some embodiments of the machine, as described in any of the preceding paragraphs of the summary of the present invention, the analyzed image includes a plurality of pixels, each having a first bit depth. In some such embodiments, each of the plurality of exposures includes a plurality of pixels, each of which has a second bit depth less than the first bit depth.

[0015] In some embodiments of the machine as described in the preceding paragraph, each pixel contained in each image captured by the camera has a third bit depth, the third bit depth being greater than the second bit depth. Obtaining multiple exposures of an exposure involves, for each exposure, capturing an image with the camera while the camera's field of view is illuminated by an illumination source, and truncating some of the most significant bits of pixels from the image captured by the camera, the number of most significant bits truncated being equal to the difference between the third bit depth and the second bit depth.

[0016] In some embodiments of the machine, as described in any of the preceding paragraphs of the summary of the present invention, a threshold illumination energy dose is required to image a feature. For each exposure of a feature, illuminating the camera's field of view with an illumination source involves activating the illumination source with power that provides an individual exposure energy dose smaller than the threshold illumination energy dose for imaging the feature when multiplied by a second duration, and a combined exposure energy dose larger than the threshold illumination energy dose for imaging the feature when multiplied by the second duration and the number of exposures in the multiple exposures.

[0017] In some embodiments of the machine, as described in any of the preceding paragraphs of the summary of the present invention, the actions performed by the controller include acquiring an image of a reference object with a camera, the reference object comprising a plurality of features having known locations. The actions performed by the controller further include creating a distortion map by performing an action which includes comparing the known locations of the plurality of features contained in the reference object with the apparent locations of the plurality of features in the image of the reference object, and applying the distortion map to each of one or more exposures of the features.

[0018] In some embodiments of the machine, as described in any of the preceding paragraphs of the summary of the present invention, the stage is mounted on the frame of the machine using ball bearings, the camera captures images using a complementary metal-oxide-semiconductor sensor, and the illumination source is a diode laser.

[0019] In some embodiments of the machine, as described in any of the preceding paragraphs of the summary of the present invention, the feature is a nanowell.

[0020] In some embodiments of the machine, as described in any of the preceding paragraphs of the summary of the present invention, the analytical image is one of a plurality of analytical images, the controller performs a plurality of sequencing cycles, each analytical image from the plurality of analytical images corresponds to a single sequencing cycle, the controller determines cluster polynucleotides for each feature in the sample container based on the plurality of analytical images, and the controller determines the complete polynucleotides of the sample associated with the sample container based on the cluster polynucleotides determined for the features from the sample container.

[0021] In some embodiments of the machine, as described in any of the preceding paragraphs of the summary of the present invention, the array of features contained in the sample container has a pitch perpendicular to the direction of movement of the sample container, which is smaller than the pitch in the direction of movement of the sample container.

[0022] In some embodiments of the machine, as described in any of the preceding paragraphs of the summary of the present invention, the machine comprises a motor for counteracting the movement of the stage by translating the camera's field of view in the direction of movement of the sample container during a period having a second duration.

[0023] Another embodiment relates to a method comprising translating a feature on a stage relative to the field of view of a camera in the direction of movement, wherein the camera has a pixel size corresponding to the distance in the direction of movement on the stage, and the feature is contained in an array of features in a sample container, the array of features having a pitch length in the direction of movement. The method further comprises generating an analytical image by performing an action, which includes obtaining one or more exposures of the feature by performing the action for each of the one or more exposures while the feature is in the field of view of the camera and moving relative to the field of view of the camera. Such an action includes illuminating the sensor of the camera for a first duration, and illuminating the field of view of the camera with an illumination source for a period having a second duration that elapses while the sensor of the camera is illuminated. In such a method, the displacement of the feature in the field of view of the camera from the start to the end of the period having the second duration is less than or equal to the pitch length in the direction of movement.

[0024] In some embodiments, in the method as described in the preceding paragraph of the summary of the present invention, the displacement of the feature in the moving direction from the start to the end of the period having the second duration is less than the pixel size.

[0025] In some embodiments, in the method as described in either of the two preceding paragraphs of the summary of the present invention, obtaining one or more exposures of the feature includes obtaining a plurality of exposures of the feature. This method further includes producing an analysis image of the feature by overlaying a plurality of exposures of the feature by performing an act including translating one or more of the plurality of exposures of the feature.

[0026] In some embodiments, in the method as described in the preceding paragraph, this method includes, for each exposure, obtaining a corresponding value for the position of the sample container when the field of view of the camera is illuminated by the illumination source, and translating one or more of the plurality of exposures of the feature based on the difference between the corresponding values of the exposures for the position of the sample container.

[0027] In some embodiments, in the method as described in the preceding paragraph, for each exposure, the corresponding value for the position of the sample container when the field of view of the camera is illuminated by the illumination source is obtained from an encoder.

[0028] In some embodiments, in the method as described in the preceding paragraph, the encoder has a resolution for distinguishing distances smaller than the distance on the stage corresponding to the pixel size, and overlaying a plurality of exposures of the spot includes registering each of the plurality of exposures with the resolution of the encoder.

[0029] In some embodiments, colregiping each of a plurality of exposures at the encoder resolution in a manner such as that described in the preceding paragraphs of the summary of the present invention includes obtaining a frequency-space representation of at least one of the one or more exposures by performing a fast Fourier transform of the exposure. Colliggiating each of a plurality of exposures at the enclosure resolution further includes translating the frequency-space representation of at least one of the one or more exposures by a distance that is not an integer multiple of the distance on the stage corresponding to the pixel size, and performing an inverse fast Fourier transform of the translated frequency-space representation.

[0030] In some embodiments, colregistrating each of a plurality of exposures in a manner such as that described in either of the preceding two paragraphs includes, for each plurality of exposures, upsampling that exposure to the resolution of the encoder based on interpolating the data between pixels, and translating one or more of the exposures after upsampling.

[0031] In some embodiments, in a method as described in any of the preceding paragraphs of the summary of the invention, the sample container includes a plurality of reference points, and the method includes translating one or more of a plurality of exposures of a feature based on the difference in the location of the reference points between the exposures.

[0032] In some embodiments, as described in any of the preceding paragraphs of the summary of the present invention, the analysis image comprises a plurality of pixels, each having a first bit depth, and each of the plurality of exposures comprises a plurality of pixels, each having a second bit depth, the second bit depth being smaller than the first bit depth.

[0033] In some embodiments, as described in the preceding paragraphs of the summary of the invention, each pixel contained in each image captured by the camera has a third bit depth, the third bit depth being greater than the second bit depth. In addition, obtaining multiple exposures of a feature includes, for each exposure, capturing an image with the camera while the camera's field of view is illuminated by an illumination source, and truncating some of the most significant bits of pixels from the image captured by the camera, the number of most significant bits truncated being equal to the difference between the third bit depth and the second bit depth.

[0034] In some embodiments, a threshold illumination energy dose is required to image a feature in a manner such as that described in any of the preceding paragraphs of the summary of the invention. In addition, in such a manner, for each exposure of a feature, illuminating the camera's field of view with an illumination source includes activating the illumination source with power that provides an individual exposure energy dose smaller than the threshold illumination energy dose for imaging the feature when multiplied by a second duration, and a combined exposure energy dose larger than the threshold illumination energy dose for imaging the feature when multiplied by the second duration and the number of exposures in the multiple exposures.

[0035] In some embodiments, the method, as described in any of the preceding paragraphs of the summary of the invention, comprises acquiring an image of a reference object with a camera, the reference object comprising a plurality of features having known locations. The method may further comprise creating a distortion map by performing an act which comprises comparing the known locations of the plurality of features contained in the reference object with the apparent locations of the plurality of features in the image of the reference object. The method may further comprise applying the distortion map to each of one or more exposures of the features.

[0036] In some embodiments, as described in any of the preceding paragraphs of the summary of the present invention, the stage is mounted on a stationary frame using ball bearings, the camera captures images using a complementary metal-oxide-semiconductor sensor, and the illumination source is a diode laser.

[0037] In some embodiments, the feature is a nanowell, as described in any of the preceding paragraphs of the summary of the present invention.

[0038] In some embodiments, the analytical image is one of a plurality of analytical images in a method as described in any of the preceding paragraphs of the summary of the invention. In some such embodiments, the method further comprises performing a plurality of sequencing cycles, wherein each analytical image from the plurality of analytical images corresponds to a single sequencing cycle; determining cluster polynucleotides for each feature in the sample container based on the plurality of analytical images; and determining the complete polynucleotide of the sample associated with the sample container based on the cluster polynucleotides determined for the features from the sample container.

[0039] In some embodiments, as described in any of the preceding paragraphs of the summary of the present invention, the array of features contained in the sample container has a pitch perpendicular to the direction of movement of the sample container, which is smaller than the pitch in the direction of movement of the sample container.

[0040] In some embodiments, the method, as described in any of the preceding paragraphs of the present invention, includes a motor that counteracts the movement of the stage by translating the camera's field of view in the direction of movement over a period having a second duration.

[0041] Another embodiment relates to a machine comprising a stage for moving a sample relative to the field of view of a camera that overlaps with the stage. The machine further comprises a camera for capturing an image containing pixels, each pixel having a pixel size corresponding to a distance on the stage. The machine further comprises an illumination source for illuminating the field of view of the camera. The machine further comprises means for acquiring analytical images of a continuously moving sample using pulsed illumination.

