Systems and methods for multicolor imaging

Abstract

In an aspect, an imaging system is provided. In a non-limiting exemplary embodiment, the imaging system includes multiple sensor arrays (e.g., two or four independent time-delay-integration [TDI] sensor arrays), two light sources (e.g., two lasers) that illuminate the sample, a first optical system that directs one excitation beam from each light source to the sample, and a second optical system that directs fluorescent radiation from the sample to each sensor array. In an embodiment, an aerial image is formed when the fluorescent radiation impinges on the sensor array. The aerial image is an image formed by the radiation in the plane of the sensor. In an embodiment, the aerial image and its movement are synchronized with the sample stage. In an embodiment, the charge generated in the sensor by the fluorescent radiation travels across the sensor array, and the transfer of the charge from one row of the sensor array to the next row is synchronized with the movement of the stage. In an embodiment, one or more pixels of the sensor array collect charge as the image sweeps over each sensor array (i.e., as the sample is scanned).

Description

[Background technology]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 128,477, filed December 21, 2020, which is incorporated herein by reference in its entirety for all purposes.

[0002] Next-generation sequencing (NGS) methods typically rely on the detection of genomic fragments immobilized on an array. For example, in sequencing by synthesis (SBS), fluorescently labeled nucleotides are added to an array of polynucleotide primers and detected upon incorporation. The extension of the nucleic acid primer along the nucleic acid template is monitored to determine the sequence of nucleotides in the template. Each detection event (e.g., feature) can be distinguished by its location within the array.

[0003] For these and other uses of polynucleotide arrays, improvements have been made in recent years to increase the density of features within the array. For example, such arrays have at least 50,000 features / cm, 100,000 features / cm, 1,000,000 features / cm, 5,000,000 features / cm, or more. Technological advances have reduced the typical distance between adjacent features so that features are slightly larger than the optical resolution scale, the camera pixel pitch, or both. The combination of high-resolution optics (e.g., an objective lens with a numerical aperture (NA) of 0.8) and the high-speed imaging methods described herein enables improved analysis and throughput in imaging systems. Summary of the Invention

[0004] Disclosed herein are solutions to, among other things, the aforementioned and other problems in the art. The present disclosure provides image analysis methods and systems useful for rapidly identifying and / or quantifying features on a substrate.

[0005] In one aspect, an imaging system is provided. In a non-limiting exemplary embodiment, the imaging system includes multiple sensor arrays (e.g., two or four independent time delay integration (TDI) sensor arrays), two light sources (e.g., two lasers) that illuminate a sample, a first optical system that directs one excitation beam from each light source onto the sample, and a second optical system that directs fluorescent emission from the sample onto each sensor array.

[0006] In another aspect, an imaging system includes a sample stage moving at a sample stage velocity, the sample stage configured to receive a sample including a first fluorophore and a second fluorophore; a first sensor array and a second sensor array; a first light source configured to provide a first excitation beam and a second light source configured to provide a second excitation beam; and a first optical system configured to direct the first excitation beam and the second excitation beam onto the sample, wherein interaction of the first excitation beam with the first fluorophore produces a first fluorescent emission and interaction of the second excitation beam with the second fluorophore produces a second fluorescent emission. An imaging system is disclosed that includes a first optical system in which a second fluorescent radiation is generated by interaction with a second fluorophore; and a second optical system configured to direct the first fluorescent radiation to a first sensor array and the second fluorescent radiation to a second sensor array, wherein the first fluorescent radiation impinges on the first sensor array to generate a first charge that travels across the first sensor array, and the second fluorescent radiation impinges on the second sensor array to generate a second charge that travels across the second sensor array, wherein the travel of at least one of the first charge and the second charge is synchronized with a sample stage velocity. In an embodiment, the imaging system further includes a third fluorophore, a fourth fluorophore, a third sensor array, and a fourth sensor array, wherein a third fluorescent radiation is generated by interaction of the first excitation beam with the third fluorophore and a fourth fluorescent radiation is generated by interaction of the second excitation beam with the fourth fluorophore; the second optical system is configured to direct the third fluorescent radiation to the third sensor array and the fourth fluorescent radiation to the fourth sensor array, wherein the third fluorescent radiation impinges on the third sensor array to generate a third charge that travels across the third sensor array, and the fourth fluorescent radiation impinges on the fourth sensor array to generate a fourth charge that travels across the fourth sensor array, and the travel of at least one of the third charge and the fourth charge is synchronized with the sample stage speed.

[0007] In another aspect, an imaging system includes: a sample stage moving at a sample stage velocity, the sample stage including a sample comprising a first fluorophore, a second fluorophore, a third fluorophore, and a fourth fluorophore; a first sensor array, a second sensor array, a third sensor array, and a fourth sensor array; a first light source configured to provide a first excitation beam, and a second light source configured to provide a second excitation beam; and a first optical system configured to direct the first excitation beam and the second excitation beam onto the sample, wherein interaction of the first excitation beam with the first fluorophore produces a first fluorescent emission, interaction of the second excitation beam with the second fluorophore produces a second fluorescent emission, and interaction of the first excitation beam with the third fluorophore produces a third fluorescent emission; An imaging system is disclosed that includes: a first optical system in which a fourth fluorescent emission is generated by interaction of a second excitation beam with a fourth fluorophore; and a second optical system configured to direct the first fluorescent emission to a first sensor array, the second fluorescent emission to a second sensor array, the third fluorescent emission to a third sensor array, and the fourth fluorescent emission to a fourth sensor array, wherein the first fluorescent emission impinges on the first sensor array and generates a first charge that travels across the first sensor array, the second fluorescent emission impinges on the second sensor array and generates a second charge that travels across the second sensor array, the third fluorescent emission impinges on the third sensor array and generates a third charge that travels across the third sensor array, and the fourth fluorescent emission impinges on the fourth sensor array and generates a fourth charge that travels across the fourth sensor array.

[0008] In another aspect, a method of imaging a sample is disclosed that includes: a) directing a first excitation beam and a second excitation beam onto the sample, the sample being on a sample stage moving at a sample stage velocity, the sample including a first fluorophore that generates a first fluorescent emission after interaction with the first excitation beam and a second fluorophore that generates a second fluorescent emission after interaction with the second excitation beam, respectively; b) directing the first fluorescent emission to impinge on a first sensor array and generate a first charge that travels across the first sensor array at a first charge velocity, and directing the second fluorescent emission to impinge on a second sensor array and generate a second charge that travels across the second sensor array at a second charge velocity, wherein at least one of the first charge velocity and the second charge velocity is synchronized with the sample stage velocity; and c) scanning the sample in a scan dimension and repeating steps a) and b) to form an image of the sample. In an embodiment, the sample further includes a third fluorophore that generates a third fluorescent emission after interaction with the first excitation beam and a fourth fluorophore that generates a fourth fluorescent emission after interaction with the second excitation beam, and the third fluorescent emission is directed to impinge on the third sensor array and generate a third charge that travels across the third sensor array at a third charge velocity, and the fourth fluorescent emission is directed to impinge on the fourth sensor array and generate a fourth charge that travels across the fourth sensor array at a fourth charge velocity.

[0009] The details of one or more variations of the subject matter described herein are set forth in the accompanying drawings and the description below. Other features and advantages of the subject matter described herein will be apparent from the description and drawings, and from the claims. [Brief explanation of the drawings]

[0010] [Figure 1] 1 is an exemplary diagram of a four-color imaging system using one camera, where the camera contains four sensor arrays (ie, image sensor arrays). [Figure 2] 1 is an exemplary diagram of a four-color imaging system using two cameras, each containing two sensor arrays (ie, image sensor arrays). DETAILED DESCRIPTION OF THE INVENTION

[0011] Nucleic acid sequencing, biomolecular imaging, and other uses of biochemical arrays require sophisticated imaging systems to achieve commercially viable data acquisition rates. The number of biochemical experiments for which data can be collected per unit time can depend on the array density and image acquisition rate, among other factors. High array density can complicate the image acquisition problem because it can be difficult to track the identity of each experiment in the image. One of the most common methods for sequencing DNA is to fluorescently label each of the four nucleotide bases in a DNA strand. Typically, the resolution of each base pair is approximately 0.5 nm laterally, and the light from their fluorescence is extremely weak. Highly sensitive image sensors and imaging systems are required.

[0012] Image acquisition speed can be increased by using multiple excitation beams to image various fluorophores in a sample (e.g., a sample containing nucleic acids for sequencing). However, imaging using multiple excitation beams may require additional optical elements (e.g., optical gratings) to separate the emission beam (e.g., fluorescence beam) from the various fluorophores. Additionally or alternatively, the excitation beams can be impinged on the sample one at a time (e.g., using time-division multiplexing). Employing additional optical elements and / or time-division multiplexing can increase the complexity of the imaging system and / or slow the speed of data acquisition. The subject matter described herein can improve image acquisition speed by using spatially separated excitation beams. This can result in emission beams that can be detected simultaneously without employing additional optics to separate the emission beams and / or detect one emission beam at a time.

[0013] In another aspect, a method of imaging a sample is disclosed. In an embodiment, the sample includes a first fluorophore and a second fluorophore (e.g., a first fluorophore type and a second fluorophore type). The sample may include a plurality of fluorophores of at least four different types, or fluorophore types with different absorption and emission profiles. In an embodiment, the sample includes a first fluorophore, a second fluorophore, a third fluorophore, and a fourth fluorophore, each fluorophore having a different fluorescence emission profile. In an embodiment, the method includes directing a first excitation beam and a second excitation beam onto the sample, wherein interaction of the first excitation beam with the first fluorophore generates a first fluorescence emission, and interaction of the second excitation beam with the second fluorophore generates a second fluorescence emission. In an embodiment, a method includes directing a first excitation beam and a second excitation beam at a sample, wherein interaction of the first excitation beam with a first fluorophore generates a first fluorescent emission, interaction of the second excitation beam with a second fluorophore generates a second fluorescent emission, interaction of the first excitation beam with a third fluorophore generates a third fluorescent emission, and interaction of the second excitation beam with a fourth fluorophore generates a fourth fluorescent emission. In an embodiment, the method includes directing a first fluorescent radiation to a first sensor array and a second fluorescent radiation to a second sensor array, wherein the first fluorescent radiation impinges on the first sensor array to generate a first charge that travels across the first sensor array (i.e., is absorbed in a photodiode), and the second fluorescent radiation impinges on the second sensor array to generate a second charge that travels across the second sensor array (i.e., is absorbed in a photodiode), and wherein the travel of at least one of the first charge and the second charge is synchronized with a sample stage velocity.In an embodiment, the method includes directing a first fluorescent radiation to a first sensor array, a second fluorescent radiation to a second sensor array, a third fluorescent radiation to a third sensor array, and a fourth fluorescent radiation to a fourth sensor array, wherein the first fluorescent radiation impinges on the first sensor array and generates a first charge that travels across the first sensor array, the second fluorescent radiation impinges on the second sensor array and generates a second charge that travels across the second sensor array, the third fluorescent radiation impinges on the third sensor array and generates a third charge that travels across the third sensor array, and the fourth fluorescent radiation impinges on the fourth sensor array and generates a fourth charge that travels across the fourth sensor array, and wherein the travel of at least one of the first charge, the second charge, the third charge, and the fourth charge is synchronized with a sample stage velocity. In an embodiment, the velocities of all four charges are synchronized with the sample stage velocity. In an embodiment, the speed of two charges (e.g., a first charge and a third charge) is synchronized with the sample stage speed. As the charges travel across the sensor array (e.g., a first charge traveling across a first sensor array), it is understood that the charges are transferred across the array according to theories currently understood in the art. For example, charge transfer originates from the thermal motion phenomenon of electrons, where electrons with sufficient thermal velocity in the direction of transfer cross a barrier on the charge transfer path (i.e., across the image sensor). The light energy incident on the sensor is converted into an electrical signal for digitization, which is transferred to a computing device.

[0014] In another aspect, an imaging system includes a sample stage moving at a sample stage velocity, the sample stage configured to receive a sample including a first fluorophore and a second fluorophore; a first sensor array and a second sensor array; a first light source configured to provide a first excitation beam and a second light source configured to provide a second excitation beam; and a first optical system configured to direct the first excitation beam and the second excitation beam onto the sample, wherein interaction of the first excitation beam with the first fluorophore produces a first fluorescent emission and interaction of the second excitation beam with the second fluorophore produces a second fluorescent emission. An imaging system is disclosed that includes a first optical system in which a second fluorescent radiation is generated by interaction with a second fluorophore; and a second optical system configured to direct the first fluorescent radiation to a first sensor array and the second fluorescent radiation to a second sensor array, wherein the first fluorescent radiation impinges on the first sensor array to generate a first charge that travels across the first sensor array, and the second fluorescent radiation impinges on the second sensor array to generate a second charge that travels across the second sensor array, wherein the travel of at least one of the first charge and the second charge is synchronized with a sample stage velocity. In an embodiment, the imaging system further includes a third fluorophore, a fourth fluorophore, a third sensor array, and a fourth sensor array, wherein a third fluorescent radiation is generated by interaction of the first excitation beam with the third fluorophore and a fourth fluorescent radiation is generated by interaction of the second excitation beam with the fourth fluorophore; the second optical system is configured to direct the third fluorescent radiation to the third sensor array and the fourth fluorescent radiation to the fourth sensor array, wherein the third fluorescent radiation impinges on the third sensor array to generate a third charge that travels across the third sensor array, and the fourth fluorescent radiation impinges on the fourth sensor array to generate a fourth charge that travels across the fourth sensor array, and wherein the travel of at least one of the third charge and the fourth charge is synchronized with the sample stage speed.

[0015] In another aspect, an imaging system includes a sample stage moving at a sample stage velocity, the sample stage including a sample comprising a first fluorophore (e.g., a plurality of fluorophores of a first type), a second fluorophore (e.g., a plurality of fluorophores of a second type), a third fluorophore (e.g., a plurality of fluorophores of a third type), and a fourth fluorophore (e.g., a plurality of fluorophores of a fourth type); a first sensor array, a second sensor array, a third sensor array, and a fourth sensor array; a first light source configured to provide a first excitation beam, and a second light source configured to provide a second excitation beam; and a first optical system configured to direct the first excitation beam and the second excitation beam onto the sample, wherein interaction of the first excitation beam with the first fluorophore generates a first fluorescent emission, interaction of the second excitation beam with the second fluorophore generates a second fluorescent emission, and interaction of the first excitation beam with the third fluorophore generates a second fluorescent emission. a first optical system configured to direct the first fluorescent radiation to the first sensor array, the second fluorescent radiation to the second sensor array, the third fluorescent radiation to the third sensor array, and the fourth fluorescent radiation to the fourth sensor array, wherein the first fluorescent radiation impinges on the first sensor array and generates a first charge traveling across the first sensor array. a second fluorescent radiation impinging on a second sensor array to generate a second charge that travels across the second sensor array; a third fluorescent radiation impinging on a third sensor array to generate a third charge that travels across the third sensor array; and a fourth fluorescent radiation impinging on a fourth sensor array to generate a fourth charge that travels across the fourth sensor array, wherein the travel of at least one of the first charge, the second charge, the third charge, and the fourth charge is synchronized with a sample stage velocity. In an embodiment, each fluorophore type is different.For example, the first fluorophore can be a cyanine dye (e.g., CY3B) and the second fluorophore can be a different cyanine dye (e.g., Alexa® Fluor 647). In embodiments, each fluorophore (e.g., the first fluorophore, the second fluorophore, the third fluorophore, and the fourth fluorophore) is spectrally distinct. In embodiments, each fluorophore includes an emission maximum of 405 nm, 470 nm, 488 nm, 514 nm, 520 nm, 532 nm, 561 nm, 633 nm, 639 nm, 640 nm, 800 nm, 808 nm, 912 nm, 1024 nm, or 1500 nm.

[0016] In embodiments, an aerial image is formed when fluorescent radiation impinges on the sensor array. The aerial image is an image formed by radiation in the plane of the sensor. In embodiments, the aerial image and its movement are synchronized with the sample stage. In embodiments, charge generated in the sensor by the fluorescent radiation travels across the sensor array, and the transfer of charge from one row of the sensor array to the next is synchronized with the movement of the stage. In embodiments, as the image sweeps across each sensor array (i.e., as the sample is scanned), one or more pixels of the sensor array collect charge. At specific time intervals, the charge of the pixels within each row of pixels is transferred to their adjacent rows in the same direction and speed as the sample scans. The accumulation of charge can be integrated during the entire time required for the row of charge to travel from one end of the sensor array to the other end of the sensor array within the imaging system.