[0042] In some embodiments, in a machine such as those described in the preceding paragraphs of the summary of the present invention, means for acquiring an analytical image of a continuously moving sample using pulsed illumination include means for translating and overlaying a plurality of subthreshold exposures.

[0043] Other features and aspects of the disclosed technology will become apparent from the following detailed description, in conjunction with the accompanying drawings illustrating the features of the disclosed technology by example. The summary of the invention is not intended to limit the scope of any protection provided in this document or any related document, which is defined by the claims and equivalents of the respective document.

[0044] It should be understood that all combinations of the aforementioned concepts (provided that such concepts are not mutually contradictory) are intended to be part of the subject matter of the invention disclosed herein. Specifically, all combinations of the claimed subject matter appearing at the end of this disclosure are intended to be part of the subject matter of the invention disclosed herein. [Brief explanation of the drawing]

[0045] This disclosure will be described in detail with reference to the following figures, according to one or more different examples. The figures are provided for illustrative purposes only and merely illustrate typical or exemplary embodiments. [Figure 1] A generalized block diagram of an exemplary image scanning system in which the systems and methods disclosed herein may be implemented is shown as an example. [Figure 2]This is a block diagram illustrating an exemplary two-channel line-scan modular optical imaging system that may be implemented in a particular embodiment. [Figure 3] This section shows an exemplary configuration of a patterned sample that can be imaged according to the embodiments disclosed herein. [Figure 4] This illustrates an exemplary scenario in which a camera is used to image a sample that is continuously moving through its field of view. [Figure 5] This illustrates an exemplary process involving a combination of multiple exposures. [Figure 6] This disclosure shows exemplary computing modules that may be used to implement various features of the embodiments described herein. [Figure 7A] This diagram shows a configuration in which illumination from a feature is focused onto a camera using a lens and mirror. [Figure 7B] This diagram shows a configuration in which illumination from a feature is focused onto a camera using a lens and mirror. [Figure 7C] This diagram shows a configuration in which illumination from a feature is focused onto a camera using a lens and mirror.

[0046] The figures are not exhaustive and do not limit this disclosure to the exact form disclosed. [Modes for carrying out the invention]

[0047] When used herein to refer to a sample, the terms “spot” or “feature” are intended to mean a point or region of a pattern that can be distinguished from other points or regions according to their relative location. Individual spots may contain one or more molecules of a particular type. For example, a spot may contain a single target nucleic acid molecule having a particular sequence, or a spot may contain several nucleic acid molecules having the same sequence (and / or complementary sequences).

[0048] When used herein to refer to a spot or feature in relation to a direction, the term “pitch” is intended to mean that a spot or feature is separated from other spots or features in that direction. For example, if a sample container has an array of features separated from each other by 650 nm in the direction in which the container moves during imaging, the “pitch” of the features in that direction may be said to be 650 nm.

[0049] As used herein, the term “xy-plane” is intended to mean a two-dimensional area defined by the linear axes x and y in the Cartesian coordinate system. When used in reference to a detector and an object observed by the detector, the area may be further specified to be orthogonal to the direction of observation between the detector and the object being detected. When used herein to refer to a line scanner, the term “y-direction” refers to the direction of the scan.

[0050] As used herein, the term “z-coordinate” is intended to mean information specifying the location of a point, line, or region along an axis perpendicular to the xy-plane. In certain embodiments, the z-axis is perpendicular to the region of the object observed by the detector. For example, the direction of the focal point of an optical system may be specified along the z-axis.

[0051] As used herein, the term “scanning a line” is intended to mean detecting a two-dimensional cross-section of an object in the xy-plane, where the cross-section is rectangular or elliptical, causing relative movement between the cross-section and the object. For example, in fluorescence imaging, a region of an object having a rectangular or elliptical shape may be specifically excited (excluding other regions), and / or emission from that region may be specifically acquired (excluding other regions) at a given point in the scan.

[0052] The embodiments disclosed herein relate to the illumination of an object being imaged during motion. The illumination may be provided for one or more short intervals, and an image may be generated by combining data corresponding to multiple short intervals of illumination.

[0053] Figure 1 shows an exemplary imaging system 100 in which the technologies disclosed herein may be implemented. The exemplary imaging system 100 may include devices for acquiring or generating images of a sample. The example outlined in Figure 1 shows an exemplary imaging configuration of an embodiment of the backlight design. Systems and methods may be described herein from time to time in the context of the exemplary imaging system 100, but it should be noted that these are merely examples in which embodiments of the illumination and imaging technologies disclosed herein may be implemented.

[0054] As can be seen in the example in Figure 1, the sample is located on a sample container 110 (e.g., a flow cell as described herein) and positioned below the objective lens 142 on a sample stage 170 mounted on a frame 190. A light source 160 and associated optical elements direct a light beam, such as laser light, to the location of the selected sample on the sample container 110. The sample fluorescence and the resulting light are collected by the objective lens 142 and directed to an image sensor of a camera system 140 for fluorescence detection. The sample stage 170 is moved relative to the objective lens 142 to position the location of the next sample on the sample container 110 at the focal point of the objective lens 142. The movement of the sample stage 170 relative to the objective lens 142 can be achieved by moving the sample stage itself, the objective lens, some other components of the imaging system, or any combination thereof. Further embodiments may also include moving the entire imaging system over a stationary sample.

[0055] The fluid delivery module or device 180 directs (and passes through) a flow of reagents (e.g., fluorescently labeled nucleotides, buffers, enzymes, cleavage reagents, etc.) to the sample container 110 and the waste valve 120. The sample container 110 may include one or more substrates on which the sample is to be provided. For example, in a system for analyzing a large number of different nucleic acid sequences, the sample container 110 may include one or more substrates on which the nucleic acids to be sequenced bind, adhere, or associate. In various embodiments, the substrate may be any inert substrate or surface to which nucleic acids can adhere, such as glass surfaces, plastic surfaces, latex, dextran, polystyrene surfaces, polypropylene surfaces, polyacrylamide gels, gold surfaces, and silicon wafers. In some applications, the substrate is located in channels or other regions at multiple locations formed in a matrix or array across the sample container 110.

[0056] In some embodiments, the sample container 110 may contain a biological sample to be imaged using one or more fluorescent dyes. For example, in certain embodiments, the sample container 110 may be implemented as a patterned flow cell containing a transparent cover plate, a substrate, and a liquid sandwiched between the transparent cover plate and the substrate, with the biological sample located on the inner surface of the transparent cover plate or the inner surface of the substrate. The flow cell may contain a large number of wells (e.g., thousands, millions, or billions) or other types of spot regions (e.g., pads, divots) patterned into defined arrays (e.g., hexagonal arrays, rectangular arrays, etc.) within the substrate. Each spot may form a cluster (e.g., a monoclonal cluster) of the biological sample, such as DNA, RNA, or another genomic material, which can be sequenced using, for example, synthetic sequencing. The flow cell may be further divided into a large number of spaced lanes (e.g., eight lanes), each lane containing a hexagonal array of clusters. Examples of flow cells that may be used in the embodiments disclosed herein are described in U.S. Patent No. 8,778,848.

[0057] The system also includes a temperature station actuator 130 and a heater / cooler 135 that can arbitrarily adjust the temperature of the fluid state in the sample container 110. A camera system 140 may be included to monitor and track the sequencing of the sample container 110. The camera system 140 may be implemented, for example, as a charge-coupled device (CCD) camera (e.g., a time-difference integral (TDI) CCD camera) and may interact with various filters in a filter switching assembly 145, an objective lens 142, and a focusing laser / focusing laser assembly 150. The camera system 140 is not limited to a CCD camera, and other camera and image sensor technologies may be used. In certain embodiments, the camera sensor may have a pixel size of about 5 to about 15 μm, but in some cases other pixel sizes such as 2.4 μm may be used.

[0058] The output data from the sensors of the camera system 140 may be communicated to a real-time analysis module (not shown) which may be implemented as a software application that analyzes image data (e.g., image quality scoring), reports or displays the characteristics of the laser beam (e.g., focus, shape, intensity, power, brightness, position) to a graphical user interface (GUI), and dynamically corrects distortions in the image data, as further described below.

[0059] Light sources 160 (e.g., excitation lasers in an assembly containing multiple lasers optionally) or other light sources may be included to illuminate the fluorescence sequencing reaction in the sample by illumination via an optical fiber interface (which may optionally include one or more re-imaging lenses, optical fiber mounting sections, etc.). In the illustrated example, a low-watt lamp 165 and a focused laser 150 are also presented. In some embodiments, the focused laser 150 may be turned off during imaging. In other embodiments, the alternative focus configuration may include a second focused camera (not shown) which may be a quadrant detector, a position-sensitive detector (PSD), or a similar detector for measuring the location of scattered beams reflected from the surface simultaneously with data acquisition.

[0060] Although illustrated as a backlit device, other examples may include light from a laser or other light source directed onto the sample on the sample container 110 through the objective lens 142. The sample container 110 may be finally 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 may have one or more actuators that allow movement in any of the three dimensions. For example, actuators may be provided that allow the stage to move in the X, Y, and Z directions relative to the objective lens from the viewpoint of a Cartesian coordinate system. This may allow one or more locations of samples on the sample container 110 to be optically aligned and positioned with respect to the objective lens 142.

[0061] This example includes a focal (z-axis) component 175, which is included to control the positioning of the optical components relative to the sample container 110 in the focal direction (typically referred to as the z-axis or z-direction). The focal component 175 may include one or more actuators physically coupled to the optical stage, the sample stage, or both, to move the sample container 110 on the 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 actuators may be physically coupled to each stage, for example, by direct or indirect mechanical, magnetic, fluid, or other connections or contacts with the stage. One or more actuators may move the stage in the z-direction while keeping the sample stage in the same plane (e.g., while maintaining a level or horizontal orientation perpendicular to the optical axis). One or more actuators may also tilt the stage. This may be done, for example, so that the sample container 110 can be dynamically flattened to account for any inclination on its surface.

[0062] System focusing generally refers to aligning the focal plane of the objective lens with the sample being imaged at a selected sample location. However, focusing can also refer to adjusting the system to obtain desired characteristics for the representation of the sample, such as a desired level of sharpness or contrast in the image of the test sample. Because the usable depth of field of the objective lens's focal plane can be small (sometimes as small as 1 μm or less), the focal component 175 closely follows the imaged surface. Since the sample container is not perfectly flat as it is fixed to the instrument, the focal component 175 may be set to follow this profile as it moves along the scanning direction (referred to herein as the y-axis).

[0063] Light emitted from the test sample at the sample location may be directed to one or more detectors of the camera system 140. An aperture may be included and positioned to allow only light emitted from the focal region to reach the detector. The aperture may be included to improve image quality by filtering out components of light emitted from regions outside the focal region. An emission filter may be included in the filter switching assembly 145 and may be selected for recording the determined emission wavelength and for cutting out any laser stray light.

[0064] Although not shown, a controller, which may be implemented as a computing module as discussed below in the context of Figure 6, may be provided to control the operation of the scanning system. The controller may be implemented to control aspects of system operation, such as focusing, stage movement, and imaging operations. In various embodiments, the controller may be implemented using hardware, algorithms (e.g., machine-executable instructions), or a combination of the foregoing. For example, in some embodiments, the controller may include a processor having one or more CPUs or associated memory. As another example, the controller may include hardware or other circuit configurations for controlling the operation of a computer processor and a non-temporary computer-readable medium storing machine-readable instructions. For example, this circuit configuration may include one or more of the following: field programmable gate arrays (FPGAs), application-specific integrated circuits (ASICs), programmable logic devices (PLDs), complex programmable logic devices (CPLDs), programmable logic arrays (PLAs), programmable array logic (PALs), or other similar processing devices or circuit configurations. In yet another example, a controller may include a combination of this circuit configuration and one or more processors.

[0065] Other imaging systems may be used when implementing the disclosed techniques. For example, Figure 2 is a block diagram showing an exemplary two-channel line-scan modular optical imaging system 200 in which embodiments of the techniques of this disclosure may be implemented. In some embodiments, the system 200 may be used for sequencing nucleic acids. Applicable techniques include those in which nucleic acids are attached to fixed locations in an array (e.g., wells of a flow cell) and the array is repeatedly imaged as it moves relative to the field of view of a camera in the imaging system 200. In such embodiments, the system 200 may acquire images of two different color channels which can be used to distinguish one nucleotide base type from another. More specifically, the system 200 may perform a process referred to as “base calling,” which generally refers to the process of determining the base call (e.g., adenine (A), cytosine (C), guanine (G), or thymine (T)) for a given spot location in an image during an imaging cycle. During a two-channel base call, the presence of one of four base types can be determined by encoding base identity as a combination of the intensities of the two images using image data extracted from the two images. For a given spot or location in each of the two images, base identity can be determined based on whether the signal identity combination is [on, on], [on, off], [off, on], or [off, off].

[0066] Referring again to the imaging system 200, the system includes a line generation module (LGM) 210 in which two light sources 211 and 212 are disposed. The light sources 211 and 212 may be coherent light sources such as laser diodes that emit laser beams. Light source 211 may emit light at a first wavelength (e.g., a red wavelength), and light source 212 may emit light at a second wavelength (e.g., a green wavelength). The light beams emitted from the laser sources 211 and 212 may be directed through a beam-forming lens(s) 213. In some embodiments, a single photo-forming lens may be used to shape the light beams emitted from both light sources. In other embodiments, a separate beam-forming lens may be used for each light beam. In some examples, the beam-forming lens is a Powell lens, so that the light beam is shaped into a line pattern. The beam-shaping lens or other optical component imaging system of the LGM210 can shape the light emitted by light sources 211 and 212 into a line pattern (for example, by using one or more Powell lenses or other beam-shaping lenses, diffraction or scattering components).

[0067] The LGM210 may further include mirrors 214 and semi-reflective mirrors 215 for directing the light beam through a single interface port to an emission optics module (EOM) 230. The light beam may pass through a shutter element 216. The EOM230 may include an objective lens 235 and a z-stage 236 for moving the objective lens 235 longitudinally towards or away from a target 250. For example, the target 250 may include a liquid layer 252 and a translucent cover plate 251, and the biological sample may be located on the inner surface of the translucent cover plate, as well as on the inner surface of a substrate layer located beneath the liquid layer. The z-stage 236 may then move the objective lens to focus the light beam onto any inner surface of the flow cell (e.g., onto a biological sample). Similarly, in some embodiments, the target 250 may be mounted on or include a stage movable in the xy plane relative to the objective lens 235. The biological sample may be DNA, RNA, protein, or other biological material that responds to optical sequencing known in the art.

[0068] The EOM230 may include a semi-reflective mirror 233 for reflecting the focus-tracking light beam emitted from the focus-tracking module (FTM) 240 to the target 250, and then reflecting the light returned from the target 250 back to the FTM240. The FTM240 may include a focus-tracking optical sensor for detecting the characteristics of the returned focus-tracking light beam and generating a feedback signal to optimize the focus of the objective lens 235 relative to the target 250.

[0069] The EOM230 may also include a semi-reflective mirror 234 for directing the light that has passed through the objective lens 235, while allowing light reflected from the target 250 to pass through. In some embodiments, the EOM230 may include a tube lens 232. Light transmitted through the tube lens 232 may pass through a filter element 231 and enter the camera module (CAM) 220. The CAM 220 may include one or more optical sensors 221 for detecting light emitted from a biological sample in response to an incident light beam (e.g., fluorescence in response to red and green light received from light sources 211 and 212).

[0070] Output data from the CAM220 sensor can be communicated to the real-time analysis module 225. In various embodiments, the real-time analysis module executes computer-readable commands for analyzing image data (e.g., image quality scoring, base call, etc.), reporting or displaying beam characteristics (e.g., focus, shape, intensity, power, brightness, position) to a graphical user interface (GUI), etc. These operations may be performed in real time during the imaging cycle to minimize downstream analysis time and provide real-time feedback and troubleshooting during the imaging run. In embodiments, the real-time analysis module may be a computing device (e.g., computing device 1000) that is communicably connected to and controls the imaging system 200. In embodiments further described below, the real-time analysis module 225 may further execute computer-readable commands for controlling the illumination of the target 250 and for optionally integrating data collected during multiple exposures of the optical sensor 221 into the image.

[0071] Figure 3 shows an exemplary configuration of a sample container 300 that can be imaged according to embodiments disclosed herein. In this example, the sample container 300 is patterned with a hexagonal array of regular spots 310 that can be imaged simultaneously during an imaging run. While a hexagonal array is shown in this example, in other embodiments, the sample container may be patterned using a linear array, a circular array, an octagonal array, or some other array pattern. For simplicity of illustration, the sample container 300 is shown as having tens to hundreds of spots 310. However, as can be understood by those skilled in the art, the sample container 300 may have thousands, millions, or billions of spots 310 to be imaged. Furthermore, in some cases, the sample container 300 may be a multiplanar sample containing multiple planes (perpendicular to the focusing direction) of spots 310 sampled during an imaging run.

[0072] In a particular embodiment, the sample container 300 may be a flow cell patterned with millions or billions of wells divided into lanes. In this particular embodiment, each well of the flow cell may contain a biological material that is sequenced using synthesis sequencing.

[0073] As described above, illumination and imaging of moving objects relative to the field of view of an imaging device have been achieved through high-precision motion stages, time-difference integral (TDI) cameras, and diode-pumped solid-state lasers. However, embodiments of the disclosed technology can achieve the same goals while relaxing the usual strict tolerances and performance requirements that are met by those types of components. For example, in some embodiments, instead of utilizing a TDI camera that continuously images the sample container as the sample container moves, a different type of camera, such as a consumer camera using a complementary metal-oxide-semiconductor (CMOS) sensor, may be used to capture an image of the sample at a specific moment in time. In such embodiments, the operation of the light source(s) of the embodiment (e.g., light source 160 in Figure 1 or light sources 211, 212 in Figure 2) may differ from the operation of the light source(s) of an embodiment that uses a camera that continuously images a moving target.