[0017] In an embodiment, the first optical system is configured to direct a first excitation beam onto a first region of the sample at a first angle of incidence and a second excitation beam onto a second region of the sample at a second angle of incidence. In an embodiment, the first excitation beam and the second excitation beam are spatially separated and impinge on the sample at different locations. As a result, the fluorescent radiation generated by the first excitation beam is spatially separated from the fluorescent radiation generated by the second excitation beam. For example, the first and third fluorescent radiations generated by the first excitation beam are spatially separated from the second and fourth fluorescent radiations generated by the second excitation beam.

[0018] In some embodiments, the first optical system is configured to direct a first excitation beam toward a first region of the sample and a second excitation beam toward a second region of the sample, the first and second regions being separated by about 10 μm to about 500 μm. In some embodiments, the first and second regions are separated by about 10 μm, about 20 μm, about 30 μm, about 40 μm, about 50 μm, about 60 μm, about 70 μm, about 80 μm, about 90 μm, or about 100 μm. In some embodiments, the first and second regions are separated by about 1 μm, about 2 μm, about 3 μm, about 4 μm, about 5 μm, about 6 μm, about 7 μm, about 8 μm, about 9 μm, or about 10 μm. In embodiments, the first and second regions are separated by about 30 μm, about 35 μm, about 40 μm, about 45 μm, about 50 μm, about 55 μm, or about 60 μm. In embodiments, the first and second regions are separated by about 50 μm. In embodiments, the first and second regions are separated by about 50 μm, about 51 μm, about 52 μm, about 53 μm, about 54 μm, about 55 μm, about 56 μm, about 57 μm, about 58 μm, about 59 μm, or about 60 μm.

[0019] In embodiments, radiation (e.g., a beam of light or multiple photons) forms an image within a particular location in space, such as, for example, within the plane of an image sensor. In embodiments, the radiation beam is absorbed on the image sensor, and the image formed by this radiation within the plane of the sensor (i.e., an aerial image, using terminology from lithography) is said to be traveled across the sensor while the sample (the object giving rise to the image) is traveled across the sample stage.

[0020] In an embodiment, the second optical system includes a first optical element including a first surface configured to reflect the first fluorescent radiation toward the first sensor array and the second fluorescent radiation toward the second sensor array, and a second surface configured to reflect the third fluorescent radiation toward the third sensor array and the fourth fluorescent radiation toward the fourth sensor array. By reflecting the first fluorescent radiation and the third fluorescent radiation (generated by the first excitation beam) from the first surface and the second surface, respectively, spatial separation between the first fluorescent radiation and the third fluorescent radiation can be achieved. By reflecting the second fluorescent radiation and the fourth fluorescent radiation (generated by the second excitation beam) from the first surface and the second surface, respectively, spatial separation between the second fluorescent radiation and the fourth fluorescent radiation can be achieved.

[0021] In an embodiment, the second optical system includes a second optical element downstream from the first optical element and is configured to focus the first fluorescent radiation, the second fluorescent radiation, the third fluorescent radiation, and the fourth fluorescent radiation.

[0022] In embodiments, the second optical system includes a bandpass filter configured to selectively transmit the first fluorescent radiation, the second fluorescent radiation, the third fluorescent radiation, and the fourth fluorescent radiation. The bandpass filter selectively passes light in a wavelength range defined by a central wavelength of maximum emitted light transmission (Tmax) and a bandwidth, and blocks the passage of light outside this range. Tmax defines the percentage of emitted light transmitted at the central wavelength. In embodiments, the bandpass filter is configured to transmit excitation beams having wavelengths of 405 nm, 470 nm, 488 nm, 514 nm, 520 nm, 532 nm, 561 nm, 633 nm, 639 nm, 640 nm, 800 nm, 808 nm, 912 nm, 1024 nm, or 1500 nm.

[0023] In embodiments, the first optical element is a dichroic wedge. In embodiments, the first optical element is a dichroic mirror. In embodiments, the first optical element includes a dichroic wedge. In embodiments, the first optical element is a dichroic filter.

[0024] In an embodiment, the detection camera includes a first sensor array, a second sensor array, a third sensor array, and a fourth sensor array. In an embodiment, the detection camera includes a first sensor array and a second sensor array. In an embodiment, the detection camera includes a third sensor array and a fourth sensor array. In an embodiment, the imaging system includes one camera. In an embodiment, the imaging system includes two cameras. In an embodiment, the imaging system includes three cameras. In an embodiment, the imaging system includes four cameras. In an embodiment, the camera includes one image sensor. In an embodiment, the camera includes two image sensors. In an embodiment, the camera includes three image sensors. In an embodiment, the camera includes four image sensors. In an embodiment, each camera includes one or more image sensors. In an embodiment, the camera includes two image sensors. In an embodiment, the imaging system includes two cameras, each camera independently including two image sensors.

[0025] In embodiments, the second optical system comprises a first optical element configured to reflect the first fluorescent radiation toward the first sensor array, transmit the third fluorescent radiation toward the third sensor array (thereby providing spatial separation between the first and third fluorescent radiation), and reflect the second fluorescent radiation toward the second sensor array and transmit the fourth fluorescent radiation toward the fourth sensor array. By configuring the first optical element to reflect the first fluorescent radiation and transmit the third fluorescent radiation, spatial separation between the first and third fluorescent radiation is achieved. By configuring the second optical element to reflect the second fluorescent radiation and transmit the fourth fluorescent radiation, spatial separation between the second and fourth fluorescent radiation is achieved. In embodiments, the imaging system spatially separates the excitation beam and the emission beam.

[0026] In an embodiment, the second optical system includes a first lens downstream from the first optical element and configured to focus the first fluorescent radiation and the third fluorescent radiation, and a second lens downstream from the first optical element and configured to focus the second fluorescent radiation and the fourth fluorescent radiation.

[0027] In an embodiment, the second optical system includes a first bandpass filter configured to selectively transmit the first fluorescent radiation and the third fluorescent radiation, and a second bandpass filter configured to selectively transmit the second fluorescent radiation and the fourth fluorescent radiation.

[0028] In an embodiment, the first detector camera includes a first sensor array and a third sensor array, and the second detector camera includes a second sensor array and a fourth sensor array.

[0029] In an embodiment, the first optical element is an optical filter.In an embodiment, the first optical element is a dichroic beam splitter.

[0030] In an embodiment, each sensor array is a TDI sensor array. A sensor array refers to a device or apparatus having multiple elements that convert the energy of contacting photons into an electrical response. The term "time delay integration" or "TDI" refers to the sequential detection of different portions of a sample by different subsets of elements of a detector array, with the transfer of charge between the subsets of elements proceeding at a speed synchronized with and in the same direction as the apparent movement of the sample being imaged. For example, TDI can be implemented by scanning a sample such that a frame transport device creates continuous video images of the sample with a stack of linear arrays aligned and synchronized with the apparent movement of the sample, so that as the image moves from one line to the next, the accumulated charge moves with it. The charge accumulation can be integrated during the entire time required for a row of charge to move from one end of the sensor array to the other end of the sensor array.

[0031] In embodiments, the sensor array (e.g., a TDI sensor array) can be configured for binning. Binning increases the sensitivity of the detector array by summing charge from multiple pixels in the array into one pixel. Exemplary types of binning that can be used include horizontal binning, vertical binning, or full binning. Horizontal binning sums pairs of adjacent pixels in each line of the detector array. Vertical binning sums pairs of adjacent pixels from two lines in the array. Full binning is a combination of horizontal and vertical binning, where four adjacent pixels are summed. For example, binning can include horizontal (1x2), vertical (2x1), or combined (2x2). In embodiments, the sensor array (e.g., a TDI sensor array) is not configured for binning.

[0032] In an embodiment, a first subset of sensors of a first sensor array are activated when the first cross-section overlaps the first subset of sensors, and a second subset of sensors of the first sensor array are activated when the first cross-section overlaps the second subset of sensors. In an embodiment, a third subset of sensors of a second sensor array are activated when the second cross-section overlaps the third subset of sensors, and a fourth subset of sensors of the second sensor array are activated when the second cross-section overlaps the fourth subset of sensors. In an embodiment, a first time difference between activation of the first subset of sensors of the first sensor array and activation of the second subset of sensors is based on a first rate of advance of the first cross-section and a separation between the first subset of sensors and the second subset of sensors. In an embodiment, a second time difference between the activation of the third subset of sensors and the activation of the fourth subset of sensors of the second sensor array is based on a second rate of progression of the second cross section and a separation between the third subset of sensors and the fourth subset of sensors.

[0033] In embodiments, the sample stage moves at a speed of about 1 mm / sec to about 50 mm / sec. In embodiments, the sample stage moves at a speed of about 10 mm / sec to about 30 mm / sec. In embodiments, the sample stage moves at a speed of about 15 mm / sec to about 25 mm / sec. In embodiments, the sample stage moves at a speed of about 20 mm / sec. The sample stage is configured to receive or support a sample, such as a flow cell, reaction vessel, or other substrate, which contains one or more objects (e.g., biomolecules) to be imaged. The sample stage is configured to move along any of the x, y, and z axes, which are oriented and / or aligned with respect to the sample stage. In embodiments, the system includes a precision mounting plate. The precision mounting plate may be fabricated with alignment surfaces, such as mounting pins, grooves, slots, grommets, tabs, magnets, datum surfaces, tooling balls, or other surfaces designed to receive subassemblies or target modules.

[0034] In embodiments, each sensor array is at least 2,000 pixels wide. In embodiments, each sensor array is at least 4,000 pixels wide. In embodiments, each sensor array is at least 8,000 pixels wide. In embodiments, each sensor array is at least 12,000 pixels wide. In embodiments, each sensor array is at least 16,000 pixels wide. In embodiments, each sensor array is at least 16 pixels long. In embodiments, each sensor array is at least 32 pixels long. In embodiments, each sensor array is at least 64 pixels long. In embodiments, each sensor array is at least 128 pixels long. In embodiments, each sensor array is at least 256 pixels long. In embodiments, each sensor array is at least 8,000 pixels wide and at least 64 pixels long. In embodiments, each sensor array is at least 8,000 pixels wide and at least 128 pixels long. In embodiments, each sensor array is at least 8,000 pixels wide and at least 256 pixels long. In embodiments, each sensory array is rectangular (i.e., two sides of an isometric rectangle are longer than the other two sides). In embodiments, each sensory array is square (i.e., all four sides of an isometric rectangle are equal).

[0035] In embodiments, each sensor array is between about 1,000 pixels wide and about 20,000 pixels wide. In embodiments, each sensor array is between about 3,000 pixels wide and about 10,000 pixels wide. In embodiments, each sensor array is between about 5,000 pixels wide and about 9,000 pixels wide. In embodiments, each sensor array is about 1,000 pixels wide. In embodiments, each sensor array is about 2,000 pixels wide. In embodiments, each sensor array is about 3,000 pixels wide. In embodiments, each sensor array is about 4,000 pixels wide. In embodiments, each sensor array is about 5,000 pixels wide. In embodiments, each sensor array is about 6,000 pixels wide. In embodiments, each sensor array is about 7,000 pixels wide. In embodiments, each sensor array is about 8,000 pixels wide. In embodiments, each sensor array is about 9,000 pixels wide. In embodiments, each sensor array is approximately 10,000 pixels wide. In embodiments, each sensor array is approximately 11,000 pixels wide. In embodiments, each sensor array is approximately 12,000 pixels wide. In embodiments, each sensor array is approximately 13,000 pixels wide. In embodiments, each sensor array is approximately 2,000 pixels wide. In embodiments, each sensor array is approximately 4,000 pixels wide. In embodiments, each sensor array is approximately 8,000 pixels wide. In embodiments, each sensor array is approximately 12,000 pixels wide. In embodiments, each sensor array is approximately 16,000 pixels wide. In embodiments, each sensor array is approximately 16 pixels long. In embodiments, each sensor array is approximately 32 pixels long. In embodiments, each sensor array is approximately 64 pixels long. In embodiments, each sensor array is approximately 128 pixels long. In embodiments, each sensor array is approximately 256 pixels long. In an embodiment, each sensor array is about 8,000 pixels wide and about 64 pixels long. In an embodiment, each sensor array is about 8,000 pixels wide and about 128 pixels long. In an embodiment, each sensor array is about 8,000 pixels wide and about 256 pixels long. In an embodiment, each sensor array is about 32 pixels long.In embodiments, each sensor array is about 64 pixels long. In embodiments, each sensor array is about 256 pixels long. In embodiments, each sensor array is about 512 pixels long. In embodiments, each sensor array is about 10 to 300 pixels long. In embodiments, each sensor array is about 32 to about 256 pixels long. In embodiments, each sensor array is about 32 to about 64 pixels long.

[0036] In an embodiment, the sample stage is a motorized translation stage. In an embodiment, the motor is a stepper motor, a piezo motor, a brushless motor, a hysteresis motor, a linear motor, or a servo motor. In an embodiment, the motor is a stepper motor. In an embodiment, the stepper motor includes an integrated ball spline. In an embodiment, the motor is a stepper motor. In an embodiment, the motor is a brushless motor. In an embodiment, the motor is a hysteresis motor. In an embodiment, the motor is a linear motor. In an embodiment, the motor is a servo motor. In an embodiment, the servo motor includes a brake mechanism. In an embodiment, the motor is a Picomotor™ actuator.

[0037] In embodiments, the sample stage is configured to receive and hold a sample. In embodiments, the sample stage is configured to receive and hold a reaction vessel containing a sample (e.g., a flow cell described herein). In embodiments, the sample stage includes a position encoder, which generates a synchronization signal that synchronizes the progression of the fluorescent emission. In embodiments, the imaging system further includes an absolute encoder. The absolute encoder provides information about the position (i.e., distance) of the camera, image sensor, and / or lens relative to the sample stage and / or sample. The absolute position encoder not only provides highly repeatable positioning, but also enables recovery of previously saved positions when rescanning a previously imaged area.

[0038] In embodiments, the sample stage includes, and optionally holds, a reaction vessel, a flow cell, a substrate, or a multi-well vessel. Those skilled in the art will recognize that a flow cell or other support structure can be used with any of a variety of arrays known in the art to achieve similar results. Such arrays can be formed by randomly arranging biological components of the sample or in a predetermined pattern on a support surface by any known technique. As used herein, the term "multi-well vessel" refers to a substrate that includes a surface, the surface including multiple reaction chambers separated from one another by interstitial regions on the surface. In embodiments, the microplate has dimensions provided and described by the American National Standards Institute (ANSI) and the Society for Laboratory Automation and Screening (SLAS), e.g., the tolerances and dimensions described in ANSI SLAS1-2004(R2012), ANSI SLAS2-2004(R2012), ANSI SLAS3-2004(R2012), ANSI SLAS4-2004(R2012), and ANSI SLAS6-2012, which are incorporated herein by reference. The microplate dimensions and reaction chamber arrangements described herein may be compatible with established formats for automated laboratory equipment.

[0039] The reaction chambers may be provided as wells (alternatively referred to as reaction chambers), e.g., a multi-well vessel may contain 2, 4, 6, 12, 24, 48, 96, 384, or 1536 sample wells. In embodiments, the 96 and 384 wells are arranged in a 2:3 rectangular matrix. In embodiments, the 24 wells are arranged in a 3:8 rectangular matrix. In embodiments, the 48 wells are arranged in a 3:4 rectangular matrix. In embodiments, the reaction chambers are microscope slides (e.g., glass slides about 75 mm by about 25 mm). In embodiments, the slides are concave slides (e.g., the slides include depressions). In embodiments, the slides include a coating (e.g., poly-L-lysine, silane, carbon nanotubes, polymers, epoxy resins, or gold) to enhance adhesion of biomolecules. In embodiments, the multi-well vessel is about 5 inches by about 3.33 inches and includes a plurality of 5 mm diameter wells. In embodiments, the multiwell vessel is approximately 5 inches by approximately 3.33 inches and contains a plurality of 6 mm diameter wells. In embodiments, the multiwell vessel is approximately 5 inches by approximately 3.33 inches and contains a plurality of 7 mm diameter wells. In embodiments, the multiwell vessel is approximately 5 inches by approximately 3.33 inches and contains a plurality of 7.5 mm diameter wells. In embodiments, the multiwell vessel is approximately 5 inches by approximately 3.33 inches and contains a plurality of 7.5 mm diameter wells. In embodiments, the multiwell vessel is approximately 5 inches by approximately 3.33 inches and contains a plurality of 8 mm diameter wells. In embodiments, the multiwell vessel is a flat glass or plastic tray formed with an array of wells, each capable of holding a few to several hundred microliters of fluidic reagents and samples.

[0040] The term "well" refers to a discrete concave feature of a substrate having a surface opening completely surrounded by an interstitial region of the surface. A well can have any of a variety of shapes at its opening in the surface, including, but not limited to, circular, oval, square, polygonal, or star-shaped (i.e., a star with any number of vertices). A cross-section of a well taken perpendicular to the surface can be curvilinear, square, polygonal, hyperbolic, conical, or angular. The wells of a multi-well container are available in different shapes, such as, for example, an F-bottom, which is a flat bottom; a C-bottom, which is a bottom with minimal rounded edges; a V-bottom, which is a V-shaped bottom; or a U-bottom, which is a U-shaped bottom. In an embodiment, the wells are substantially square. In an embodiment, the wells are square. In an embodiment, the wells are F-bottom. In an embodiment, the multi-well container includes 24 substantially round, flat-bottom wells. In an embodiment, the multi-well container includes 48 substantially round, flat-bottom wells. In embodiments, the multi-well vessel contains 96 substantially round, flat-bottom wells. In embodiments, the multi-well vessel contains 384 substantially square, flat-bottom wells.

[0041] The discrete regions (i.e., features, wells) of a multi-well container can have defined locations within a regular array, which may correspond to a linear pattern, a circular pattern, a hexagonal pattern, or the like. In embodiments, the pattern of wells includes concentric circles, such as regions, spiral patterns, linear patterns, hexagonal patterns, and the like. In embodiments, the pattern of wells is arranged in a linear or hexagonal pattern. Such a regular array of regions is advantageous for detection of signals and data analysis collected from the array during analysis. These discrete regions are separated by interstitial regions. As used herein, the term "interstitial region" refers to an area within a substrate or on a surface that separates other regions of the substrate or surface. For example, an interstitial region can separate one concave feature of an array from another concave feature of the array. Two regions that are separated from each other can be discrete and do not contact each other. In another example, an interstitial region can separate a first portion of a feature from a second portion of a feature. In embodiments, the interstitial regions are continuous while the features are discrete, such as in the case of an array of wells on an otherwise continuous surface. The separation provided by the interstitial regions can be partial or complete. In embodiments, the interstitial regions have a surface material that is different from the surface material of the wells (e.g., the interstitial regions contain photoresist and the surfaces of the wells are glass). In embodiments, the interstitial regions have a surface material that is the same as the surface material of the wells (e.g., both the surface of the interstitial regions and the surface of the wells contain a polymer or copolymer).

[0042] In an embodiment, the imaging system further comprises one or more of a collimating lens, a beam shaping lens, a mirror, or a cylindrical lens.

[0043] In embodiments, the imaging system further includes one or more line generators. In embodiments, the imaging system further includes two line generators. In embodiments, one or more line generators (e.g., 2, 4, 6, 8, or 10 lines) are used to illuminate the sample. The one or more line generators may be configured to create excitation lines having a shape at the sample that is rectangular or oblong. Exemplary shapes include, but are not limited to, rectangular, elliptical, or oval. In embodiments, the one or more excitation lines contact the sample to illuminate and / or excite one or more biomolecules in the sample. In embodiments, the lines are rectangular having a height and a width. In embodiments, the height of each line is about 1, 2, 3, 4, or 5 mm. In embodiments, the height of each line is about 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9 mm, or 2.0 mm. In an embodiment, the width of each line is about 5 μm, 10 μm, 15 μm, 20 μm, 25 μm, or about 30 μm. In an embodiment, the width of each line is 1.5 mm to about 2.0 mm, and the height of each line is about 10 μm to about 30 μm. In an embodiment, the width of each line is 1.6 mm to about 1.7 mm, and the height of each line is about 15 μm to about 25 μm. In an embodiment, the width of each line is 1.7 mm, and the height of each line is about 20 μm.

[0044] In embodiments, the imaging system is located within a microfluidic device. In an aspect, a microfluidic device is provided, the microfluidic device comprising the imaging system described herein. In embodiments, the microfluidic device includes one or more reaction vessels or solid supports with which reagents interact and are imaged. Exemplary systems having fluidic components that can be readily modified for use within the systems herein include, but are not limited to, those described in U.S. Patent Nos. 8,241,573, 8,039,817, or U.S. Patent Application Publication No. 2012 / 0270305A1, each of which is incorporated herein by reference. In embodiments, the microfluidic device further includes one or more excitation lasers. In embodiments, the imaging system is located within a bioanalytical instrument. In embodiments, the bioanalytical instrument further includes a light source and an integrated fluidic system including one or more interconnected chambers, ports, and channels in fluid communication with the light source and configured to perform an analytical reaction or process. In embodiments, the devices described herein detect scattered light from a sample. In embodiments, the devices described herein detect diffracted light from a sample. In embodiments, the devices described herein detect reflected light from the sample. In embodiments, the devices described herein detect absorbed light from the sample. In embodiments, the devices described herein detect refracted light from the sample. In embodiments, the devices described herein detect transmitted light that is not absorbed by the sample. In embodiments, the devices further include at least one reservoir physically coupled to the structure. In embodiments, the reservoir is configured to store one or more reagents or to hold waste. In some embodiments, the reagents include fluids such as water, buffers (e.g., imaging buffers containing ascorbic acid), target capture reagents, or nucleic acid amplification reagents. In some embodiments, the reagent container compartments may be configured to maintain the contents of such containers at a predetermined storage temperature and / or agitate such containers to maintain the contents of the containers in solution or suspension.In embodiments, at least one reservoir contains reaction reagents, such as nucleic acid amplification reagents (e.g., polymerase and nucleotides necessary for amplification) and / or nucleic acid sequencing reagents. In embodiments, at least one reservoir includes at least one of a waste reservoir, a sequencing reagent reservoir, a clustering reagent reservoir, and a wash solution reservoir. In embodiments, the device includes multiple sequencing reagent reservoirs and clustering reagent reservoirs. In embodiments, the clustering reagent reservoir contains amplification reagents (e.g., an aqueous buffer containing enzymes, salts, and nucleotides, denaturants, crowding agents, etc.).

[0045] In embodiments, the imaging system may generate image data, for example, at a resolution of 0.1 to 50 microns, which is then transferred to a control / processing system within the bioanalytical instrument. The control / processing system may perform various operations, such as analog-to-digital conversion, scaling, filtering, and correlation of data across multiple frames, to properly and accurately image multiple sites at specific locations on the sample. The control / processing system may store the image data and ultimately transfer it to a post-processing system where the data is further analyzed. For example, further analysis may include determining nucleotide sequence information from the image data. In embodiments, the control / processing system may include hardware, firmware, and software designed to control the operation of the bioanalytical instrument. The image data may be analyzed by the bioanalytical instrument itself or may be stored for analysis by another system and at a different time after imaging. In embodiments, the camera includes an objective lens with a high numerical aperture (NA). The image data acquired by the optical assembly may have a resolution of 0.1 to 50 microns, or more specifically, 0.1 to 10 microns. In embodiments, the numerical aperture of the camera is at least 0.2. In embodiments, the camera has a numerical aperture of 0.8 or less. In embodiments, the camera has a numerical aperture of 0.5 or less. The imaging systems described herein may have a resolution sufficient to individually resolve features or regions separated by distances of 10 μm, 5 μm, 2 μm, 1.5 μm, 1.0 μm, 0.8 μm, 0.5 μm, or less. In embodiments, the imaging systems described herein may have a resolution sufficient to individually resolve features or regions separated by distances of up to 100 μm. Depending on the sample, e.g., microwells or nanowells in a multi-well container, the imaging systems described herein may be configured for wide-field detection. The field of view diameter of the imaging system may be, for example, at least 0.5 mm, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, or more. By selecting suitable optical components, the field of view diameter can similarly be limited to a maximum area, such that the field of view diameter can be, for example, 5 mm, 4 mm, 3 mm, 2 mm, or 1 mm or less.For example, in embodiments, the image acquired by the imaging system may have an area in the range of about 0.25 mm 2 to about 25 mm 2 .

[0046] In another aspect, a method of imaging a sample is disclosed that includes: a) directing a first excitation beam and a second excitation beam onto the sample, the sample being on a sample stage moving at a sample stage velocity, the sample including a first fluorophore that generates a first fluorescent emission after interaction with the first excitation beam and a second fluorophore that generates a second fluorescent emission after interaction with the second excitation beam, respectively; b) directing the first fluorescent emission to impinge on a first sensor array and generate a first charge that travels across the first sensor array at a first charge velocity, and directing the second fluorescent emission to impinge on a second sensor array and generate a second charge that travels across the second sensor array at a second charge velocity, wherein at least one of the first charge velocity and the second charge velocity is synchronized with the sample stage velocity; and c) scanning the sample in a scan dimension and repeating steps a) and b) to form an image of the sample. In an embodiment, the sample further includes a third fluorophore that generates a third fluorescent emission after interaction with the first excitation beam and a fourth fluorophore that generates a fourth fluorescent emission after interaction with the second excitation beam, and the third fluorescent emission is directed to impinge on a third sensor array and generate a third charge that travels across the third sensor array at a third charge velocity, and the fourth fluorescent emission is directed to impinge on a fourth sensor array and generate a fourth charge that travels across the fourth sensor array at a fourth charge velocity. In an embodiment, the sum of the charges in each pixel travels across the sensor array.

[0047] The sample stage is configured to move along any of the x, y, and z axes, which are oriented and / or aligned with respect to the sample stage. To establish a standard coordinate system and frame of reference, it is useful to provide a description of the axes. In a traditional description of three-dimensional space using Cartesian coordinates, there are six degrees of freedom. Each degree of freedom corresponds to a translation and rotation along three perpendicular axes: X, Y, and Z. The first degree of freedom can be defined as moving left and right along the X axis. The second degree of freedom can be defined as moving forward and backward along the Y axis. The third degree of freedom can be defined as moving up and down along the Z axis. The fourth degree of freedom can be defined as rotation around the "roll" axis, which is alternatively referred to as the X axis, or the longitudinal axis. The fifth degree of freedom can be defined as rotation around the "pitch" axis, which is alternatively referred to as the lateral axis. Pitch and roll, used interchangeably throughout, can also be referred to as tip and tilt. The sixth degree of freedom can be defined as rotation about the Z-axis, or "yaw." A plane refers to a two-dimensional (2D) area defined by two axes (e.g., x and y together form the xy plane). When used in reference to a detection device (e.g., an image sensor) and an object (e.g., a sample) observed by the detector, the xy plane can be defined as orthogonal to the direction of observation between the detector and the detected object. An image plane is a projection of an image onto a two-dimensional plane. For example, in an embodiment, the image plane is a projection of an image onto the surface of the image sensor. In an embodiment, the scan axis is the x-axis. In an embodiment, the scan axis is the y-axis.

[0048] In embodiments, the travel speed of the first radiation and the travel speed of the second radiation are synchronized with the sample stage speed. Synchronizing the sample stage with the travel of the charge across the array allows for higher precision and more accurate imaging (e.g., versus controls such as not synchronizing the sample stage). Controlling the flow rate of the sample when there are many different biomolecules and components in the sample that modulate viscosity and thermal sensitivity is undesirable due to the challenges associated with accurately controlling the flow rate and maintaining synchronization.

[0049] In embodiments, the method includes illuminating the sample and detecting light from the sample (e.g., fluorescent excitation events, scattered light, transmitted light, or reflected light) using an imaging system described herein. In embodiments, the method includes scanning the sample (i.e., translating the sample relative to a camera). In embodiments, the method includes illuminating the sample to generate fluorescent events and detecting one or more fluorescent events using an imaging system described herein. In embodiments, the method includes detecting clusters (e.g., amplified colonies of nucleic acids) on a solid support. In embodiments, the method includes detecting fluorescently labeled nucleotides incorporated into template nucleic acids. In embodiments, the method includes sequencing one or more nucleic acid templates. In embodiments, the method includes amplifying one or more nucleic acid templates on a solid support, thereby generating clusters or colonies of a plurality of nucleic acid templates at the feature. In embodiments, the method includes incorporating a labeled nucleotide into a primer hybridized to one or more of the nucleic acid templates and detecting the incorporated nucleotide. In embodiments, the detecting includes imaging the feature using an imaging system described herein. In embodiments, the sample includes one or more biomolecules. A variety of biomolecules can be present in a sample. Exemplary biomolecules include, but are not limited to, nucleic acids such as DNA or RNA, proteins such as enzymes or receptors, polypeptides, nucleotides, amino acids, sugars, coenzymes, metabolites, or derivatives of these naturally occurring components. While the systems and methods described herein relate to biomolecules, it will be understood that other samples or components can be used as well. For example, synthetic samples can be used, such as combinatorial libraries or libraries of compounds with species known or suspected to have a desired structure or function. In embodiments, the sample includes one or more fluorescent labels. In embodiments, the sample includes one or more fluorescently labeled biomolecules.

[0050] In embodiments, the method includes imaging a sample containing four different fluorophores simultaneously, i.e., collecting fluorescence emission information for the four different fluorophores simultaneously.

[0051] In embodiments, the first excitation beam and the second excitation beam comprise excitation lines (e.g., excitation beams provided by line generators). In embodiments, one or more line generators (e.g., 2, 4, 6, 8, or 10 lines) are used to illuminate the sample. The one or more line generators may be configured to create excitation lines having a shape at the sample that is rectangular or oblong. Exemplary shapes include, but are not limited to, rectangular, elliptical, or oval. In embodiments, the one or more excitation lines contact the sample to illuminate and / or excite one or more biomolecules in the sample.

[0052] In embodiments, scanning the sample includes moving a sample stage. In embodiments, scanning the sample includes moving the sample stage at a constant velocity. The sample stage movement can occur in one or more dimensions, including, for example, one or both of the dimensions orthogonal to the direction of propagation of the fluorescent radiation, typically denoted as the x and y dimensions. The system may further include a scanning element, which may be a mechanical component, an electromechanical component, a software component, or a combination thereof, configured to scan the sample along a direction, which may correspond to the scan direction. In embodiments, the scan direction is orthogonal to the excitation direction of the sample. In embodiments, the scan direction is non-orthogonal to the excitation beam direction, and orthogonal projected components directly contribute to the final image reconstruction. The term "scanning element" is intended to mean an element capable of sequentially detecting different portions of the sample. The scanning element can operate by changing the position of one or more components of the system, including, for example, the light source, the objective lens, the image sensor, or the sample. Exemplary scanning elements include, but are not limited to, a galvanometer configured to move the beam (e.g., excitation beam) across the sample or a translation stage configured to move the sample across the beam. In embodiments, the sample is scanned at about 1 mm / sec, 1.5 mm / sec, 5 mm / sec, 10 mm / sec, 50 mm / sec, or 100 mm / sec. In embodiments, the sample is scanned at 10 mm / sec, 20 mm / sec, 30 mm / sec, 40 mm / sec, or 50 mm / sec. In embodiments, the sample is scanned at about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mm / sec. In embodiments, the sample is scanned at about 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mm / sec. In an embodiment, the sample is scanned at at least 20 mm / sec. In an embodiment, the camera is dynamically adjusted as the sample is scanned (e.g., continuously scanned in a scan axis, such as the x-axis).In embodiments, the camera is initially adjusted (e.g., during a configuration or first cycle of a series of cyclical experiments) as the sample is scanned (e.g., sequentially scanned in a scan axis such as the x-axis). For example, when scanning a sample stage over a series of imaging cycles (e.g., sequencing cycles), an initial configuration scan of the sample adjusts the camera to maximize focus on the sample, and for the remaining imaging cycles, the camera orientation and / or position remains stationary.

[0053] In an embodiment, the method further comprises storing a data representation of the image of the sample in a computer readable memory.

[0054] In embodiments, the excitation beam comprises UV radiation, VIS radiation, or IR radiation, hi embodiments, the excitation beam comprises an excitation beam having a wavelength of 405 nm, 470 nm, 488 nm, 514 nm, 520 nm, 532 nm, 561 nm, 633 nm, 639 nm, 640 nm, 800 nm, 808 nm, 912 nm, 1024 nm, or 1500 nm.