[0074] To illustrate why the operation of the light source(s) can be modified in embodiments using a camera that captures an image of a single moment in time, consider the scenario in Figure 4, in which the camera used to image the sample has a frame rate that allows it to capture three exposures while the feature of the sample container (e.g., a nanowell) is in its field of view, and a resolution that allows it to divide its field of view into six pixels in the direction of the sample container's movement. In such a scenario, if the sample container is illuminated continuously while it is in the camera's field of view (i.e., continuously illuminated from time T1 to time T6), two blurred images may be produced, as the exposure captured in the first frame may include photons from when the feature is in the first, second, and third pixels out of the field of view, while the exposure captured in the second frame may include photons from when it is in the fourth, fifth, and sixth pixels out of the field of view. This blurring may render the image unusable. For example, as shown in Figure 4, if a sample container contains three features, and each of these features is separated by a single pixel's distance, the camera's frame rate may mix photons from multiple features (for example, in frame 1, photons from feature 1 at T1 may be mixed with photons from feature 3 at T3). This can prevent individual features from being distinguished from each other in the resulting image.

[0075] As described above, various measures can be taken to address blurring. In some embodiments, the distance between features on the sample container in the direction of the sample container's movement may be increased, so that, given the camera's frame rate, photons from multiple features are not mixed. This increase in the distance between features in the direction of movement may be accompanied by an increase in the spacing perpendicular to the direction of movement, or it may be done only in the direction of movement, with the spacing perpendicular to the direction of movement remaining unchanged (or being changed in some other way). If this approach is applied to the scenario in Figure 4, such as increasing the distance between features from 1 pixel to 2 pixels in the direction of the sample container's movement, it may be possible to distinguish individual features despite the blurring caused by the camera's frame rate. Similarly, in some embodiments, the speed of the sample container's movement may be reduced. For example, if the speed of movement in the scenario in Figure 4 is reduced by 50%, individual features may be distinguishable from each other in the image captured by the camera, regardless of the blurring caused by the frame rate.

[0076] Furthermore, the effects of blurring can be avoided by using short-term illumination rather than continuous illumination of the sample container. For example, in embodiments using one or more laser light sources, such as light source 160 in Figure 1 or light sources 211 and 212 in Figure 2, these light sources may be implemented using pulsed lasers rather than continuous-wave lasers, or the light sources may be equipped with additional components such as optical choppers that enable short-term illumination even when operating in continuous-wave mode. As described below, these types of approaches may enable the use of a camera that captures an image of the sample container at separate moments in time with a continuously moving sample container, even if the resulting image could be unusable due to blurring of a single pixel. The following description explains how the use of non-continuous illumination may enable the avoidance of pixel blurring, but the same techniques may be used in embodiments where blurring of more than one pixel is acceptable. For example, as described above, one approach to mitigating blurring may be to increase the pitch of features in the direction of movement of the sample container. If this type of pitch expansion is insufficient to avoid photon mixing from different features (for example, if the distance of the blur in the frame is greater than the pitch), the pitch expansion approach may be combined with an approach using short bursts of illumination to address this further blur. Therefore, the following discussion should be understood as illustrating an approach to address blur using short bursts of illumination, and should not be interpreted as meaning that this approach is only applicable when blur of one pixel or less is acceptable.

[0077] One approach to avoid the aforementioned blurring while imaging a continuously moving sample container is to illuminate the sample container with pulses of sufficient intensity to allow the necessary photons for the image to be collected during a sufficiently short period, such that the distance the sample container moves while illuminated is less than one pixel. For example, if this type of approach is applied to the scenario in Figure 4, the sample container may be illuminated only during T1 (or some other period of the same or shorter duration), rather than being illuminated from T1 to T6, as may be the case in embodiments using a TDI camera or similar device designed to continuously image a moving target. In addition, the intensity of the illumination may be set so that the dose provided during period T1 is the same as the dose that may be provided from T1 to T6 in embodiments using a TDI camera or similar device designed to continuously image a moving target. In this way, embodiments following this approach can avoid the blurring that may result from attempts to create an image from photons collected across pixels, while still collecting enough photons to enable the image captured by the camera to be usable for its intended purpose (for example, to enable an imaging system, such as those considered in the context of Figures 1 and 2, to sequence a sample).

[0078] Other variations are possible that use short illumination periods to avoid blurring. For example, in some embodiments, the sample container may be illuminated with multiple pulses while it is in the camera's field of view, with each pulse illuminating the container for such a short period that the distance the container travels while illuminated by that pulse is less than one pixel. This may be done, for example, to avoid requiring a laser with enough power to fully illuminate a feature for a time shorter than the time it takes to travel the distance of one pixel, to consider the saturation limit of the dye used to sequence the imaged sample, or for other reasons that may be applicable in specific situations. In some embodiments following this approach, the sample container may be illuminated once per camera frame while it is in the field of view. In this way, multiple exposures may be generated, each exposure based only on photons collected from illumination periods so short that the sample container cannot travel a full pixel. For example, if this approach is applied to the scenario in Figure 4, the sample container may be illuminated during periods T1 and T4. The intensity of the illumination may also be increased in a manner similar to that described above. In other words, the intensity of the illumination can be set such that the photons collected from each illumination period allow each exposure to provide a usable image.

[0079] The approaches described above, and the examples illustrating how they may be applied, are for illustrative purposes only. It should be understood that other approaches and variations of the described approaches are possible and may be applied in several embodiments. For example, consider the intensity of illumination provided in an embodiment in which a sample container is illuminated with multiple short pulses while it is within the camera's field of view. In some embodiments of this type, the illumination intensity may be set to a level that does not allow for the collection of a sufficient number of photons for each exposure to provide a usable image. For example, the illumination may be set to a lower intensity to mitigate the risk of photodamage caused by repeatedly exposing the sample to high-peak-power laser illumination, or to avoid reaching photosaturation of the phosphorescent dye used for sequencing the sample. In this type of embodiment, data from multiple exposures may be combined using a process such as that shown in Figure 5 to obtain (at least) one usable image of the sample.

[0080] Figure 5 is a flowchart illustrating an exemplary method 500 that may be implemented to derive a usable image from multiple exposures. In method 500 of Figure 5, an exposure may be captured in block 501. This may be done, for example, by exposing a camera sensor and illuminating a moving sample container for a short period of time while it is in the camera's field of view, as described above in the context of approaches to avoid blurring. In block 502, the position of the sample container at the time the exposure was captured may be determined. This may be done, for example, by mounting the sample container on a precision motion control stage that moves at a constant speed, and multiplying the known speed of the stage by the amount of time elapsed in the scanning process when the exposure was captured. This position information, along with the exposure itself, may be stored in block 503. This process may be repeated and cycled as the scan progresses, with each iteration of blocks 501, 502, and 503 preferably corresponding to a single frame of the camera used to capture the exposure.

[0081] After the scan is complete, method 500, as shown in Figure 5, can continue in block 504, which defines a reference position. This can be done, for example, by defining the position of the sample container when the first exposure is captured as the reference position. Next, with the reference position defined, the offset of exposures not processed in blocks 505-507 can be determined in block 505. This can be done, for example, by taking the difference between the position stored for the exposure to be processed and the reference position previously stored in block 504. In block 506, the exposure to be processed can be translated by the offset. This can be done, for example, by adding the offset determined in block 505 to the coordinates of the data in the exposure to be processed. The translated exposure can then be overlaid in block 507 with data from any previously processed overlays. This can be done, for example, by summing the data from the translated exposure and the data from any previously processed exposures pixel by pixel, taking advantage of the fact that translation can place all exposures in a consistent coordinate system defined by the reference position. The operations in blocks 505, 506, and 507 may then be repeated for each exposure. Once all exposures have been overlaid, method 500, as shown in Figure 5, may end in block 508, and the overlay with the combined data from all the processed exposures may be processed as an image of the sample container for further analysis.

[0082] Modifications and alterations of method 500 in Figure 5 are also possible. For example, in some embodiments, instead of translating the exposure by the offset through the additions described above in the context of block 506, some embodiments may utilize other types of translation. For example, in embodiments where the position of the sample container at the time the exposure is captured can be determined with sub-pixel precision, the translation of the exposure, as in the case of block 506, can be achieved by performing a Fourier transform of the translated exposure, multiplying the representation of the exposure in frequency space by a complex exponent defined as exp(-idk) (where d is the translation amount and k is the position in frequency space), and then performing an inverse fast Fourier transform on the translated frequency space representation. Similarly, in some embodiments where the position of the sample container at the time the exposure is captured can be determined with sub-pixel precision, the translation that can be performed in block 506 may include performing linear interpolation to determine how the sub-pixel measurements can be transformed into all-pixel measurements in a coordinate system defined by the reference position. Other variations are also possible and may be used in some embodiments, such as expanding the exposure to have a pixel resolution that matches the positional resolution, and using interpolation (e.g., linear, bilinear, or cubic interpolation) to fill in the positional data between pixels in the original image before translation. Therefore, the above examples of various translational approaches should be understood as merely illustrative and not treated as limitations.