[0055] In embodiments, the illuminator or light source is a radiation source (i.e., a source or generator of propagated electromagnetic energy) that provides incident light to the sample. Radiation sources can include irradiation sources that produce electromagnetic radiation in the ultraviolet (UV) range (approximately 200-390 nm), visible (VIS) range (approximately 390-770 nm), or infrared (IR) range (approximately 0.77-25 microns), or other ranges of the electromagnetic spectrum. In embodiments, the illuminator or light source is a lamp, such as an arc lamp or a quartz halogen lamp. In embodiments, the illuminator or light source is a coherent light source. In embodiments, the light source is a laser, an LED (light emitting diode), a mercury or tungsten lamp, or a supercontinuous diode. In embodiments, the light source provides an excitation beam having a wavelength between 200 nm and 1500 nm. In embodiments, the laser provides an excitation beam having a wavelength of 405 nm, 470 nm, 488 nm, 514 nm, 520 nm, 532 nm, 561 nm, 633 nm, 639 nm, 640 nm, 800 nm, 808 nm, 912 nm, 1024 nm, or 1500 nm. In embodiments, the laser provides an excitation beam having a wavelength of 405 nm, 488 nm, 532 nm, or 633 nm.

[0056] In embodiments, the light source provides one or more excitation beams. Excitation beam is intended to mean electromagnetic energy propagated toward a sample or sample region. The excitation beam may be shaped so that a collection of electromagnetic waves or particles propagates in a uniform direction, with a two-dimensional cross section perpendicular to the propagation direction being rectangular or oblong. Exemplary two-dimensional cross sections of the excitation beam may include rectangular, elliptical, or oval shapes. The cross-sectional width of the excitation beam may have one or both dimensions ranging, for example, from about 0.5 μm to about 50 μm. For example, the excitation beam dimensions may be at least about 0.05 μm, 0.1 μm, 0.5 μm, 1 μm, 5 μm, or 10 μm. Furthermore, the excitation beam dimensions may be, for example, at most about 0.1 μm, 0.2 μm, 0.5 μm, 1 μm, 5 μm, or 10 μm. In embodiments, the excitation beam dimensions are from about 0.2 μm to about 50 μm. In embodiments, the excitation beam has a dimension of about 10 μm to about 30 μm. In embodiments, the excitation beam has a dimension of about 20 μm to about 30 μm. In embodiments, the excitation beam has a dimension of about 20 μm. It should be understood that these dimensions are merely exemplary and excitation beams having other dimensions can be used as desired.

[0057] In embodiments, the light source is a laser (e.g., a laser such as a solid-state laser or a gas laser). In embodiments, the light source includes one or more vertical cavity surface-emitting lasers (VCSELs), vertical external cavity surface-emitting lasers (VECSELs), or diode-pumped solid-state (DPSS) lasers. In embodiments, the light source is a continuous wave (CW) laser or a pulsed laser. In embodiments, the light source is a pulsed laser. In embodiments, the light source is an ultrashort pulsed laser. An ultrashort laser is a laser that can produce an excitation beam for a duration of picoseconds or less. An ultrashort laser typically includes additional components such as a pulse controller, a pulse shaper, and a spatial light modulator to control the pulses of the excitation beam. In embodiments, the ultrashort laser provides an excitation beam for a duration of femtoseconds or picoseconds. In embodiments, the light source is a femtosecond or picosecond pulsed laser. In embodiments, the laser is a titanium sapphire laser, a dye laser, or a fiber laser. In embodiments, the system includes two or more light sources (e.g., lasers). In an embodiment, the first light source is configured to emit light at a red wavelength and the second light source is configured to emit light at a green wavelength. In an embodiment, the system includes two or more lasers.

[0058] In embodiments, the sample comprises modified nucleotides (e.g., nucleotides comprising a reversible terminator and / or a label). In embodiments, the sample comprises nucleotides attached to a substrate. In embodiments, the sample comprises a surface-immobilized polynucleotide (e.g., a polynucleotide primer or polynucleotide template covalently attached to a substrate). In embodiments, the 5' end of the polynucleotide contains a functional group that is tethered to a solid support. Non-limiting examples of covalent linkages include amine-modified polynucleotides reacting with epoxy or isothiocyanate groups on a solid support, succinylated polynucleotides reacting with aminophenyl or aminopropyl functional groups on a solid support, dibenzocycloctine-modified polynucleotides reacting with azide functional groups on a solid support (or vice versa), transcyclooctyne-modified polynucleotides reacting with tetrazine or methyltetrazine groups on a solid support (or vice versa), disulfide-modified polynucleotides reacting with mercapto functional groups on a solid support, and the like. These include thiol-modified polynucleotides, amine-functionalized polynucleotides that react with carboxylic acid groups on the core via 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) chemistry, thiol-modified polynucleotides that attach to the solid support via a disulfide or maleimide bond, alkyne-modified polynucleotides that attach to the solid support via a copper-catalyzed click reaction to azide functional groups on the solid support, and acrydite-modified polynucleotides that are polymerized on the solid support with free acrylic acid monomers to form polyacrylamide or react with thiol groups on the solid support.

[0059] In embodiments, the substrate is glass or quartz, such as a microscope slide, with a uniformly silanized surface. This can be achieved using conventional protocols, e.g., Beattie et al. (1995), Molecular Biotechnology, 4:213. Such surfaces are easily treated to allow terminal attachment of oligonucleotides (e.g., forward and reverse primers, and / or splint primers) prior to amplification. In embodiments, the solid support surface further comprises a polymer coating containing functional groups capable of immobilizing polynucleotides. In some embodiments, the solid support comprises a patterned surface suitable for immobilizing polynucleotides in an ordered pattern. A patterned surface refers to the arrangement of different regions in or on the exposed layer of the solid support. For example, one or more of the regions can be features in which one or more primers are present. The features can be separated by interstitial regions in which no capture primers are present. In some embodiments, the pattern can be an xy format of features in rows and columns. In some embodiments, the pattern can be a repeating arrangement of features and / or interstitial regions. In some embodiments, the pattern can be a random arrangement of features and / or interstitial regions. In some embodiments, the primers are randomly distributed on the solid support. In some embodiments, the primers are distributed on a patterned surface.

[0060] In embodiments, the sample comprises an array having a plurality of individual sites (e.g., a microarray or multi-well container). A typical microarray contains sites, sometimes referred to as features, each containing a population of targets. The sites or features of the array are typically discrete and separated by spaces between them. The size of the features and the spacing between features can be varied such that the array can be high-density, medium-density, or low-density. High-density arrays are characterized by sites separated by less than about 15 μm. Medium-density arrays have sites separated by about 15-30 μm, while low-density arrays have sites separated by more than 30 μm. In embodiments, the sample is an array containing features separated by less than 100 μm, 50 μm, 10 μm, 5 μm, 1 μm, or 0.5 μm. Other exemplary samples include, but are not limited to, biological specimens (e.g., nucleic acids, proteins, cells, viruses, or tissues), nanoparticles, or electronic chips (e.g., microprocessor chips). A sample refers to an object or location intended to be detected. In embodiments, a sample includes multiple distinct features that are targets for imaging. In some embodiments, a sample includes a non-planar structure having a surface, such as a bead or well, to which target nucleic acids are attached as target features. In embodiments, the sample is held by a sample holder. The sample holder can be a multiwell plate. In some instances, the multiwell plate has 16, 24, 48, 96, 384, or more sample wells. In some of these instances, the array of light sources, e.g., LEDs, has 16, 24, 48, 96, 384, or more corresponding light sources. In some instances, the multiwell plate is a standard microwell plate for biological analysis. In embodiments, the sample holder is at least internally coated with a material to prevent adhesion of biological materials to the sample holder, such as a fluoropolymer or BSA. In embodiments, the sample includes genomic material that can be sequenced. In embodiments, the sample includes labeled nucleotides, e.g., nucleotides containing different labels that correspond to different wavelengths of light. The labels can be, for example, fluorescent, chemiluminescent, or bioluminescent labels.For example, in gene sequencing (or DNA sequencing), embodiments can be used to determine the precise order of nucleotide bases within a nucleic acid polynucleotide (e.g., a strand of DNA). The nucleotide bases can be labeled with specific fluorescent labels (e.g., adenine (A), guanine (G), cytosine (C), or thymine (T)). Alternatively, for example, one-color, two-color, or three-color sequencing methods can be used. With regard to fluorescence, each of the nucleotide bases can be determined in turn by sequentially exciting the nucleic acid with excitation light. The nucleic acid can absorb the excitation light and transmit emitted light of different wavelengths onto an image sensor, as described herein. The image sensor can measure the wavelength of the emitted light and the intensity received by the photodiode. When excited by excitation light of a specific wavelength and / or intensity, each nucleotide (e.g., a fluorescently labeled nucleotide) can emit light of a specific wavelength and / or intensity to the image sensor, allowing identification of the presence of a specific nucleotide base at a specific position in the nucleic acid. Once that particular nucleotide base is determined, it can be removed from the nucleic acid so that the next consecutive nucleotide base can be determined following a similar process.

[0061] In embodiments, the sample comprises a microplate array comprising a substrate comprising a surface, the surface comprising a plurality of wells separated from one another by interstitial regions on the surface, one or more wells comprising a sample (e.g., a cell or tissue sample), particles, or nucleic acid. In embodiments, the sample comprises cells. In embodiments, the sample comprises particles. In embodiments, the sample comprises nucleic acid. In embodiments, the sample is a tissue sample. In embodiments, the sample comprises cells. In embodiments, the surface is substantially free of oligonucleotides. In embodiments, the microplate array comprises 2, 4, 6, 12, 24, 48, 96, 384, or 1536 wells. In embodiments, the microplate array comprises 24, 48, 96, or 384 wells. In embodiments, the microplate array comprises 24 wells. In embodiments, the microplate array comprises 48 wells. In embodiments, the microplate array comprises 96 wells. In embodiments, the microplate array comprises 384 wells. In embodiments, the dimensions of the microplate comply with standards provided by the American National Standards Institute (ANSI) and the Society for Laboratory Automation and Screening (SLAS), such as the tolerances and dimensions set forth in ANSI SLAS1-2004(R2012), ANSI SLAS2-2004(R2012), ANSI SLAS3-2004(R2012), ANSI SLAS4-2004(R2012), and ANSI SLAS 6-2012. In embodiments, the microplate has a rectangular shape, measuring 127.7 mm±0.5 mm long by 85.4 mm±0.5 mm wide, and contains 6, 12, 24, 48, or 96 wells. In embodiments, the microplate has a rectangular shape, measuring 127.7 mm ± 0.5 mm long by 85.4 mm ± 0.5 mm wide, and contains 6, 12, 24, 48, or 96 wells, each with an average diameter of about 5-7 mm.In embodiments, the microplate has a rectangular shape, 127.7 mm ± 0.5 mm long by 85.4 mm ± 0.5 mm wide, and contains 6, 12, 24, 48, or 96 wells, each with an average diameter of about 6 mm.

[0062] A general overview of an exemplary workflow is provided in Figures 1 and 2. Described herein is an imaging system that includes at least one CCD-CMOS sensor array (alternatively referred to herein as a TDI array or hybrid TDI line-scan sensor). Hybrid TDI sensors (e.g., CCD-CMOS sensor arrays) combine CCD pixel structures with CMOS technology, enabling more sensitive, ultra-fast image capture compared to traditional CCD and / or traditional CMOS sensors. In embodiments, the CCD-CMOS sensor array captures multiple (e.g., hundreds of) lines, each line capturing successive snapshots of the sample as it passes, enabling the accumulation of multiple images that can result in very low-noise images of dark or difficult-to-image objects.

[0063] In embodiments, the sample is an array (e.g., a microarray). A typical microarray contains sites, sometimes referred to as features, each having a population of targets. The sites or features of an array are typically discrete and separated by spaces between them. The size of the features and the spacing between features can be varied such that the array can be high-density, medium-density, or low-density. High-density arrays are characterized by sites separated by less than about 15 μm. Medium-density arrays have sites separated by about 15-30 μm, while low-density arrays have sites separated by more than 30 μm. In embodiments, the sample is an array containing features separated by less than 100 μm, 50 μm, 10 μm, 5 μm, 1 μm, or 0.5 μm. Other exemplary samples include, but are not limited to, biological specimens (e.g., nucleic acids, proteins, cells, viruses, or tissues), nanoparticles, or electronic chips (e.g., microprocessor chips). In embodiments, the sample comprises a microplate array comprising a substrate comprising a surface, the surface comprising a plurality of wells separated from one another by interstitial regions on the surface, one or more wells comprising a sample (e.g., a cell or tissue sample), particles, or nucleic acid. In embodiments, the sample comprises cells. In embodiments, the sample comprises particles. In embodiments, the sample comprises nucleic acid. In embodiments, the sample is a tissue sample. In embodiments, the sample comprises cells. In embodiments, the surface is substantially free of oligonucleotides. In embodiments, the microplate array comprises 2, 4, 6, 12, 24, 48, 96, 384, or 1536 wells. In embodiments, the microplate array comprises 24, 48, 96, or 384 wells. In embodiments, the microplate array comprises 24 wells. In embodiments, the microplate array comprises 48 wells. In embodiments, the microplate array comprises 96 wells. In embodiments, the microplate array comprises 384 wells.In embodiments, the dimensions of the microplate comply with standards provided by the American National Standards Institute (ANSI) and the Society for Laboratory Automation and Screening (SLAS), such as the tolerances and dimensions set forth in ANSI SLAS1-2004(R2012), ANSI SLAS2-2004(R2012), ANSI SLAS3-2004(R2012), ANSI SLAS4-2004(R2012), and ANSI SLAS 6-2012. In embodiments, the microplate has a rectangular shape, measuring 127.7 mm±0.5 mm long by 85.4 mm±0.5 mm wide, and contains 6, 12, 24, 48, or 96 wells. In embodiments, the microplate has a rectangular shape, measuring 85.4 mm ± 0.5 mm wide and 127.7 mm ± 0.5 mm long, and contains 6, 12, 24, 48, or 96 wells, each with an average diameter of about 5-7 mm. In embodiments, the microplate has a rectangular shape, measuring 127.7 mm ± 0.5 mm long and 85.4 mm ± 0.5 mm wide, and contains 6, 12, 24, 48, or 96 wells, each with an average diameter of about 6 mm.

[0064] In an embodiment, the imaging system includes one camera and one CCD-CMOS sensor array per channel. In an embodiment, the imaging system includes one camera and four CCD-CMOS sensors. In an embodiment, the imaging system includes two cameras and two CCD-CMOS sensor arrays. In an embodiment, the imaging system architecture includes an upright, infinity-corrected epifluorescence microscope using two cameras, each responsible for capturing two of the four color channels. In an embodiment, the imaging path includes a beam splitter that routes the four fluorescent bands to the first camera or the second camera. In an embodiment, each branch of the imaging path includes a dual bandpass filter specifically designed to transmit the pair of color channels intended for that branch and block excitation light and out-of-band fluorescence.

[0065] A typical implementation of multicolor image acquisition relies on two sequential exposures with red and green excitation light to capture images in multiple channels. In contrast, the imaging system described herein images all channels in parallel and relies on spatial separation of excitation light instead of temporal separation; see Figures 1 and 2. An advantage of the systems and methods described herein is that they provide rapid and efficient detection of multiple target nucleic acids in parallel.

[0066] In a first configuration workflow, as shown in FIG. 1 , a first light source 101 provides a first excitation beam 103, and a second light source 102 provides a second excitation beam 104. The excitation beam 103 is directed to a dichroic filter 140 by mirrors 112, 113, and lens 110. The excitation beam 104 is directed to a dichroic filter 140 by mirrors 111, 113, and lens 110. The dichroic filter 140 directs the excitation beams 103 and 104 through an objective lens 130 and onto a sample 120. Interaction of the excitation beams with multiple fluorophores in the sample generates fluorescence emissions 105, 106, 107, and 108. The fluorescence emissions 105, 106, 107, and 108 are reflected by a dichroic wedge 141 and transmitted through a tube lens 115 and further through a bandpass filter 145. Fluorescent radiation 105 is transmitted toward sensor array 151, fluorescent radiation 106 is transmitted toward sensor array 152, fluorescent radiation 107 is transmitted toward sensor array 153, and fluorescent radiation 108 is transmitted toward sensor array 154. In some implementations, mirrors 111 and 112 can be oriented with respect to first excitation beam 103 and second excitation beam 104, respectively, such that first excitation beam 103 and second excitation beam 104 are spatially separated. This can allow first excitation beam 103 to impinge on a first location of sample 120 and second excitation beam 104 to impinge on a second location of sample 120. As a result, fluorescent radiation 105 and 106 (produced by excitation beam 103) are spatially separated from radiation 107 and 108 (produced by excitation beam 104). In an embodiment, the first excitation beam 103 and the second excitation beam 104 are spatially separated by about 10 μm to about 500 μm. In an embodiment, the first excitation beam 103 and the second excitation beam 104 are spatially separated by about 10 μm, about 20 μm, about 30 μm, about 40 μm, about 50 μm, about 60 μm, about 70 μm, about 80 μm, about 90 μm, or about 100 μm.In embodiments, the first excitation beam 103 and the second excitation beam 104 are spatially separated by about 1 μm, about 2 μm, about 3 μm, about 4 μm, about 5 μm, about 6 μm, about 7 μm, about 8 μm, about 9 μm, or about 10 μm. In embodiments, the first excitation beam 103 and the second excitation beam 104 are spatially separated by about 30 μm, about 35 μm, about 40 μm, about 45 μm, about 50 μm, about 55 μm, or about 60 μm. In embodiments, the first excitation beam 103 and the second excitation beam 104 are spatially separated by about 50 μm. In embodiments, the first excitation beam 103 and the second excitation beam 104 are spatially separated by about 50 μm, about 51 μm, about 52 μm, about 53 μm, about 54 μm, about 55 μm, about 56 μm, about 57 μm, about 58 μm, about 59 μm, or about 60 μm.