[0083] Modifications may also be implemented to provide optimization of exposure representation and / or processing. For example, consider an embodiment in which an image of a sample container is captured by a 10-megapixel camera with a frame rate of 1000 Hz and a bit depth of 12 bits. In such a case, the data to be processed may be generated at a rate of 120 gigabits per second. To help mitigate the difficulties caused by transferring, storing, and processing this amount of data, some embodiments may truncate the bit depth of the output provided by the camera based on the amount of illumination provided to each exposure. For example, if the relationship between the camera's frame rate and the sample container's speed is such that 125 exposures of the sample container's features can be captured while it is in the camera's field of view, the illumination may be set to a level that provides each exposure with 1 / 125 of the illumination required for the usable image. As a result of this lower level of illumination, no pixel from any exposure can have more than 6 bits of data, and therefore the 6 most significant bits of data may be truncated from each pixel of the camera's output, which is then processed or stored as described above in the context of Figure 5. Next, when the exposure is overlaid in block 507, the overlaid image is encoded with a bit depth of 12 bits / pixel so that it can reflect all photons captured by the camera, even if none of the individual exposures are stored or encoded in a way that can store their data. Similarly, in some embodiments, several additional or alternative types of compression may be applied. For example, in embodiments where bit expansion sequences are generally repeated (e.g., sequences of zeros), these sequences may be replaced by a more compact representation, such as using Huffman coding or other types of substitution to reduce the amount of data required to represent the associated data.

[0084] Another type of variation that may be included in some embodiments is the addition of additional processing actions to further consider the requirements of the components that may be used. For example, in some embodiments, the camera may use high-precision, low-distortion optical elements to capture an image of the sample container. However, in other embodiments, additional processing actions may be performed to account for defects that may be introduced by capturing the exposure using a camera with a lens manufactured to lower precision rather than using a low-distortion optical element. For example, in some embodiments, a calibration process may be performed before using a method such as the one shown in Figure 5 for imaging the sample container, in which a calibration target patterned by lithography with multiple holes in a known configuration may be imaged using the method in Figure 5 (e.g., the pattern of feature 310 shown in Figure 3). The pattern of holes in the image of the target captured by the camera may then be compared with the pattern of known holes, and a polynomial map representing the distortion introduced by defects in the camera lens may be constructed from this comparison. Next, when the camera is used to image the sample container, this polynomial map is applied to invert the distortion introduced by the lens into the image(s) of the sample container, thereby potentially allowing for a relaxation of the tolerances typically required for low-distortion optical elements in the system, as shown in Figure 1 or Figure 2, in some implementations. As another example, in some embodiments, the light source may illuminate the sample container with multiple pulses before the distance the sample container travels reaches the size of a camera pixel capturing an image of the same object. This can be used, for example, when it is not possible to provide continuously sufficient intensity illumination during the time required for the sample container to travel one pixel's distance, but it is possible to provide multiple shorter, higher-intensity pulses during that time.

[0085] Additional components may also be included in some embodiments to address and / or mitigate the limitations imposed by discontinuous illumination. For example, in some cases, image stabilization techniques may be used to keep the sample container stationary within the camera's field of view, thereby reducing the effects of container movement and potentially increasing the amount of time the container can be illuminated during any given frame. This may be done, for example, by using a motor to shift the camera (or the camera's lens) in a manner synchronized with the stage movement, thereby moving the camera's field of view during a frame so that the sample container remains stationary (or moves by a distance of less than one pixel). Alternatively, in some cases using this type of image stabilization approach, a piezo or garbo mirror may be placed in the light emission path from the sample container, effectively allowing the camera's field of view to be moved to counteract the stage movement during parts of the frame when the sample container is illuminated. When the sample container is no longer illuminated, the motor can reset the field of view for the next frame, and this process may be repeated over the duration of the imaging run.

[0086] To illustrate potential embodiments showing how image stabilization can be used to mitigate the limitations imposed by discontinuous illumination, consider Figures 7A–7C. In those figures, Figure 7A shows the relationship between a feature 701, a camera 702 whose field of view is divided into 12 pixels, and a movable mirror (e.g., a Garbo mirror) 703 used for image stabilization. In that figure, as light is emitted from the feature, it is directed by a first lens 704 to the mirror 703, reflected from the mirror 703 to a second lens 705, and focused by the second lens 705 to a first pixel on the camera 702, where it is detected. Figure 7B shows the result of the feature 701 moving over a distance greater than the size of a pixel on the camera 702, when the feature is illuminated continuously, the mirror 703 remains stationary, and the camera 702 captures only a single exposure during its movement. As shown in Figure 7B, this result may cause the signal from feature 701 to spread to multiple pixels on camera 702, potentially resulting in a single overlap if a second feature (not shown in Figure 7B) is adjacent to feature 701 shown in Figures 7A and 7B. In contrast, Figure 7C shows the result of moving mirror 703 while feature 701 moves a distance greater than one pixel on camera 702. As shown in Figure 7C, by moving mirror 703 to compensate for the movement of feature 701, the light emitted from the feature can be continuously focused to a single pixel, thereby avoiding the blurring shown in Figure 7B. As previously mentioned, this can also be achieved through the movement of lenses used to direct or focus illumination from camera 702 or a feature (e.g., the first lens 704 or the second lens 705 shown in Figures 7A–7C). Therefore, the specific configurations and components shown in Figures 7A–7C should be understood as illustrative and not as limitations.

[0087] While the above examples and discussions focus on variations of illumination and image capture components, it should be understood that variations of other types of components may also be used in some embodiments. For example, consider a stage for moving a sample container through the field of view of an imaging device. In some embodiments, the stage may be implemented with components such as cross-roller bearings to allow its movement to be precisely controlled (e.g., embodiments that determine the position of the sample container when exposure is captured based on assumptions about the uniformity of the stage's movement). However, in other embodiments, stages with less precise motion control, such as friction-based stages or stages mounted on the frame of the imaging system with ball bearings, and additional components such as encoders may be introduced to determine the position of the stage at a particular point in time when the exposure of the sample container is captured. In such embodiments, determining the position of exposure, rather than positioning based on time, as shown in block 502 of Figure 5, may be done by querying the encoder for the position of the stage when the sample container is illuminated for exposure. Alternative positioning features may also be used. For example, in some variations, the sample container may be provided with a set of bright beads that can act as reference points, allowing the relative positions of the sample containers captured in different images to be determined, and as a result, the features of those images can be collated with each other as described above.

[0088] Naturally, variations of this are also possible. For example, in some embodiments, exposure may be stored with time information rather than its position, as described in block 503. In this type of embodiment, the actual position of exposure may be determined subsequently only when it is necessary to calculate the offset, such as by multiplying it by a known travel speed as described above in the context of block 502, or by matching the exposure time to time-stamped position information collected from the encoder during scanning. Various variations may also be possible in some implementations, such as capturing multiple locations for each illumination pulse (e.g., at the beginning and end of the pulse) and then averaging them to obtain the corresponding exposure location for the pulse, or omitting position determination and determining the exposure offset by comparing the locations of reference points. Therefore, the above examples should be understood as merely illustrative and not treated as limitations.

[0089] Some embodiments may also feature methods that deviate from the overall structure of the method shown in Figure 5. For example, in some embodiments, instead of capturing and storing multiple exposures and then overlaying previously captured and stored exposures, offset determination, offset translation, and overlay of the translated exposure may be performed in real time for each exposure as it is captured. In such embodiments, the actions described above in the context of blocks 501, 502, 504, 505, 506, and 507 may be performed repeatedly during the scan, potentially allowing the user to see the progress of imaging the sample as exposures are captured.

[0090] To further illustrate how the embodiments of the disclosed technology may be applied in practice, consider a scenario in which a biological sample is divided into clusters in nanowells within an array having a pitch length of 624 nm in the direction of movement, and the data captured from the nanowells is used for DNA sequencing using synthetic sequencing. In such a case, if the sample container is imaged while moving at 10 mm / s through a 1 × 1 mm field of view of a 1000 Hz camera, and each pixel in the camera corresponds to a distance of 0.3 μm in the field of view, an embodiment using the method shown in Figure 5 may capture 100 exposures of each nanowell while it is in the camera's field of view per sequencing cycle, based on the camera's frame rate, the camera's field of view, and the speed at which the sample container is moving (i.e., exposure = camera's frame rate). * (Field of view length / movement speed). In addition, in such embodiments, the sample container can be illuminated for 0.03 milliseconds or less per exposure, based on the size of the camera pixels, the movement speed of the sample, and the frame rate of the camera (i.e., illumination time = (pixel size / movement speed)). * (frame rate). In such scenarios, 1-5 J / cm² is needed to accurately image each nanowell. 2 When a threshold dose of such magnitude is required, an embodiment of the method shown in Figure 5 can obtain a usable image of the sample for each sequencing cycle by illuminating the sample using a combination of a laser having a continuous wave power in the range of 3.3 to 16.5 W and an optical chopper for controlling the illumination duration, based on the required dose, field of view (FOV), and duration of each illumination pulse (i.e., power = dose). *(FOV area / pulse duration). This can be provided in various ways, including the aforementioned diode-excited solid-state (DPSS) laser, or using less expensive components such as diode lasers. These images can then be used to identify the sequences of nucleotides within clusters in each nanowell, and each cluster can then be coupled to existing sequencing by synthesis, which may be performed using data acquired using continuous illumination of the sample.

[0091] Figure 6 shows exemplary computing components that may be used to implement various features of the systems and methods disclosed herein, including the aforementioned features and functions of one or more embodiments of methods 400 and 450. For example, a computing component may be implemented as a real-time analysis module 225.

[0092] As used herein, the term "module" may describe a given functional unit that can be implemented according to one or more embodiments of this application. As used herein, a module may be implemented using any form of hardware, software, or a combination thereof. For example, a module may consist of one or more processors, controllers, ASICs, PLAs, PALs, CPLDs, FPGAs, logic components, software routines, or other mechanisms. In embodiments, the various modules described herein may be implemented as separate modules, or the functions and features described may be shared in whole or in part among one or more modules. In other words, as will be apparent to those skilled in the art after reading this description, the various features and functions described herein may be implemented in any given application and may be implemented in one or more separate modules or shared modules in various combinations and permutations. Although elements of various features or functions may be described or claimed individually as separate modules, those skilled in the art will understand that these features and functions may be shared among one or more common software and hardware elements, and that such descriptions do not require or imply that separate hardware or software components are used to implement such features or functions.

[0093] When application components or modules are implemented whole or partially using software, in one embodiment, these software elements may be implemented to operate on a computing or processing module capable of performing the functions described therein. One such exemplary computing module is shown in Figure 6. Various embodiments are described in reference to this exemplary computing module 1000. After reading this description, those skilled in the art will be able to see how to implement the application using other computing modules or architectures.

[0094] Referring here to Figure 6, the computing module 1000 may represent computing or processing power found in, for example, desktop, laptop, notebook, and tablet computers, handheld computing devices (tablets, PDAs, smartphones, mobile phones, palmtops, etc.), mainframes, supercomputers, workstations, or servers, or any other type of dedicated or general-purpose computing device that may be desirable or appropriate for a given application or environment. The computing module 1000 may also represent computing power embedded in or otherwise available in a given device. For example, the computing module may be found in other electronic devices such as, for example, digital cameras, navigation systems, mobile phones, portable computing devices, modems, routers, WAPs, terminals, and other electronic devices that may include some form of processing power.

[0095] The computing module 1000 may include, for example, one or more processors, controllers, control modules, or other processing devices such as processor 1004. Processor 1004 may be implemented using a general-purpose or dedicated processing engine, such as a microprocessor, controller, or other control logic. In the illustrated example, processor 1004 is connected to bus 1002, but any communication medium may be used to facilitate interaction with other components of the computing module 1000 or to communicate with the outside world.

[0096] The computing module 1000 may also include one or more memory modules, which are referred to herein as main memory 1008. For example, preferably random access memory (RAM) or other dynamic memory may be used to store information and instructions executed by the processor 1004. The main memory 1008 may also be used to store temporary variables or other intermediate information during the execution of instructions executed by the processor 1004. The computing module 1000 may also include read-only memory ("ROM") or other static storage devices coupled to the bus 1002 for storing static information and instructions to the processor 1004.

[0097] The computing module 1000 may also include one or more different forms of information storage mechanisms 1010, for example, a media drive 1012 and a storage unit interface 1020. The media drive 1012 may include a drive or other mechanism for supporting a fixed or removable storage medium 1014. For example, a hard disk drive, a solid-state drive, a magnetic tape drive, an optical disc drive, a CD or DVD drive (R or RW), or other removable or fixed media drive may be provided. Thus, the storage medium 1014 may include, for example, a hard disk, a solid-state drive, a magnetic tape, a cartridge, an optical disc, a CD, a DVD, or a Blu-ray, or other fixed or removable media that are read, written to, or accessed by the media drive 1012. As these examples show, the storage medium 1014 may include a computer-usable storage medium in which computer software or data is stored.

[0098] In alternative embodiments, the information storage mechanism 1010 may include other similar means for enabling computer programs or other instructions or data to be loaded into the computing module 1000. Such means may include, for example, fixed or removable storage units 1022 and interfaces 1020. Examples of such storage units 1022 and interfaces 1020 may include program cartridges and cartridge interfaces, removable memory (e.g., flash memory or other removable memory modules) and memory slots, PCMCIA slots and cards, and other fixed or removable storage units 1022 and interfaces 1020 that enable software and data to be transferred from the storage unit 1022 to the computing module 1000.

[0099] The computing module 1000 may also include a communication interface 1024. The communication interface 1024 may be used to enable the transfer of software and data between the computing module 1000 and external devices. Examples of the communication interface 1024 include a modem or soft modem, a network interface (such as Ethernet, a network interface card, WiMedia, IEEE 802.XX, or other interfaces), a communication port (e.g., a USB port, an IR port, an RS232 port, a Bluetooth® interface, or other ports), or other communication interfaces. The software and data transferred via the communication interface 1024 may be transmitted as signals, which may be electronic, electromagnetic (including optical), or other signals that can be exchanged by a given communication interface 1024. These signals may be provided to the communication interface 1024 via a channel 1028, which may transmit signals and may be implemented using a wired or wireless communication medium. Some examples of channels include telephone lines, cellular links, RF links, optical links, network interfaces, local or wide area networks, and other wired or wireless communication channels.

[0100] In this specification, the terms “computer-readable medium,” “computer-usable medium,” and “computer program medium” are used generally to refer to volatile or non-volatile, non-temporary media such as memory 1008, storage unit 1022, and medium 1014. These and various other forms of computer program mediums or computer-usable media may be involved in transmitting one or more sequences of one or more instructions to an execution processing device. Such instructions embodied on a medium are generally referred to as “computer program code” or “computer program product” (which may be grouped in the form of computer programs or other groupings). When executed, such instructions may enable computing module 1000 to perform features or functions of the present application as discussed herein.

[0101] In the claims, the phrase "means for acquiring analytical images of a continuously moving sample using pulsed illumination" should be understood as means with the functional limitations set out in Section 112 of the U.S. Patent Act, which is acquiring analytical images of a continuously moving sample using pulsed illumination, and the corresponding structure is an illumination source, camera, moving stage, and computer, as described in the context of Figure 4, for generating subpixel illumination pulses and avoiding blurring that may otherwise occur with continuous illumination.

[0102] In the claims, the phrase “means for translating and overlaying multiple subthreshold exposures” should be understood as means with functional limitations as defined in Section 112 of the U.S. Patent Act, the function being “translating and overlaying multiple subthreshold exposures,” and the corresponding structures are computers for performing the actions described in the context of blocks 505–507 of Figure 5, as well as variations of the actions described above, which are included in some embodiments.

[0103] While various embodiments and configurations have been described above, it should be understood that the various features, aspects, and functions described in one or more of the individual embodiments are not limited to the specific embodiment in which they are described, but rather may be applied individually or in various combinations to one or more of the other embodiments of this application, regardless of whether such embodiments are described or whether such features are presented as part of the described embodiment. Accordingly, the scope and extent of protection provided by this document or any related documents should not be limited by any of the embodiments described above.

[0104] It should be understood that all combinations of the aforementioned concepts (provided that such concepts are not mutually contradictory) are intended to be part of the subject matter of the invention disclosed herein. Specifically, all combinations of the claimed subject matter appearing at the end of this disclosure are intended to be part of the subject matter of the invention disclosed herein.

[0105] The terms “substantially” and “about” as used throughout this disclosure, including the claims, are used to describe and explain small variations, such as those resulting from variations in processing. For example, these terms may be expressed as ±5% or less, ±2% or less, ±1% or less, ±0.5% or less, ±0.2% or less, ±0.1% or less, ±0.05% or less, etc.

[0106] To the extent applicable, terms such as “first,” “second,” and “third” used herein are used solely to indicate the respective objects described by these terms as distinct entities and do not imply a chronological meaning unless otherwise specifically stated herein.

[0107] The terms and phrases used in this document, as well as their variations, should be interpreted as unrestrictive, as opposed to restrictive, unless otherwise specified. For example, the term “includes” should be read as meaning “includes but not restrictive.” The term “example” is used to provide examples of the item in consideration, rather than as an exhaustive or restrictive list. The term “one (a or an)” should be read as meaning “at least one,” “one or more.” Adjectives such as “existing,” “conventional,” “ordinary,” “standard,” and “known,” and similar terms, should not be interpreted as limiting the items described to items available during a given period or at a given point in time, but rather as encompassing existing, conventional, ordinary, or standard technologies that may be available or known at any point in time, now or in the future. Similarly, where this document refers to a technology that may be obvious or known to a person skilled in the art, such technology encompasses what is obvious or known to a person skilled in the art at any point in time, now or in the future.

[0108] In some cases, the presence of broader words and phrases such as “one or more,” “at least,” “but not limited to,” or other similar terms should not be interpreted as meaning that a narrower case is intended or required in the absence of such broader terms. The use of the term “module” does not mean that all components or functions described or claimed as part of a module are comprised of a common package. In fact, any or all of the various components of a module, whether control logic or other components, may be combined within a single package, maintained separately, or further distributed within multiple groups or packages or across multiple locations.

[0109] In addition, the various embodiments described herein are described with respect to block diagrams, flowcharts, and other figures. As will become apparent to those skilled in the art after reading this document, the illustrated embodiments and their various alternatives can be implemented without limiting themselves to the examples shown. For example, the block diagrams and their accompanying descriptions should not be construed as obligating a particular architecture or configuration.

[0110] While various embodiments of this disclosure have been described above, it should be understood that these are presented merely as examples and are not limiting. Similarly, various diagrams may show exemplary architectures or other configurations for this disclosure, which are provided to aid in understanding the features and functions that may be included in this disclosure. This disclosure is not limited to the exemplary architectures or configurations shown, but desired features may be implemented using various alternative architectures and configurations. In fact, how alternative functional, logical, or physical partitioning and configurations may be implemented to implement desired features of this disclosure will be obvious to those skilled in the art. Furthermore, numerous different configuration module names other than those shown herein may be applied to various partitions. Moreover, with respect to flowcharts, operation descriptions, and claims of methods, the order in which the actions are presented herein does not obligate various embodiments to be implemented to perform the enumerated functions in the same order, unless otherwise indicated by the context.