[0067] In the second configuration workflow, as shown in FIG. 2 , a first light source 201 provides a first excitation beam 203, and a second light source 202 provides a second excitation beam 204. Excitation beam 203 is directed to dichroic filter 240 by mirror 212, mirror 213, and lens 210. Excitation beam 204 is directed to dichroic filter 240 by mirror 211, mirror 213, and lens 210. Dichroic filter 240 directs excitation beam 203 and excitation beam 204 through objective lens 230 and onto sample 220. Interaction of the excitation beams with multiple fluorophores in the sample generates fluorescence emissions 205, 206, 207, and 208. Fluorescence emissions 205 and 207 are reflected by dichroic beam splitter 241 through tube lens 215 and further through bandpass filter 245. Fluorescent radiation 205 is transmitted toward sensor array 251, and fluorescent radiation 207 is transmitted toward sensor array 253. Fluorescent radiation 206 and 208 are reflected by dichroic beam splitter 241 through tube lens 216 and bandpass filter 246. Fluorescent radiation 206 is transmitted toward sensor array 252, and fluorescent radiation 208 is transmitted toward sensor array 254. In some implementations, mirrors 211 and 212 can be oriented relative to first excitation beam 203 and second excitation beam 204, respectively, such that first excitation beam 203 and second excitation beam 204 are spatially separated. This can allow first excitation beam 203 to impinge on sample 220 at a first location and second excitation beam 204 to impinge on sample 220 at a second location. As a result, fluorescent emissions 205 and 206 (produced by excitation beam 203) are spatially separated from emissions 207 and 208 (produced by excitation beam 204).

[0068] In embodiments, the objective lens is a microscope objective lens. Exemplary telecentric objective lenses useful in the present invention include those described in U.S. Patent No. 5,847,400, which is incorporated herein by reference. In embodiments, the objective lens is an air objective lens. In embodiments, the objective lens is an immersion objective lens. In embodiments, the objective lens has a large numerical aperture (NA) (e.g., an NA in the range of 0.95 to 1.5) and images via air immersion or liquid immersion (e.g., water, oil, or other immersion fluid). For example, the NA may be at least about 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, or higher. Those skilled in the art will appreciate that the NA, depending on the refractive index of the medium in which the lens operates, may be higher, including, for example, up to 1.0 for air, 1.33 for pure water, or higher for other media such as oil. However, other embodiments may have lower NA values ​​than the above examples. The image data acquired by the optical assembly may have a resolution of 0.1 to 50 microns, or more specifically, 0.1 to 10 microns. In embodiments, the numerical aperture of the camera is at least 0.2. In embodiments, the numerical aperture of the camera is 0.8 or less. In embodiments, the numerical aperture of the camera is 0.5 or less. The imaging systems described herein may have a resolution sufficient to individually resolve features or regions separated by distances of 10 μm, 5 μm, 2 μm, 1.5 μm, 1.0 μm, 0.8 μm, 0.5 μm, or less. In embodiments, the imaging systems described herein may have a resolution sufficient to individually resolve features or regions separated by distances of up to 100 μm.

[0069] In an embodiment, each camera includes at least one CCD-CMOS sensor array. The light-sensitive pixels are CCD, allowing noiseless charge transfer from line to line. The readout circuitry is CMOS, reading out the columns in parallel, allowing for high line speeds. Each line successively captures snapshots of a passing object, allowing for the accumulation of multiple images, resulting in very low-noise images of dark or difficult-to-image objects. Combined with the power efficiency of CMOS technology, CCD-CMOS sensors allow for ultra-high-speed imaging with excellent light sensitivity.

[0070] In an embodiment, the CCD-CMOS sensor array is at least 8,000 pixels wide. In an embodiment, the CCD-CMOS sensor array is at least 64 pixels long. In an embodiment, the CCD-CMOS sensor array is at least 128 pixels long. In an embodiment, each pixel has a width and a height in two dimensions (e.g., corresponding to the x and y axes). In an embodiment, each pixel is 3 μm wide. In an embodiment, each pixel is 4 μm wide. In an embodiment, each pixel is 5 μm wide. In an embodiment, each pixel is 2 μm wide. In an embodiment, each pixel is 1 μm wide. In an embodiment, each pixel is 3 μm high. In an embodiment, each pixel is 4 μm high. In an embodiment, each pixel is 5 μm high. In an embodiment, each pixel is 2 μm high. In an embodiment, each pixel is 1 μm high. In an embodiment, the sensor array comprises a plurality of pixels. In an embodiment, each pixel is approximately square (i.e., each pixel has equal horizontal and vertical sampling pitches). In an embodiment, each pixel is approximately rectangular (i.e., each pixel has unequal horizontal and vertical sampling pitches, resulting in an oblong shape).

[0071] In an embodiment, a sensor array generates an output signal. The output signal of an image sensor originates from electrons generated by light incident (e.g., radiation) on the photodiode of each pixel. The output voltage of the output signal depends on the migration of signal electrons from the photodiode to a readout node. The shape and size of the photodiode are known to control the charge migration speed. For example, large photodiodes reduce the lateral electric field that pushes electrons into the transfer transistor, making it difficult to fully extract the generated electrons. Adjusting the shape of the photodiode is known to enhance migration (e.g., a triangular-shaped photodiode increases the lateral electric field).

[0072] In an embodiment, the CCD-CMOS sensor array consists of rows of pixels extending transverse to the scan direction and is capable of transferring charge from one row to the next at a rate determined by an input synchronization signal. The synchronization signal is generated by a position encoder of a motion stage that moves the object to be imaged (e.g., a sample) under an imaging lens. For example, the sample is translated under the objective lens at a constant speed. An excitation source illuminates a rectangular area on the sample. As the scan progresses, a fluorescent image of a specific object in the sample is translated onto the camera chip (e.g., a CCD-CMOS sensor array) at a speed that is double the scan speed of the stage. In an embodiment, the sample is scanned at approximately 1 mm2 / s, 1.5 mm2 / s, 5 mm2 / s, 10 mm2 / s, 50 mm2 / s, or 100 mm2 / s. In an embodiment, the sample is scanned at 10 mm2 / s, 20 mm2 / s, 30 mm2 / s, 40 mm2 / s, or 50 mm2 / s. In an embodiment, the sample is scanned at at least 20 mm2 / sec.

[0073] In embodiments, the system may further include a scanning element, which may be a mechanical component, an electromechanical component, a software component, or a combination thereof configured to scan the sample along a direction, which may correspond to the scan direction. In embodiments, the scan direction is orthogonal to the excitation direction of the sample. In embodiments, the scan direction is non-orthogonal to the excitation beam direction, and orthogonal projected components directly contribute to the final image reconstruction. The term "scanning element" is intended to mean an element capable of sequentially detecting different portions of the sample. The scanning element may operate by changing the position of one or more components of the system, including, for example, the light source, the objective lens, the image sensor, or the sample. Exemplary scanning elements include, but are not limited to, a galvanometer configured to move a beam (e.g., an excitation beam) across the sample or a translation stage configured to move the sample across the beam.

[0074] TDI array (e.g., CCD-CMOS sensor array) imaging systems can also be configured to sequentially detect different portions of a sample with different subsets of elements of the detector array, with the transfer of charge between the subsets of elements proceeding in the same direction and at a speed synchronized with the apparent motion of the sample being imaged. For example, a CCD-CMOS sensor array imaging system can scan a sample such that a frame transfer device creates a continuous video image of the sample with a stack of linear arrays aligned and synchronized with the apparent motion of the sample, so that as the image moves from one line to the next, the accumulated charge moves with it. The charge accumulation can be integrated during the entire time required for a row of charge to move from one end of the detector to a serial register (or, in the case of a frame transfer CCD, to a storage area of ​​the device).

[0075] The system may also include other components, including a group of lenses (such as collimating lenses, beam-shaping lenses (e.g., Powell lenses), and cylindrical lenses), mirrors (e.g., dichroic mirrors), beam splitters, one or more pinhole apertures, excitation filters, or combinations thereof. For example, the direction, size, and / or polarization of the light source may be adjusted using lenses, mirrors, and / or polarizers. In embodiments, one or more of the system's components may be adjusted or operated automatically. The automatic control device may include a motorized translation stage, an actuation device, one or more piezo stages, and / or one or more automatic switches and flipping mirrors and lenses. In embodiments, the system includes one or more optical components (e.g., beam-shaping lenses) configured to shape light emitted from one or more light sources into a desired pattern. For example, in some embodiments, the optical component may shape the light into a line pattern (e.g., by using one or more Powell lenses or other beam-shaping lenses, diffractive components, or scattering components). In embodiments, the optical component includes a line generator.

[0076] In embodiments, the optical component includes a Powell lens, a microlens, or a microlens array. In embodiments, the optical component includes a microlens fabricated on glass, metal, or plastic. In embodiments, the excitation beam may be directed through one beam-shaping lens or multiple beam-shaping lenses. In some embodiments, a single beam-shaping lens may be used to shape excitation beams output from multiple light sources (e.g., two light sources). In some embodiments, a separate beam-shaping lens may be used for each light beam. In embodiments, the beam-shaping lens is a Powell lens, alternatively referred to as a Powell prism. The shape of the beam may be shaped into an appropriate geometric shape according to known techniques, e.g., line, cone, hyper-Gaussian, ring, donut, Bessel-Gaussian, Hermite-Gaussian, Laguerre-Gaussian, hypergeometric Gaussian, Ins-Gaussian, etc. In embodiments, the beam is uniform within acceptable limits (e.g., less than 30% intensity variation across the beam). In embodiments, the beam is shaped or includes a gradient.

[0077] A sample refers to an object or location intended to be detected. In embodiments, the sample includes multiple distinct features that are targets for imaging. In some embodiments, the sample includes a non-planar structure having a surface, such as a bead or well, to which target nucleic acids are attached as target features. In embodiments, the sample is held by a sample holder. The sample holder can be a multiwell plate. In some instances, the multiwell plate has 16, 24, 48, 96, 384, or more sample wells. In some of these instances, the array of light sources, e.g., LEDs, has 16, 24, 48, 96, 384, or more corresponding light sources. In some instances, the multiwell plate is a standard microwell plate for biological analysis.

[0078] In an aspect, a nucleic acid sequencing system is provided, the genetic sequencing system including the imaging system described herein. The genetic sequencing system utilizes an excitation beam to excite labeled nucleotides in a DNA-containing sample, enabling analysis of base pairs present within the DNA. High-speed sequencing employs rapid scanning to deliver the excitation beam to a DNA fluorophore, stimulating sufficient emission of reactive photons from the DNA sample that are detected by an image sensor. Many next-generation sequencing (NGS) technologies use a form of sequencing-by-synthesis (SBS), in which modified nucleotides are used in conjunction with enzymes to read the sequence of a DNA template in a controlled manner. In embodiments, sequencing involves a sequencing-by-synthesis process in which individual nucleotides are iteratively identified as they are polymerized to form a growing complementary strand. In embodiments, the nucleotides added to the growing complementary strand contain both a label and a reversible chain terminator that prevents further extension, so that the nucleotides can be identified by their label before removing the terminator to add and identify additional nucleotides. Such reversible chain terminators include removable 3' protecting groups, as described, for example, in U.S. Patent Nos. 10,738,072, 7,541,444, and 7,057,026. When such a modified nucleotide is incorporated into a growing polynucleotide strand complementary to the sequenced template region, no free 3'-OH group is available to guide further sequence extension, and therefore the polymerase cannot add additional nucleotides. Once the identity of the base incorporated into the growing strand is determined, the 3' reversible terminator can be removed to allow the addition of the next nucleotide in a sequence. In embodiments, the gene sequencing system utilizes the detection of four different nucleotides containing four different labels.

[0079] In some embodiments, the nucleic acid sequencing system utilizes detection of four different nucleotides using fewer than four different labels. As a first example, a pair of nucleotide types can be detected at the same wavelength, but can be distinguished based on differences in signal conditions, such as intensity, for one member of the pair compared to the other, or based on a change to one member of the pair (e.g., via chemical, photochemical, or physical modification) that causes an apparent signal to appear or disappear compared to the signal detected for the other member of the pair. As a second example, three of the four different nucleotide types can be detected under certain conditions, while the fourth nucleotide type lacks a detectable label or is minimally detected under those conditions. The incorporation of the first three nucleotide types into a nucleic acid can be determined based on the presence of their respective signals, and the incorporation of the fourth nucleotide type into a nucleic acid can be determined based on the absence or minimal detection of any signal. As a third example, one nucleotide type can contain a label that is detected in two different channels, while the other nucleotide type is detected in only one channel.

[0080] In an aspect, a cellular imaging system is provided, the cellular imaging system comprising an imaging system as described herein, which utilizes an excitation beam to detect radiation (e.g., diffracted, reflected, refracted light) from a sample containing cells (e.g., from a tissue of interest, or from a biopsy, blood sample, or cell culture). Non-limiting examples of cell-containing samples, including fluids or tissues from a subject, include, but are not limited to, blood or blood products (e.g., serum, plasma, platelets, buffy coat, or the like), umbilical cord blood, chorionic villi, amniotic fluid, cerebrospinal fluid, spinal fluid, lavage fluid (e.g., lung, stomach, peritoneal, breast duct, ear, arthroscopic), biopsy sample, abdominal paracentesis sample, cells (blood cells, lymphocytes, placental cells, stem cells, bone marrow-derived cells, embryonic or fetal cells) or portions thereof (e.g., mitochondria, nuclei, extracts, or the like), urine, stool, sputum, saliva, nasal mucus, prostatic fluid, lavage, semen, lymph, bile, tears, sweat, breast milk, milk, the like, or combinations thereof. Non-limiting examples of tissues include organ tissue (e.g., liver, kidney, lung, thymus, adrenal gland, skin, bladder, reproductive organs, intestine, colon, spleen, brain, and the like, or portions thereof), epithelial tissue, hair, hair follicles, ducts, tubes, bone, eye, nose, mouth, throat, ear, nail, and the like, portions thereof, or combinations thereof. Samples may include cells or tissues that are normal, healthy, diseased (e.g., infected), and / or cancerous (e.g., cancer cells). Samples obtained from a subject may include cells or cellular material (e.g., nucleic acids) from multiple organisms (e.g., viral nucleic acid, fetal nucleic acid, bacterial nucleic acid, parasitic nucleic acid).

[0081] In an aspect, a cellular imaging system is provided, the cellular imaging system comprising an imaging system as described herein. The tissue imaging system utilizes an excitation beam to detect radiation (e.g., diffracted, reflected, refracted light) from a sample comprising tissue (e.g., from a tissue of interest, or from a biopsy, blood sample, or cell culture).

[0082] In embodiments, a system (e.g., a nucleic acid sequencing system, a cellular imaging system, or a tissue imaging system) includes an integrated system of one or more interconnected chambers, ports, and channels in fluid communication and configured to perform an analytical reaction or process, either alone or in conjunction with an appliance or instrument that provides a supporting function. A reagent aspiration manifold and / or a reagent dispensing manifold are in fluid communication with the fluidic system. The fluidic system may store fluids for washing or cleaning the device's fluidic network and for diluting reactants. For example, the fluidic system may include various reservoirs for storing reagents, enzymes, other biomolecules, buffer solutions, aqueous solutions, and non-polar solutions. Additionally, the fluidic system may also include a waste reservoir for receiving waste products. As used herein, a fluid may be a liquid, gel, gas, or a mixture thereof. A fluid may also be a mixture of two or more fluids. A fluidic network may include multiple fluidic components (e.g., fluid lines, pumps, aspirators, nozzles, valves, or other fluidic devices, manifolds, reservoirs) configured to have one or more fluids flowing therethrough. In embodiments, the system includes one or more peristaltic pumps. In embodiments, the system includes one or more syringe pumps. In embodiments, the support functions include at least one of sample introduction, fluid and / or reagent drive means, temperature control, a detection system, and a data collection and integration system, and are configured to determine the nucleic acid sequence of a template polynucleotide (e.g., a target polynucleotide, optionally including a barcode). The device can use, for example, pressure-driven flow control utilizing valves and pumps, to manipulate the flow of reagents, molecules, or enzymes in one or more directions and / or into one or more channels of the device.