[0111] [Implementation Method] (1) A machine, A camera for capturing an image containing pixels, wherein each of the pixels has a pixel size corresponding to the distance on the stage in the direction of movement of the sample container, The stage is for moving the sample container with respect to the field of view of the camera that overlaps with the stage, wherein the sample container includes an array having a pitch length in the direction of movement of the sample container, A light source for illuminating the field of view of the camera, A controller for acquiring an analysis image by performing an action, wherein the action is performed for each exposure of one or more features while a feature from the array of features is within the field of view of the camera and while it is moving relative to the field of view of the camera. Illuminating the sensor of the aforementioned camera for a first duration, A controller comprising: obtaining one or more exposures of the features by performing an action including: illuminating the field of view of the camera with the illumination source during a period having a second duration that elapses while the sensor of the camera is illuminated; A machine in which the displacement of the feature within the field of view of the camera from the start to the end of the period having the second duration is less than or equal to the pitch length in the direction of movement of the sample container. (2) The machine according to Embodiment 1, wherein the displacement of the feature in the direction of movement of the sample container within the field of view of the camera from the start to the end of the period having the second duration is less than or equal to the pixel size. (3) Obtaining exposure of one or more of the features includes obtaining multiple exposures of the features, The machine according to any one of embodiments 1 to 2, wherein the action performed by the controller includes overlaying the multiple exposures of the feature based on translating one or more of the multiple exposures of the feature. (4) The actions performed by the controller include, for each exposure, obtaining a corresponding value of the position of the sample container when the field of view of the camera is illuminated by the illumination source, The machine according to Embodiment 3, wherein the controller translates one or more of the plurality of exposures of the feature based on the difference between the corresponding values ​​of the exposure at the position of the sample container. (5) The machine is equipped with an encoder for providing a value for the position of the sample container, The machine according to Embodiment 4, wherein the controller obtains the corresponding value of the position of the sample container when the field of view of the camera is illuminated by the illumination source for each exposure, from the encoder.

[0112] (6) The encoder has a resolution for distinguishing distances smaller than the distance on the stage corresponding to the pixel size, The machine according to Embodiment 5, wherein overlaying the plurality of exposures of the features includes coregistrating each of the plurality of exposures at the resolution of the encoder. (7) Collisionizing each of the plurality of exposures at the resolution of the encoder means that for at least one of the one or more exposures, By performing a fast Fourier transform of the exposure, a frequency-space representation is obtained, Translating the frequency space representation by a distance that is not an integer multiple of the distance on the stage corresponding to the pixel size, The machine according to Embodiment 6, comprising performing an inverse fast Fourier transform on the translated frequency space representation. (8) Collisionizing each of the plurality of exposures at the resolution of the encoder means that for each of the plurality of exposures, Based on interpolating data between pixels, the exposure is upsampled to the resolution of the encoder, The machine according to Embodiment 6, comprising translating one or more of the exposures after upsampling. (9) The sample container includes a plurality of reference points, The machine according to Embodiment 3, wherein the controller translates one or more of the plurality of exposures of the feature based on the difference in the location of the reference point between exposures. (10) The analysis image includes a plurality of pixels, each having a first bit depth, The machine according to any one of embodiments 3 to 9, wherein each of the plurality of exposures includes a plurality of pixels, each having a second bit depth, the second bit depth being smaller than the first bit depth.

[0113] (11) Each pixel in each image captured by the camera has a third bit depth, the third bit depth being greater than the second bit depth. Obtaining the aforementioned multiple exposures of the aforementioned features means that for each exposure, While the field of view of the camera is illuminated by the light source, the camera captures an image, The machine according to Embodiment 10, comprising truncating some of the most significant bits of the pixels from the image captured by the camera, wherein the number of the most significant bits truncated is equal to the difference between the third bit depth and the second bit depth. (12) A threshold illumination energy dose is required to image the above features, For each of the one or more exposures of the aforementioned features, the illumination source illuminates the field of view of the camera, When multiplied by the second duration, it provides individual exposure energy doses that are smaller than the threshold illumination energy dose for imaging the features, The machine according to any one of embodiments 1 to 11, comprising starting the illumination source with power that provides a combined exposure energy dose greater than the threshold illumination energy dose for imaging the feature when multiplied by the second duration and the number of exposures within the plurality of exposures of the feature. (13) The actions performed by the controller are: The camera acquires an image of a reference object, wherein the reference object includes a plurality of features having known locations. Creating a distortion map by performing an action that includes comparing the known locations of the plurality of features contained in the reference object with the apparent locations of the plurality of features in the image of the reference object, A machine according to any one of embodiments 1 to 12, comprising applying the distortion map to each of the one or more exposures of the aforementioned features. (14) The stage is mounted on the frame of the machine using ball bearings, The camera captures images using a complementary metal-oxide-semiconductor sensor. The machine according to any one of embodiments 1 to 13, wherein the illumination source is a diode laser. (15) The machine according to any one of embodiments 1 to 14, wherein the feature is a nanowell.

[0114] (16) The aforementioned analysis image is one of several analysis images, The controller executes multiple sequencing cycles, and each analysis image from the multiple analysis images corresponds to a single sequencing cycle. The controller determines cluster polynucleotides for each feature in the sample container based on the multiple analysis images, The machine according to any one of embodiments 1 to 15, wherein the controller determines the complete polynucleotide of a sample associated with the sample container based on the cluster polynucleotide determined for the characteristics from the sample container. (17) The machine according to any one of embodiments 1 to 16, wherein the array of features contained in the sample container has a pitch perpendicular to the direction of movement of the sample container, and is smaller than the pitch in the direction of movement of the sample container. (18) The machine according to any one of embodiments 1 to 17, wherein the machine comprises a motor for counteracting the movement of the stage by translating the field of view of the camera in the direction of movement of the sample container during the period having the second duration. (19) A method, The method involves translating features on a stage relative to the camera's field of view in the direction of movement, wherein the camera has a pixel size corresponding to the distance in the direction of movement on the stage, the features are included in an array of features in a sample container, and the array of features has a pitch length in the direction of movement. While the feature is within the field of view of the camera and is moving relative to the field of view of the camera, for each exposure of the feature, The sensor of the aforementioned camera is illuminated for a first duration, The process includes generating an analytical image by performing an action that includes obtaining one or more exposures of the features by performing an action that includes illuminating the field of view of the camera with an illumination source during a period having a second duration that elapses while the sensor of the camera is illuminated, A method wherein the displacement of the feature of the camera's field of view from the start to the end of the period having the second duration is less than or equal to the pitch length in the direction of movement. (20) The method according to Embodiment 19, wherein the displacement of the feature in the direction of movement from the start to the end of the period having the second duration is less than or equal to the pixel size.

[0115] (21) Obtaining exposure of one or more of the features includes obtaining multiple exposures of the features, The method according to any one of embodiments 19 to 20, wherein the method comprises overlaying the multiple exposures of the feature to produce the analytical image of the feature by performing an action that includes translating one or more of the multiple exposures of the feature. (22) The above method is For each exposure, the corresponding value of the position of the sample container when the camera's field of view is illuminated by the illumination source is obtained, The method according to Embodiment 21, comprising translating one or more of the plurality of exposures of the features based on the difference between corresponding values ​​of the exposures at the position of the sample container. (23) The method according to Embodiment 22, wherein, for each exposure, the corresponding value of the position of the sample container when the field of view of the camera is illuminated by the illumination source is obtained from an encoder. (24) The encoder has a resolution for distinguishing distances smaller than the distance on the stage corresponding to the pixel size, The method according to Embodiment 23, wherein overlaying the plurality of exposures of the features includes coregistrating each of the plurality of exposures at the resolution of the encoder. (25) Collisionizing each of the plurality of exposures at the resolution of the encoder means that for at least one of the one or more exposures, By performing a fast Fourier transform of the exposure, a frequency-space representation is obtained, Translating the frequency space representation by a distance that is not an integer multiple of the distance on the stage corresponding to the pixel size, The method according to Embodiment 24, comprising performing an inverse fast Fourier transform on the translated frequency space representation.

[0116] (26) Collisionizing each of the plurality of exposures with the resolution of the encoder means that for each of the plurality of exposures, Based on interpolating data between pixels, the exposure is upsampled to the resolution of the encoder, The method according to Embodiment 24, comprising translating one or more of the exposures after upsampling. (27) The sample container includes a plurality of reference points, The method according to Embodiment 21, comprising translating one or more of the plurality of exposures of the feature based on the difference in the location of the reference point between exposures. (28) The analysis image includes a plurality of pixels, each having a first bit depth, The method according to any one of embodiments 21 to 27, wherein each of the plurality of exposures includes a plurality of pixels, each having a second bit depth, the second bit depth being smaller than the first bit depth. (29) Each pixel in each image captured by the camera has a third bit depth, the third bit depth being greater than the second bit depth. Obtaining the aforementioned multiple exposures of the aforementioned features means that for each exposure, While the field of view of the camera is illuminated by the light source, the camera captures an image, The method according to Embodiment 28, comprising truncating some of the most significant bits of the pixels from the image captured by the camera, wherein the number of most significant bits truncated is equal to the difference between the third bit depth and the second bit depth. (30) A threshold illumination energy dose is required to image the above features, For each of the one or more exposures of the aforementioned features, the illumination source illuminates the field of view of the camera, When multiplied by the second duration, it provides individual exposure energy doses that are smaller than the threshold illumination energy dose for imaging the features, The method according to any one of embodiments 19 to 28, comprising activating the illumination source with power that, when multiplied by the second duration and the number of exposures in the plurality of exposures, provides a combined exposure energy dose greater than the threshold illumination energy dose for imaging the feature.

[0117] (31) The above method is The camera acquires an image of a reference object, wherein the reference object includes a plurality of features having known locations. Creating a distortion map by performing an action that includes comparing the known locations of the plurality of features contained in the reference object with the apparent locations of the plurality of features in the image of the reference object, The method according to any one of embodiments 19 to 30, comprising applying the distortion map to each of the one or more exposures of the aforementioned features. (32) The stage is mounted on a stationary frame using ball bearings, The camera captures images using a complementary metal-oxide-semiconductor sensor. The method according to any one of embodiments 19 to 31, wherein the illumination source is a diode laser. (33) The method according to any one of embodiments 19 to 32, wherein the feature is a nanowell. (34) The aforementioned analysis image is one of several analysis images, The aforementioned method, This involves executing multiple sequencing cycles, wherein each analysis image from the multiple analysis images corresponds to a single sequencing cycle. Based on the aforementioned multiple analysis images, cluster polynucleotides are determined for each characteristic within the sample container, The method according to any one of embodiments 19 to 33, comprising determining the complete polynucleotide of a sample associated with the sample container based on the cluster polynucleotide determined with respect to the characteristics from the sample container. (35) The method according to any one of embodiments 19 to 34, wherein the array of features contained in the sample container has a pitch perpendicular to the direction of movement of the sample container, which is smaller than the pitch in the direction of movement of the sample container.

[0118] (36) The method according to any one of embodiments 19 to 35, wherein the method includes a motor that counteracts the movement of the stage by translating the field of view of the camera in the direction of movement during the period having the second duration. (37) A machine, A stage for moving the sample relative to the camera's field of view, which overlaps with the stage, A camera for capturing an image including pixels, wherein each of the pixels has a pixel size corresponding to a distance on the stage, A light source for illuminating the field of view of the camera, A machine comprising means for acquiring analytical images of a continuously moving sample using pulsed illumination. (38) The machine according to embodiment 37, wherein the means for acquiring the analytical image of the continuously moving sample using pulsed illumination includes means for translating and overlaying a plurality of subthreshold exposures.

Claims

1. It is a machine, A camera for capturing an image containing pixels, wherein each of the pixels has a pixel size corresponding to the distance on the stage in the direction of movement of the sample container, The stage is for moving the sample container with respect to the field of view of the camera that overlaps with the stage, wherein the sample container includes an array having a pitch length in the direction of movement of the sample container, A light source for illuminating the field of view of the camera, A controller for acquiring an analysis image by performing an action, wherein the action is performed for each exposure of one or more features while a feature from the array of features is within the field of view of the camera and while it is moving relative to the field of view of the camera. Illuminating the sensor of the aforementioned camera for a first duration, A controller includes obtaining one or more exposures of the features by performing an action which includes illuminating the field of view of the camera with the illumination source during a period having a second duration that elapses while the sensor of the camera is illuminated, The displacement of the feature within the camera's field of view from the start to the end of the period having the second duration is less than or equal to the pitch length in the direction of movement of the sample container. The pitch length is the distance between adjacent features that are separated from each other in the direction of movement, in a machine.

2. The machine according to claim 1, wherein the displacement of the feature in the direction of movement of the sample container within the field of view of the camera from the start to the end of the period having the second duration is less than or equal to the pixel size.

3. Obtaining exposure of one or more of the aforementioned features includes obtaining multiple exposures of the aforementioned features. The machine according to claim 1, wherein the action performed by the controller includes overlaying the plurality of exposures of the feature, based on translating one or more of the plurality of exposures of the feature.

4. The aforementioned analysis image includes a plurality of pixels, each having a first bit depth. The machine according to claim 3, wherein each of the plurality of exposures comprises a plurality of pixels, each having a second bit depth, the second bit depth being smaller than the first bit depth.

5. Each pixel in each image captured by the camera has a third bit depth, and the third bit depth is greater than the second bit depth. Obtaining the aforementioned multiple exposures of the aforementioned features means that for each exposure, While the field of view of the camera is illuminated by the light source, the camera captures an image, The machine according to claim 4, comprising truncating some of the most significant bits of the pixels from the image captured by the camera, wherein the number of most significant bits truncated is equal to the difference between the third bit depth and the second bit depth.

6. A threshold illumination energy dose is required to image the aforementioned features. For each of the one or more exposures described above, the illumination source illuminates the field of view of the camera. When multiplied by the second duration, it provides individual exposure energy doses that are smaller than the threshold illumination energy dose for imaging the features, The machine according to claim 1, comprising activating the illumination source with power that, when multiplied by the second duration and multiplied by the number of exposures of the feature within the plurality of exposures, provides a combined exposure energy dose greater than the threshold illumination energy dose for imaging the feature.

7. The stage is mounted on the frame of the machine using ball bearings. The camera captures images using a complementary metal-oxide-semiconductor sensor. The machine according to claim 1, wherein the illumination source is a diode laser.

8. The aforementioned analysis image is one of several analysis images. The controller executes multiple sequencing cycles, and each analysis image from the multiple analysis images corresponds to a single sequencing cycle. The controller determines cluster polynucleotides for each feature in the sample container based on the multiple analysis images, The machine according to claim 1, wherein the controller determines the complete polynucleotide of a sample associated with the sample container based on the cluster polynucleotide determined for the characteristics from the sample container.

9. The machine according to claim 1, wherein the machine comprises a motor for counteracting the movement of the stage by translating the field of view of the camera in the direction of movement of the sample container during the period having the second duration.

10. It is a method, The method involves translating features on a stage relative to the camera's field of view in the direction of movement, wherein the camera has a pixel size corresponding to the distance in the direction of movement on the stage, the features are included in an array of features in a sample container, and the array of features has a pitch length in the direction of movement. While the feature is within the field of view of the camera and is moving relative to the field of view of the camera, for each exposure of the feature, The sensor of the aforementioned camera is illuminated for a first duration, The process includes generating an analytical image by performing an action that includes illuminating the field of view of the camera with an illumination source during a period having a second duration that elapses while the sensor of the camera is illuminated, thereby obtaining one or more exposures of the features, The displacement of the feature of the camera's field of view from the start to the end of the period having the second duration is less than or equal to the pitch length in the direction of movement. A method wherein the pitch length is the distance between adjacent features that are separated from each other in the direction of movement.

11. The method according to claim 10, wherein the displacement of the feature in the direction of movement from the start to the end of the period having the second duration is less than or equal to the pixel size.

12. Obtaining exposure of one or more of the aforementioned features includes obtaining multiple exposures of the aforementioned features. The method according to claim 10, further comprising overlaying the multiple exposures of the feature to produce the analytical image of the feature by performing an action that includes translating one or more of the multiple exposures of the feature.

13. The aforementioned analysis image includes a plurality of pixels, each having a first bit depth. The method according to claim 12, wherein each of the plurality of exposures includes a plurality of pixels, each having a second bit depth, the second bit depth being smaller than the first bit depth.

14. Each pixel in each image captured by the camera has a third bit depth, and the third bit depth is greater than the second bit depth. Obtaining the aforementioned multiple exposures of the aforementioned features means that for each exposure, While the field of view of the camera is illuminated by the light source, the camera captures an image, The method according to claim 13, comprising truncating some of the most significant bits of the pixels from the image captured by the camera, wherein the number of most significant bits truncated is equal to the difference between the third bit depth and the second bit depth.

15. A threshold illumination energy dose is required to image the aforementioned features. For each of the one or more exposures described above, the illumination source illuminates the field of view of the camera. When multiplied by the second duration, it provides individual exposure energy doses that are smaller than the threshold illumination energy dose for imaging the features, The method according to claim 10, comprising activating the illumination source with power that, when multiplied by the second duration and multiplied by the number of exposures in the plurality of exposures, provides a combined exposure energy dose greater than the threshold illumination energy dose for imaging the feature.

16. The aforementioned stage is mounted on a stationary frame using ball bearings. The camera captures images using a complementary metal-oxide-semiconductor sensor. The method according to claim 10, wherein the illumination source is a diode laser.

17. The aforementioned analysis image is one of several analysis images. The aforementioned method, This involves executing multiple sequencing cycles, wherein each analysis image from the multiple analysis images corresponds to a single sequencing cycle. Based on the aforementioned multiple analysis images, cluster polynucleotides are determined for each characteristic within the sample container, The method according to claim 10, comprising determining the complete polynucleotide of a sample associated with the sample container based on the cluster polynucleotide determined with respect to the characteristics from the sample container.

18. The method according to claim 10, further comprising a motor that counteracts the movement of the stage by translating the field of view of the camera in the direction of movement during the period having the second duration.

Images