[0083] In an aspect, a method of imaging a cellular sample (e.g., a tissue sample containing cells) is provided. In an embodiment, the method includes providing a sample containing cells, illuminating the sample using an imaging system described herein, detecting emission from the sample (e.g., fluorescence excitation events, scattered light, transmitted light, or reflected light) with an active pixel sensor array, and scanning the sample in a synchronized manner (i.e., the transfer of both the first and second charges is synchronized with the sample stage velocity).

[0084] In embodiments, the method further includes acquiring a two-dimensional or three-dimensional photograph, image, video, or other representation of the sample's physical form or structure. This representation can be obtained via light-field, fluorescence, or other microscopy techniques. In embodiments, the method further includes an additional imaging or immunohistochemistry modality (e.g., immunostaining). Immunohistochemistry (IHC) is a powerful technique that exploits the specific binding between antibodies and antigens to detect and localize specific antigens in cells and tissues, typically detected and examined by light microscopy. Known IHC modalities may be used, such as the protocol described in Magaki, S., Hojat, SA, Wei, B., So, A., & Yong, WH (2019). Methods in molecular biology (Clifton, NJ), 1897, 289-298, which is incorporated herein by reference. In embodiments, the additional imaging modality includes bright-field microscopy, phase-contrast microscopy, Nomarski differential interference contrast microscopy, or dark-field microscopy. In embodiments, the method further includes determining cell morphology (e.g., cell boundary or cell shape) of a sample containing one or more cells. For example, determining cell boundary includes comparing pixel values ​​of the image to a single intensity threshold, which can be rapidly determined using a histogram-based approach described in Carpenter, A. et al., Genome Biology 7, R100 (2006) and Arce, S., Sci Rep 3, 2266 (2013). Comparison of this representation with spatially resolved nucleic acid detection results can be used to localize genetic information with recognizable features of tissue. An exemplary method for spatial detection of nucleic acids that can be modified for use in the systems and methods described herein is described in US 2014 / 0066318, which is incorporated herein by reference. In embodiments, the method includes acquiring two-dimensional planes of images by scanning along one axis (e.g., the z direction). For example, multiple two-dimensional planes can be acquired for the same sample in the xy plane, whereby detection events can occur on different z planes.In embodiments of the methods provided herein, the methods include imaging through each of a plurality of two-dimensional planes with sufficient resolution to distinguish one imaging plane from an adjacent imaging plane. In embodiments, the methods and devices described herein simultaneously obtain multiple depth-resolved optical cross-sectional images.

[0085] In embodiments, the method includes performing additional image processing techniques (e.g., filtering, masking, smoothing, unsharp mask filter (USM), deconvolution, or maximum intensity projection (MIP)). In embodiments, the method includes computationally filtering the radiation using a linear or non-linear filter that amplifies high frequency components of the radiation. For example, a USM method applies a Gaussian blur to a copy of the original image and then compares it to the original image. If the difference is greater than a threshold setting, the images are subtracted. In embodiments, the method includes maximum intensity projection (MIP). Maximum intensity projection is a visualization technique that combines three-dimensional data (e.g., radiation from various depths within a sample) into a single two-dimensional image. For example, the projection takes the brightest pixel (voxel) at each depth and displays that pixel intensity value in the final two-dimensional image. Various machine learning approaches may be used, such as those described in Lugagne et al., Sci Rep 8, 11455 (2018) and Pattarone, G., et al., Sci Rep 11, 10304 (2021), each of which is incorporated herein by reference. In an embodiment, the method includes focus stacking (e.g., z-stacking), which combines multiple images taken at different focal lengths to produce a resulting image with a greater depth of field (DOF) than any of the individual source images.

[0086] One or more aspects or features of the subject matter described herein may be implemented in digital electronic circuitry, integrated circuits, specially designed ASICs (application-specific integrated circuits), computer hardware, firmware, software, and / or combinations thereof. These various implementations may be special purpose or general purpose, and may include implementation in one or more computer programs executable and / or interpretable on a programmable system including at least one programmable processor coupled to receive data and instructions from and send data and instructions to a storage system, at least one input device (e.g., a mouse, touch screen, etc.), and at least one output device. The methods and systems described herein may be implemented or performed by a machine such as a processor configured with specific instructions, a digital signal processor (DSP), an application-specific integrated circuit (ASIC), a field-programmable gate array (FPGA) or other programmable logic device, discrete gate or transistor logic, discrete hardware components, or any combination thereof designed to perform the functions described herein. The processor may be a microprocessor. A processor may also be implemented as a combination of computing devices, e.g., a combination of a DSP and a microprocessor, a plurality of microprocessors, one or more microprocessors in combination with a DSP core, or any other such configuration. Elements of the methods or processes described herein may be implemented in computer hardware, in software modules executed by a processor, or a combination of the two. A software module may reside in RAM memory, flash memory, ROM memory, EPROM memory, EEPROM memory, registers, hard disk, a removable disk, a CD-ROM, or any other form of computer-readable storage medium known in the art.

[0087] A computer program, which may also be referred to as a program, software, software application, application, component, or code, includes machine instructions for a programmable processor and may be implemented in a high-level procedural language, an object-oriented programming language, a functional programming language, a logic programming language, and / or an assembly / machine language. As used herein, the term “machine-readable medium” refers to any computer program product, apparatus, and / or device used to provide machine instructions and / or data to a programmable processor, such as, for example, a magnetic disk, an optical disk, a memory, and a programmable logic device (PLD), including a machine-readable medium that receives machine instructions as a machine-readable signal. The term “machine-readable signal” refers to any signal used to provide machine instructions and / or data to a programmable processor. A machine-readable medium may store such machine instructions non-transitoryly, such as, for example, a non-transitory solid-state memory or a magnetic hard drive or any equivalent storage medium. Alternatively or additionally, the machine-readable medium may store such machine instructions in a transitory manner, such as, for example, a processor cache or other random access memory associated with one or more physical processor cores. The computer may run any one of a variety of operating systems, such as, for example, Windows, or MacOS, or any one of several versions of Unix, or Linux.

[0088] In certain aspects, to provide for user interaction, the subject matter described herein can be implemented on a computer having a display device, such as, for example, a cathode ray tube (CRT) or liquid crystal display (LCD) monitor, for displaying information to the user, and a keyboard and pointing device, such as, for example, a mouse or trackball, through which the user can provide input to the computer. Other types of devices can also be used to provide for user interaction. For example, feedback provided to the user can be any form of sensory feedback, such as, for example, visual feedback, auditory feedback, or tactile feedback, and input from the user can be received in any form, including, but not limited to, acoustic, audio, or tactile input. Other possible input devices include, but are not limited to, touchscreens or other touch-sensitive devices such as single-point or multi-point resistive or capacitive trackpads, voice recognition hardware and software, optical scanners, optical pointers, digital image capture devices and associated interpretation software, etc.

[0089] The subject matter described herein may be implemented in a computing system including back-end components (e.g., as a data server), or a computing system including middleware components (e.g., an application server), or a front-end component (e.g., a client computer having a graphical user interface or a web browser through which a user may interact with an implementation of the subject matter described herein), or any combination of such back-end, middleware, or front-end components. The components of the system may be interconnected by any form or medium of digital data communication (e.g., a communications network). Examples of communications networks include a local area network ("LAN"), a wide area network ("WAN"), the Internet, WiFi (IEEE 802.11 standard), NFC, BLUETOOTH, ZIGBEE, and the like.

[0090] A computing system may include clients and servers. Clients and servers are generally remote from each other and typically interact through a communication network. The relationship of client and server arises by virtue of computer programs running on the respective computers and having a client-server relationship to each other.

[0091] The subject matter described herein may be embodied in systems, devices, methods, and / or articles, depending on the desired configuration. The implementations described in the foregoing description do not represent all implementations consistent with the subject matter described herein. Rather, they are merely some examples consistent with aspects related to the subject matter described. While some variations have been described in detail above, other modifications or additions are possible. In particular, additional features and / or variations may be provided in addition to those described herein. For example, the above-described implementations may be directed to various combinations and subcombinations of the disclosed features and / or combinations and subcombinations of certain additional features disclosed above. Furthermore, the logic flow depicted in the accompanying drawings and / or described herein does not necessarily require the particular order shown, or sequential order, to achieve desirable results. Other implementations may be within the scope of the following claims.

[0092] Embodiment Embodiment 1. An imaging system comprising: a sample stage moving at a sample stage velocity, the sample stage configured to receive a sample including a first fluorophore and a second fluorophore; a first sensor array and a second sensor array; a first light source configured to provide a first excitation beam and a second light source configured to provide a second excitation beam; and a first optical system configured to direct the first excitation beam and the second excitation beam onto the sample, wherein interaction of the first excitation beam with the first fluorophore produces a first fluorescent emission and a second excitation beam. a first optical system in which a second fluorescent radiation is generated by interaction of the excitation beam with a second fluorophore; and a second optical system configured to direct the first fluorescent radiation to a first sensor array and the second fluorescent radiation to a second sensor array, wherein the first fluorescent radiation impinges on the first sensor array to generate a first charge that migrates across the first sensor array, and the second fluorescent radiation impinges on the second sensor array to generate a second charge that migrates across the second sensor array, wherein migration of both the first charge and the second charge is synchronized with a sample stage velocity.

[0093] Embodiment 2. The imaging system of embodiment 1, further comprising a third fluorophore, a fourth fluorophore, a third sensor array, and a fourth sensor array, wherein a third fluorescent radiation is generated by an interaction of the first excitation beam with the third fluorophore and a fourth fluorescent radiation is generated by an interaction of the second excitation beam with the fourth fluorophore; wherein a second optical system is configured to direct the third fluorescent radiation to the third sensor array and the fourth fluorescent radiation to the fourth sensor array, wherein the third fluorescent radiation impinges on the third sensor array to generate a third charge that migrates across the third sensor array, and the fourth fluorescent radiation impinges on the fourth sensor array to generate a fourth charge that migrates across the fourth sensor array, and wherein migration of at least one of the third charge and the fourth charge is synchronized with a sample stage speed.

[0094] Embodiment 3. An imaging system comprising: a sample stage moving at a sample stage velocity, the sample stage comprising a sample including a first fluorophore, a second fluorophore, a third fluorophore, and a fourth fluorophore; a first sensor array, a second sensor array, a third sensor array, and a fourth sensor array; a first light source configured to provide a first excitation beam, and a second light source configured to provide a second excitation beam; and a first optical system configured to direct the first excitation beam and the second excitation beam onto the sample, wherein interaction of the first excitation beam with the first fluorophore produces a first fluorescent emission, interaction of the second excitation beam with the second fluorophore produces a second fluorescent emission, interaction of the first excitation beam with the third fluorophore produces a third fluorescent emission, and interaction of the second excitation beam with the fourth fluorophore produces a third fluorescent emission. a first optical system configured to direct the first fluorescent radiation to the first sensor array, the second fluorescent radiation to the second sensor array, the third fluorescent radiation to the third sensor array, and the fourth fluorescent radiation to the fourth sensor array, wherein the first fluorescent radiation impinges on the first sensor array and generates a first charge that migrates across the first sensor array, and the second fluorescent radiation interacts with the second sensor array to generate a fourth fluorescent radiation. a third fluorescent radiation impinging on the third sensor array to generate a third charge that migrates across the third sensor array; a fourth fluorescent radiation impinging on the fourth sensor array to generate a fourth charge that migrates across the fourth sensor array; and migration of at least one of the first charge, the second charge, the third charge, and the fourth charge is synchronized with a sample stage velocity.

[0095] Embodiment 4. An imaging system of any one of embodiments 1 to 3, wherein the first optical system is configured to direct a first excitation beam toward a first region of the sample and a second excitation beam toward a second region of the sample, and the first region and the second region are separated by about 10 μm to about 500 μm.

[0096] Embodiment 5. An imaging system of any one of embodiments 2 to 4, wherein the second optical system comprises a first optical element including a first surface configured to reflect the first fluorescent radiation toward the first sensor array and the third fluorescent radiation toward the third sensor array, and a second surface configured to reflect the second fluorescent radiation toward the second sensor array and the fourth fluorescent radiation toward the fourth sensor array.

[0097] Embodiment 6. The imaging system of embodiment 5, wherein the second optical system is downstream from the first optical element and comprises a second optical element configured to focus the first fluorescent radiation, the second fluorescent radiation, the third fluorescent radiation, and the fourth fluorescent radiation.

[0098] Embodiment 7. The imaging system of embodiment 6, wherein the second optical system comprises a bandpass filter configured to selectively transmit the first fluorescent radiation, the second fluorescent radiation, the third fluorescent radiation, and the fourth fluorescent radiation.

[0099] Embodiment 8. The imaging system of embodiment 7, wherein the detection camera includes a first sensor array, a second sensor array, a third sensor array, and a fourth sensor array.

[0100] Embodiment 9. The imaging system of embodiment 5, wherein the first optical element is a dichroic wedge.

[0101] Embodiment 10. An imaging system of any one of embodiments 2 to 4, wherein the second optical system comprises a first optical element configured to reflect the first fluorescent radiation toward the first sensor array, reflect the third fluorescent radiation toward the third sensor array, transmit the second fluorescent radiation toward the second sensor array, and transmit the fourth fluorescent radiation toward the fourth sensor array.

[0102] Embodiment 11. An imaging system of embodiment 10, wherein the second optical system comprises a first lens downstream from the first optical element and configured to focus the first fluorescent radiation and the third fluorescent radiation, and a second lens downstream from the first optical element and configured to focus the second fluorescent radiation and the fourth fluorescent radiation.

[0103] Embodiment 12. An imaging system of embodiment 11, wherein the second optical system comprises a first bandpass filter configured to selectively transmit the first fluorescent radiation and the third fluorescent radiation, and a second bandpass filter configured to selectively transmit the second fluorescent radiation and the fourth fluorescent radiation.

[0104] Embodiment 13. The imaging system of embodiment 12, wherein the first detector camera includes a first sensor array and a third sensor array, and the second detector camera includes a second sensor array and a fourth sensor array.

[0105] Embodiment 14. The imaging system of embodiment 10, wherein the first optical element is a dichroic beam splitter.

[0106] Embodiment 15. The imaging system of one of embodiments 1 to 14, wherein each sensor array is a TDI sensor array.

[0107] Embodiment 16. An imaging system according to one of embodiments 1 to 15, wherein each sensor array is approximately 1,000 to 20,000 pixels wide.

[0108] Embodiment 17. An imaging system according to one of embodiments 1 to 15, wherein each sensor array is approximately 3,000 to 10,000 pixels wide.

[0109] Embodiment 18. An imaging system according to one of embodiments 1 to 15, wherein each sensor array is approximately 5,000 to 8,000 pixels wide.

[0110] Embodiment 19. The imaging system of one of embodiments 1 to 15, wherein each sensor array is approximately 5,500 pixels wide.

[0111] Embodiment 20. The imaging system of one of embodiments 1 to 15, wherein each sensor array is approximately 1,000 to 20,000 pixels wide and approximately 10 to 300 pixels long.

[0112] Embodiment 21. An imaging system according to one of embodiments 1 to 15, wherein each sensor array is approximately 3,000 to 10,000 pixels wide and approximately 10 to 300 pixels long.

[0113] Embodiment 22. An imaging system according to one of embodiments 1 to 15, wherein each sensor array is approximately 5,000 to 10,000 pixels wide and approximately 30 to 300 pixels long.

[0114] Embodiment 23. The imaging system of one of embodiments 1 to 15, wherein each sensor array is approximately 8,000 pixels wide and approximately 128 pixels long.

[0115] Embodiment 24. The imaging system of one of embodiments 1 to 23, wherein the sample stage is a motorized translation stage.

[0116] Embodiment 25. An imaging system according to one of embodiments 1 to 24, wherein the sample stage is provided with a position encoder, and the position encoder generates a synchronization signal for synchronizing the transfer of charge.

[0117] Embodiment 26. The imaging system of one of embodiments 1 to 25, further comprising a collimating lens, a beam-shaping lens, or a cylindrical lens.

[0118] Embodiment 27. The imaging system of one of embodiments 1 to 26, further comprising one or more line generators.

[0119] Embodiment 28. The imaging system of one of embodiments 1 to 26, further comprising two line generators.

[0120] Embodiment 29. A method for imaging a sample, comprising: a) directing a first excitation beam and a second excitation beam onto a sample, said sample being on a sample stage moving at a sample stage velocity, said sample comprising a first fluorophore that generates a first fluorescent emission after interaction with the first excitation beam and a second fluorophore that generates a second fluorescent emission after interaction with the second excitation beam, respectively; b) directing said first fluorescent emission to impinge on a first sensor array and generate a first charge that migrates across the first sensor array at a first charge velocity, and directing said second fluorescent emission to impinge on a second sensor array and generate a second charge that migrates across the second sensor array at a second charge velocity, wherein at least one of the first charge velocity and the second charge velocity is synchronized with the sample stage velocity; and c) scanning the sample in a scanning dimension, and repeating steps a) and b) to form an image of the sample.

[0121] Embodiment 30. The method of embodiment 29, wherein the sample further comprises a third fluorophore that generates a third fluorescent emission after interaction with the first excitation beam and a fourth fluorophore that generates a fourth fluorescent emission after interaction with the second excitation beam, and wherein said third fluorescent emission is directed to impinge on a third sensor array and generate a third charge that migrates across the third sensor array at a third charge velocity, and said fourth fluorescent emission is directed to impinge on a fourth sensor array and generate a fourth charge that migrates across the fourth sensor array at a fourth charge velocity.

[0122] Embodiment 31. The method of embodiment 30, wherein the method comprises imaging a sample containing four different fluorophores simultaneously.

[0123] Embodiment 32. The method of embodiment 30 or 31, wherein the first excitation beam and the second excitation beam comprise excitation lines.

[0124] Embodiment 33. The method of one of embodiments 30 to 32, wherein scanning the sample includes moving a sample stage.

[0125] Embodiment 34. The method of any one of embodiments 30 to 33, wherein scanning the sample includes moving the sample stage at a constant velocity.

[0126] Embodiment 35. The method of one of embodiments 30 to 34, further comprising storing a data representation of said image of said sample in a computer-readable memory.

[0127] Embodiment 36. The method of one of embodiments 30 to 35, wherein the excitation beam comprises UV radiation, VIS radiation, or IR radiation.

[0128] Embodiment 37. The method of one of embodiments 30 to 36, wherein the excitation beam comprises an excitation beam having a wavelength of 405 nm, 470 nm, 488 nm, 514 nm, 520 nm, 532 nm, 561 nm, 633 nm, 639 nm, 640 nm, 800 nm, 808 nm, 912 nm, 1024 nm, or 1500 nm.

[0129] Embodiment 38. The method of one of embodiments 30 to 37, wherein the sample comprises modified nucleotides.

[0130] Embodiment 39. The method of one of embodiments 30 to 38, wherein the sample comprises an array having a plurality of individual sites.

[0131] I. Definition All patents, patent applications, articles and publications mentioned herein, both supra and infra, are hereby expressly incorporated by reference in their entirety.

[0132] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Various scientific dictionaries, including the terms contained herein, are well known and accessible to those skilled in the art. Although any methods and materials similar or equivalent to those described herein find use in practicing or testing the present disclosure, several preferred methods and materials are described. Therefore, the terms defined below are more fully described by reference to the entire specification. It is understood that this disclosure is not limited to the specific methodology, protocols, and reagents described, as these may vary depending on the context in which they are used by those skilled in the art. The following definitions are provided to facilitate understanding of certain terms frequently used herein and are not intended to limit the scope of the present disclosure.

[0133] As used herein, the singular terms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Throughout this specification, for example, "one embodiment," "an embodiment," "another embodiment," "a particular embodiment," "related embodiment," "a particular embodiment," "an additional embodiment," or "a further embodiment," or combinations thereof, means that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment of the present disclosure. Thus, the appearances of such phrases in various places throughout the specification do not necessarily all refer to the same embodiment. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0134] Throughout this specification, for example, the terms "one embodiment," "an embodiment," "another embodiment," "a particular embodiment," "a related embodiment," "a particular embodiment," "an additional embodiment," or "a further embodiment," or combinations thereof, mean that the particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, the appearances of such phrases in various places throughout the specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0135] As used herein, the term "about" refers to a range of values ​​that includes the specified value and that one of ordinary skill in the art would consider to be reasonably similar to the specified value. In embodiments, the term "about" refers to within a standard deviation using measurements generally accepted in the art. In embodiments, about refers to a range that extends to ±10% of the specified value. In embodiments, about refers to the specified value.

[0136] Throughout this specification, unless the context requires otherwise, the terms "comprise," "comprises," and "comprising" will be understood to imply the inclusion of the specified step or element or group of steps or elements, but not the exclusion of other steps or elements or group of steps or elements. "Consisting of" means including and limited to what follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are necessary or mandatory, and that other elements may not be present. "Consisting essentially of" means including any elements listed after the phrase, and is limited to other elements that do not interfere with or contribute to the activity or function specified in this disclosure for the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are necessary or mandatory, but that other elements are not optional and may not be present depending on whether they affect the activity or function of the listed elements.

[0137] As used herein, the term "nucleic acid" refers to nucleotides (e.g., deoxyribonucleotides or ribonucleotides) and polymers thereof, or their complements, in either single-, double-, or multi-stranded form. The terms "polynucleotide," "oligonucleotide," "oligo," or the like refer, in their ordinary and accustomed sense, to a sequence of nucleotides. The term "nucleotide," in its ordinary and accustomed sense, refers to a single unit, e.g., a monomer, of a polynucleotide. A nucleotide can be a ribonucleotide, a deoxyribonucleotide, or modified versions thereof. Examples of polynucleotides contemplated herein include single- and double-stranded DNA, single- and double-stranded RNA, and hybrid molecules having mixtures of single- and double-stranded DNA and RNA with linear or circular frameworks. Non-limiting examples of polynucleotides include genes, gene fragments, exons, introns, intergenic DNA (including but not limited to heterochromatic DNA), messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA sequences, isolated RNA sequences, nucleic acid probes, and primers. Polynucleotides useful in the methods of the disclosure may include naturally occurring nucleic acid sequences and variants thereof, artificial nucleic acid sequences, or combinations of such sequences.

[0138] A polynucleotide typically consists of a specific sequence of four nucleotide bases: adenine (A), cytosine (C), guanine (G), and thymine (T) (or uracil (U) for thymine (T) if the polynucleotide is RNA). Thus, the term "polynucleotide sequence" is the alphabetical representation of a polynucleotide molecule; alternatively, the term can apply to the polynucleotide molecule itself. This alphabetical representation can be input into a database on a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching. A polynucleotide can optionally include one or more non-standard nucleotides, nucleotide analogs, and / or modified nucleotides.

[0139] As used herein, the term "polynucleotide template" refers to any polynucleotide molecule that can be bound by a polymerase and used as a template for nucleic acid synthesis. As used herein, the term "polynucleotide primer" or "primer" refers to any polynucleotide molecule that can hybridize to a polynucleotide template, be bound by a polymerase, and be extended in a template-directed process of nucleic acid synthesis, such as a PCR or sequencing reaction. A polynucleotide primer attached to a core polymer within a core is referred to as a "core polynucleotide primer."

[0140] Generally, the term "target polynucleotide" refers to a nucleic acid molecule or polynucleotide in a starting population of nucleic acid molecules having a target sequence, the presence, amount, and / or nucleotide sequence, or changes in one or more thereof, of which it is desired to determine. Generally, the term "target sequence" refers to a nucleic acid sequence on a single strand of nucleic acid. A target sequence can be a portion of a gene, a regulatory sequence, genomic DNA, including mRNA, miRNA, rRNA, cDNA, RNA, or other portion. A target sequence can also be a target sequence from a sample or a secondary target, such as the product of an amplification reaction. A target polynucleotide is not necessarily any single molecule or sequence. For example, a target polynucleotide can be any one of multiple target polynucleotides in a reaction, or all polynucleotides in a given reaction, depending on the reaction conditions. For example, in a nucleic acid amplification reaction using random primers, all polynucleotides in the reaction can be amplified. As a further example, a group of targets can be assayed simultaneously using polynucleotide primers directed to multiple targets in a single reaction. As yet another example, all or a subset of the polynucleotides in a sample can be modified by the addition of primer binding sequences (such as by ligation of an adaptor containing the primer binding sequences), and each modified polynucleotide can be made into a target polynucleotide in a reaction with a corresponding primer polynucleotide.

[0141] As used herein, the term "flow cell" refers to a reaction vessel in a nucleic acid sequencing device. A flow cell is typically a glass slide containing small fluidic channels (e.g., a 75 mm x 25 mm x 1 mm glass slide with one or more channels) through which sequencing solutions (e.g., polymerase, nucleotides, and buffers) can traverse. While typically glass, suitable flow cell materials can include polymeric materials, plastics, silicon, quartz (fused silica), Borofloat® glass, silica, silica-based materials, carbon, metals, optical fibers or fiber optic bundles, sapphire, or plastic materials such as COC and epoxy. Particular materials can be selected based on properties desired for a particular use. For example, materials that are transparent to radiation of a desired wavelength are useful for analytical techniques that utilize radiation of the desired wavelength. Conversely, it may be desirable to select a material that does not allow radiation of a particular wavelength to pass through (e.g., is opaque, absorptive, or reflective). In embodiments, the flow cell material is selected for its ability to conduct thermal energy. In embodiments, the flow cell includes an inlet port and an outlet port and a flow channel extending therebetween.

[0142] As used herein, a "line generator" refers to an optical component configured to generate a diffraction-limited or near-diffraction-limited excitation beam in a plane perpendicular to the optical axis of propagation, with a substantially uniform intensity distribution along the horizontal axis of the line. Exemplary line generators include, but are not limited to, one-dimensional diffusers with angular uniformity, cylindrical microlens arrays, diffractive elements, or aspheric refractive lenses such as Powell lenses.

[0143] As used herein, the term "substrate" refers to a solid support material. Substrates can be non-porous or porous. Substrates can be rigid or flexible. Non-porous substrates generally provide a seal against bulk flow of liquids or gases. Exemplary solid supports include, but are not limited to, glass and modified or functionalized glass, plastics (such as acrylics, polystyrene, and copolymers of styrene with other materials, polypropylene, polyethylene, polybutylene, polyurethane, Teflon™, cyclic olefin copolymers, polyimides, etc.), nylon, ceramics, resins, Zeonor, silica or silica-based materials, including silicon and modified silicon, carbon, metals, inorganic glass, fiber optic bundles, photopatternable dry film resists, UV-curable adhesives, and polymers. Particularly useful solid supports for some embodiments have at least one surface located within a flow cell. The term "surface" is intended to mean an outer portion or layer of a substrate. A surface can be in contact with another material, such as a gas, liquid, gel, polymer, organic polymer, a second surface of a similar or different material, metal, or coating. The surface, or a region thereof, can be substantially flat. The substrate and / or surface can have surface features such as wells, pits, channels, ridges, raised regions, pegs, posts, or the like. The term "well" refers to a discrete concave feature of a substrate having a surface opening completely surrounded by an interstitial region of the surface. A well can have any of a variety of shapes at its opening in the surface, including, but not limited to, circular, elliptical, square, polygonal, or star-shaped (e.g., a star-shaped with any number of points). A cross-section of a well taken perpendicular to the surface can be curved, square, polygonal, hyperbolic, conical, or angular.

[0144] As used herein, the terms "sequencing," "sequencing," "determining a nucleotide sequence," and the like include determining partial or complete sequence information (e.g., sequence) of a polynucleotide being sequenced, particularly the physical process for generating such sequence information. That is, the term includes sequence comparison, consensus sequencing, contig assembly, fingerprinting, and similar levels of information regarding a target polynucleotide, as well as explicitly identifying and ordering nucleotides in a target polynucleotide. The term also includes identifying, ordering, and determining the location of one, two, or three of four nucleotides within a target polynucleotide. In some embodiments, the sequencing process described herein involves contacting a template and annealed primers with a suitable polymerase under conditions suitable for polymerase extension and / or sequencing. Sequencing methods are preferably performed with target polynucleotides arrayed on a solid substrate within a flow cell (e.g., within a channel of a flow cell). In embodiments, the sequencing is sequencing-by-synthesis (SBS). Briefly, SBS methods involve contacting a target nucleic acid with one or more labeled nucleotides (e.g., fluorescently labeled) in the presence of a DNA polymerase. Optionally, the labeled nucleotides can further include a reversible termination feature that stops elongation upon incorporation of the nucleotide. Thus, for embodiments using reversible termination, a cleavage solution can be delivered to the flow cell (before or after detection occurs). Washing can be performed between various delivery steps. This cycle can then be repeated n times to extend the primer with n nucleotides and detect a sequence of length n. Exemplary SBS procedures and detection platforms that can be readily adapted for use with the methods of the present disclosure are described, for example, in Bentley et al., Nature 456:53-59 (2008), WO 2004 / 018497, and WO 2007 / 123744, each of which is incorporated herein by reference in its entirety. In embodiments, the sequencing is pH-based DNA sequencing.The concept of pH-based DNA sequencing is described in documents including US2009 / 0026082 and Pourmand et al., Proc.Natl.Acad.Sci.,103:6466-6470(2006), which are incorporated by reference in their entirety.Other sequencing procedures that use circular reaction, such as pyrosequencing, can be used.Sequencing reaction by ligation is also useful, for example, as described in Shendure et al., Science 309:1728-1732(2005).

[0145] As used herein, the term "feature" refers to a point or area of ​​a pattern that can be distinguished from other points or areas according to its relative location. An individual feature can contain one or more polynucleotides. For example, a feature can contain a single target nucleic acid molecule having a particular sequence, or a feature can contain several nucleic acid molecules having the same sequence (and / or its complementary sequence). Different molecules in different features of a pattern can be distinguished from each other according to the location of the feature in the pattern. Non-limiting examples of features include wells in a substrate, particles (e.g., beads) in or on a substrate, polymers in or on a substrate, protrusions from a substrate, ridges on a substrate, or channels in a substrate.

[0146] The term "image" is used according to its ordinary meaning and refers to a representation of all or part of an object. The representation may be an optically detected reproduction. For example, an image may be obtained from fluorescence, luminescence, scattering, or absorption signals. The portion of the object present in the image may be the surface of the object or other xy plane. Typically, an image is a two-dimensional representation of a three-dimensional object. An image may include signals of different intensities (i.e., signal levels). An image may be provided in a computer-readable format or medium. An image is derived from a collection of focused points of light rays coming from an object (e.g., a sample), which may be detected by any image sensor.

[0147] As used herein, the term "signal" is intended to include, for example, fluorescence, luminescence, scattering, or absorption impulses or electromagnetic waves transmitted or received. Signals can be detected in the ultraviolet (UV) range (approximately 200-390 nm), visible (VIS) range (approximately 391-770 nm), infrared (IR) range (approximately 0.771-25 microns), or other ranges of the electromagnetic spectrum. The term "signal level" refers to the amount or quantity of energy or encoded information detected. For example, a signal can be quantified by its intensity, wavelength, energy, frequency, power, brightness, or a combination thereof. Other signals can be quantified according to characteristics such as voltage, current, electric field strength, magnetic field strength, frequency, power, temperature, etc. Absence of signal is understood to be a signal level of zero or a signal level that is not significantly distinguishable from noise.

[0148] The term "x-y coordinates" refers to information specifying the location, size, shape, and / or orientation of an x-y plane. The information can be, for example, numerical coordinates in a Cartesian system. The coordinates can be provided relative to one or both of the x- and y-axes, or can be provided relative to another location within the x-y plane (e.g., a reference plane). The term "x-y plane" refers to the two-dimensional area defined by linear axes x and y. When used in connection with a detection device and an object observed by a detector, the x-y plane can be specified as being orthogonal to the observation direction between the detector and the detected object. The terms "z-axis" and "z-direction" are intended to be used consistently with their use in the art of microscopy and imaging systems generally, and the z-axis refers to the focal axis. Thus, z-axis translation results in an increase or decrease in the length of the focal axis. Z-axis translation can be performed, for example, by moving the sample stage relative to the optical stage (e.g., by moving the sample stage or optical elements, or both).

[0149] As used herein, the terms "cluster" and "colony" are used interchangeably to refer to discrete sites on a solid support containing a plurality of immobilized polynucleotides and a plurality of immobilized complementary polynucleotides. The term "clustered array" refers to an array formed from such clusters or colonies. In this context, the term "array" should not be understood to require an ordered arrangement of clusters. The term "array" is used according to its ordinary meaning in the art to refer to a collection of different molecules attached to one or more solid substrates such that the different molecules can be distinguished from one another based on their relative locations. A flow cell can include an array, each containing different molecules located at different addressable features on the solid substrate. The molecules in the array can be nucleic acid primers, nucleic acid probes, nucleic acid templates, or nucleic acid enzymes such as polymerases or ligases. Arrays useful in the present invention can have densities ranging from about two features to millions, billions, or more. Array densities can range from two to as many as one billion or more different features per square centimeter. For example, the array can have at least about 100 features / cm2, at least about 1,000 features / cm2, at least about 10,000 features / cm2, at least about 100,000 features / cm2, at least about 10,000,000 features / cm2, at least about 100,000,000 features / cm2, at least about 1,000,000,000 features / cm2, at least about 2,000,000,000 features / cm2 or more. In embodiments, arrays have features at any of a variety of densities, including, for example, at least about 10 features / cm, 100 features / cm, 500 features / cm, 1,000 features / cm, 5,000 features / cm, 10,000 features / cm, 50,000 features / cm, 100,000 features / cm, 1,000,000 features / cm, 5,000,000 features / cm, or more. Clustering refers to the process of generating clusters (i.e., solid-phase amplification of polynucleotides).

[0150] The term "nucleic acid sequencing device" refers to an integrated system of one or more interconnected and fluidly communicating chambers, ports, and channels designed to perform an analytical reaction or process, either alone or in cooperation with an appliance or instrument that provides supporting functions such as sample introduction, fluid and / or reagent delivery means, temperature control, detection systems, data collection, and / or integration systems, for the purpose of determining the nucleic acid sequence of a template polynucleotide. Nucleic acid sequencing devices may further include valves, pumps, and specialized functional coatings on their interior walls. Nucleic acid sequencing devices may include a receiving unit, or platen, that orients the flow cell so that the maximum surface area of ​​the flow cell is exposed to the optical lens. Other nucleic acid sequencing devices include those offered by Illumina™, Inc. (e.g., HiSeq™, MiSeq™, NextSeq™, or NovaSeq™ systems), Life Technologies™ (e.g., ABI PRISM™ or SOLiD™ systems), Pacific Biosciences (e.g., systems using SMRT™ technology such as the Sequel™ or RS II™ systems), or Qiagen (e.g., the Genereader™ system). Nucleic acid sequencing devices may further include fluid reservoirs (e.g., bottles), valves, pressure sources, pumps, sensors, control systems, valves, pumps, and special functional coatings on their interior walls. In embodiments, the device includes multiple sequencing reagent reservoirs and multiple clustering reagent reservoirs. In embodiments, the clustering reagent reservoirs include amplification reagents (e.g., aqueous buffers containing enzymes, salts, and nucleotides, denaturants, crowding agents, etc.). In embodiments, the reservoirs contain sequencing reagents (e.g., an aqueous buffer containing enzymes, salts, and nucleotides), a wash solution (aqueous buffer), a cleavage solution (aqueous buffer containing a cleavage agent such as a reducing agent), or a wash fluid (dilute bleach solution, dilute NaOH solution, dilute HCl solution, dilute antibacterial solution, or water). The fluid in each reservoir can be varied.The fluid may contain, for example, a buffer (e.g., saline-sodium citrate (SSC), ascorbic acid, tris(hydroxymethyl)aminomethane or "Tris"), an aqueous salt (e.g., KCl or (NH4)2SO4)), nucleotides, a polymerase, a cleaving agent (e.g., tri-n-butyl-phosphine, triphenylphosphine and its sulfonated version (i.e., tris(3-sulfophenyl)-phosphine, TPPTS), and tri(carboxyethyl)phosphine (TCEP) and its salts), a cleaving agent scavenger compound (e.g., 2'-diaminobenzyl phosphate ... The reservoir may be an aqueous solution that may contain a reagent (e.g., thiobisetanamine or 11-Azido-3,6,9-trioxaundecan-1-amine), a chelating agent (e.g., EDTA), a detergent, a surfactant, a crowding agent, or a stabilizer (e.g., PEG, Tween, BSA). Non-limiting examples of the reservoir include a cartridge, a pouch, a vial, a container, and an Eppendorf tube. In embodiments, the device is configured to perform fluorescence imaging. In embodiments, the device includes one or more light sources (e.g., one or more lasers). In embodiments, the illuminator or light source is a radiation source (i.e., a source or generator of propagating electromagnetic energy) that provides incident light to the sample. Radiation sources can include illumination sources that produce electromagnetic radiation in the ultraviolet (UV) range (approximately 200-390 nm), visible (VIS) range (approximately 390-770 nm), or infrared (IR) range (approximately 0.77-25 microns), or other ranges of the electromagnetic spectrum. In embodiments, the illuminator or light source is a lamp, such as an arc lamp or a quartz halogen lamp. In embodiments, the illuminator or light source is a coherent light source. In embodiments, the light source is a laser. In embodiments, the illuminator or light source is a light emitting diode (LED), a mercury or tungsten lamp, or a supercontinuous diode. In embodiments, the light source provides an excitation beam having a wavelength between 200 nm and 1500 nm. In embodiments, the laser provides an excitation beam having a wavelength of 405 nm, 470 nm, 488 nm, 514 nm, 520 nm, 532 nm, 561 nm, 633 nm, 639 nm, 640 nm, 800 nm, 808 nm, 912 nm, 1024 nm, or 1500 nm. In embodiments, the illuminator or light source is a light emitting diode (LED).The LED can be, for example, an organic light-emitting diode (OLED), a thin-film electroluminescent device (TFELD), or a quantum dot-based inorganic-organic LED. The LED can include a phosphorescent OLED (PHOLED). In embodiments, the nucleic acid sequencing device includes an imaging system (e.g., an imaging system as described herein). The imaging system can excite one or more distinguishable labels (e.g., fluorescent labels) linked to the nucleotides and then obtain image data of the distinguishable labels. The image data (e.g., detection data) can be analyzed by another component within the device. The imaging system can include a system described herein and can include a fluorescence spectrophotometer including an objective lens and / or a solid-state imaging device. The solid-state imaging device can include a charge-coupled device (CCD) and / or a complementary metal-oxide semiconductor (CMOS).

[0151] As used herein, the term "label" or "labels" generally refers to a molecule that can generate or provide, directly or indirectly, a detectable signal by itself or through interaction with another molecule. The labeling moiety can be any moiety that allows for detection of a sample using, for example, spectroscopy. Exemplary labeling moieties are fluorescent labels, mass labels, chemiluminescent labels, electrochemical labels, detectable labels, and the like. Non-limiting examples of detectable labels include labels comprising fluorescent dyes, biotin, digoxin, haptens, and epitopes. Generally, a dye is a molecule, compound, or substance that can provide an optically detectable signal, such as a colorimetric signal, a luminescent signal, a bioluminescent signal, a chemiluminescent signal, a phosphorescent signal, a fluorescent signal, or the like. In embodiments, the dye is a fluorescent dye. Non-limiting examples of dyes, some of which are commercially available, include CF dyes (Biotium, Inc.), Alexa Fluor dyes (Thermo Fisher), DyLight dyes (Thermo Fisher), Cy dyes (GE Healthscience), IRDyes (Li-Cor Biosciences, Inc.), and HiLyte dyes (Anaspec, Inc.). In embodiments, the label is a fluorophore. Examples of detectable agents (e.g., labels) include imaging agents that contain fluorescent and luminescent substances, molecules, or compositions, including, but not limited to, various organic or inorganic small molecules commonly referred to as "dyes," "labels," or "indicators." Examples include fluorescein, rhodamine, acridine dyes, Alexa dyes, and cyanine dyes. In embodiments, the detectable moiety is a fluorescent molecule (e.g., an acridine dye, a cyanine dye, a fluorine dye, an oxazine dye, a phenanthridine dye, or a rhodamine dye). In embodiments, the detectable moiety is a fluorescent molecule (eg, an acridine dye, a cyanine dye, a fluorine dye, an oxazine dye, a phenanthridine dye, or a rhodamine dye).

[0152] Where a range of values ​​is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of that range, unless the context clearly indicates otherwise, and any other stated or unstated intervening value within a smaller range of values ​​within that stated range, is encompassed within the invention. The upper and lower limits of any such smaller range (within a broader recited range) may independently be included in the smaller range or as a particular value itself, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0153] The term "optical filter" refers to a device for selectively passing or rejecting radiation in a wavelength-, polarization-, or frequency-dependent manner. In embodiments, the optical filter is a dichroic or dielectric filter. Fluorescence from different fluorophores can be further separated by dichroic optics and projected onto the sensor array in spatially separated lines. To further suppress background from out-of-focus fluorescence signals, an optical filter with a stripe pattern can be placed in front of the camera to pass only selected fluorescence lines and reject unwanted ones. The term "optical filter" is used in accordance with its entirely ordinary meaning in the art to refer to a device for selectively passing or rejecting the passage of light having a particular wavelength, polarization, or frequency. The term can include interference filters, in which multiple layers of dielectric material pass or reflect light depending on whether the interference between reflections from the various layers is constructive or destructive. Interference filters are also referred to in the art as dichroic or dielectric filters. The term can also include absorptive filters, which prevent the passage of light having a selective wavelength or range of wavelengths by absorption. Absorptive filters include, for example, colored glass or colored liquids. Filters can have one or more specific filter transmission characteristics, including, for example, bandpass, shortpass, and longpass. Bandpass filters selectively pass light in a wavelength range defined by a central wavelength of maximum radiation transmission (Tmax) and a bandwidth, blocking the passage of light outside this range. Tmax defines the percentage of radiation transmitted at the central wavelength. Bandwidth is typically described as the full width at half maximum (FWHM), which is the range of wavelengths passed by the filter at a transmission value that is half Tmax. Bandpass filters can have FWHMs of 10 nanometers (nm), 20 nm, 30 nm, 40 nm, or 50 nm. Longpass filters selectively pass light of higher wavelengths, defined by Tmax and a cut-on wavelength. The cut-on wavelength is the wavelength at which light transmittance is half Tmax; as the wavelength increases above the cut-on wavelength, transmittance increases, and as the wavelength decreases below the cut-on wavelength, transmittance decreases.A shortpass filter selectively passes lower wavelength radiation defined by Tmax and a cutoff wavelength. The cutoff wavelength is the wavelength at which light transmittance is half Tmax; as wavelengths increase beyond the cutoff wavelength, transmittance decreases, and as wavelengths decrease below the cutoff wavelength, transmittance increases. Filters can have a Tmax of 50-100%, 60-90%, or 70-80%.

[0154] The term "synchronized" is used according to its ordinary meaning and refers to simultaneous events. For example, the sample stage is configured to synchronize with charge migration across the sensor array such that the sample stage is configured to move in concert with the charge migration. In embodiments, synchronism does not require the sample stage to move at the same speed as the charge migration. In embodiments, the sample stage moves at a stage velocity, and charges migrate across the sensor array at a charge migration velocity, and the stage velocity and charge migration velocity are related (e.g., the stage velocity is a fraction or ratio of the charge migration velocity). In embodiments, synchronized events occur simultaneously. In embodiments, the sample stage is configured to move at approximately the same speed as the charge migration. In embodiments, the charge migration velocity is 1000 ns (nanoseconds) / μm, 500 ns (nanoseconds) / μm, 100 ns (nanoseconds) / μm, 50 ns (nanoseconds) / μm, or less (e.g., 40 ns (nanoseconds) / μm). In embodiments, the sample is scanned at about 1 mm2 / sec, 1.5 mm2 / sec, 5 mm2 / sec, 10 mm2 / sec, 50 mm2 / sec, or 100 mm2 / sec, where the scan rate is related to the charge transfer rate.

[0155] It is understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or variations in light thereof will be suggested to those skilled in the art and are to be included within the spirit and scope of this application and the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety and for all purposes.

Claims

1. 1. An imaging system, comprising: a sample stage moving at a sample stage velocity, the sample stage comprising a sample including a first fluorophore, a second fluorophore, a third fluorophore, and a fourth fluorophore; a first sensor array, a second sensor array, a third sensor array, and a fourth sensor array; a first light source configured to provide a first excitation beam having a first wavelength and a second light source configured to provide a second excitation beam having a second wavelength different from the first wavelength; a first optical system configured to direct a first excitation beam toward a first region of a sample and direct a second excitation beam toward a second region of a sample such that the first excitation beam and the second excitation beam are spatially separated, wherein an interaction of the first excitation beam with the first fluorophore generates a first fluorescent emission, an interaction of the second excitation beam with the second fluorophore generates a second fluorescent emission, an interaction of the first excitation beam with a third fluorophore generates a third fluorescent emission, and an interaction of the second excitation beam with a fourth fluorophore generates a fourth fluorescent emission, wherein the first and third fluorescent emissions generated by the first excitation beam are spatially separated from the second and fourth fluorescent emissions generated by the second excitation beam; a second optical system configured to direct the first fluorescent radiation to the first sensor array, the second fluorescent radiation to the second sensor array, the third fluorescent radiation to the third sensor array, and the fourth fluorescent radiation to the fourth sensor array, wherein the first fluorescent radiation impinges on the first sensor array and generates a first charge that migrates across the first sensor array, the second fluorescent radiation impinges on the second sensor array and generates a second charge that migrates across the second sensor array, the third fluorescent radiation impinges on the third sensor array and generates a third charge that migrates across the third sensor array, and the fourth fluorescent radiation impinges on the fourth sensor array and generates a fourth charge that migrates across the fourth sensor array; an imaging system, wherein the transitions of the first charge, the second charge, the third charge, and the fourth charge are synchronized with the sample stage velocity;

2. An imaging system as described in claim 1, wherein the first region and the second region are separated by 10 μm to 500 μm.

3. the second optical system includes a first surface configured to reflect the first fluorescent radiation toward the first sensor array and the third fluorescent radiation toward the third sensor array; a second surface configured to reflect the second fluorescent radiation toward the second sensor array and to reflect the fourth fluorescent radiation toward the fourth sensor array.

4. 4. The imaging system of claim 3, wherein the second optical system comprises a second optical element downstream from the first optical element and configured to focus the first fluorescent radiation, the second fluorescent radiation, the third fluorescent radiation, and the fourth fluorescent radiation.

5. 5. The imaging system of claim 4, wherein the second optical system comprises a bandpass filter configured to selectively transmit the first fluorescent radiation, the second fluorescent radiation, the third fluorescent radiation, and the fourth fluorescent radiation.

6. The imaging system of claim 5 , wherein a detection camera includes the first sensor array, the second sensor array, the third sensor array, and the fourth sensor array.

7. The imaging system of claim 3 , wherein the first optical element is a dichroic wedge.

8. the second optical system reflects the first fluorescent radiation toward the first sensor array and reflects the third fluorescent radiation toward the third sensor array; and 2. The imaging system of claim 1, comprising a first optical element configured to transmit the second fluorescent radiation toward the second sensor array and transmit the fourth fluorescent radiation toward the fourth sensor array.

9. the second optical system: a first lens downstream from the first optical element and configured to focus the first fluorescent radiation and the third fluorescent radiation; a second lens downstream from the first optical element and configured to focus the second fluorescent radiation and the fourth fluorescent radiation.

10. the second optical system: a first bandpass filter configured to selectively transmit the first fluorescent radiation and the third fluorescent radiation; a second bandpass filter configured to selectively transmit the second fluorescent radiation and the fourth fluorescent radiation.

11. The imaging system of claim 8 , wherein a first detector camera includes the first sensor array and the third sensor array, and a second detector camera includes the second sensor array and the fourth sensor array.

12. The imaging system of claim 8 , wherein the first optical element is a dichroic beam splitter.

13. The imaging system of claim 1 , wherein each sensor array is a TDI sensor array.

14. 10. The imaging system of claim 1, wherein each sensor array is between 1,000 and 20,000 pixels wide.

15. 10. The imaging system of claim 1, wherein each sensor array is between 1,000 and 20,000 pixels wide and between 10 and 300 pixels long.

16. 10. The imaging system of claim 1, wherein each sensor array is 8,000 pixels wide and 128 pixels long.

17. The imaging system of claim 1 , wherein the sample stage is a motorized translation stage.

18. The imaging system of claim 1 , wherein the sample stage comprises a position encoder, the position encoder generating a synchronization signal that synchronizes the transfer of the electric charge.

19. The imaging system of claim 1 , further comprising a collimating lens, a beam shaping lens, or a cylindrical lens.

20. The imaging system of claim 1 further comprising one or more line generators.

21. 1. A method of imaging a sample, comprising: a) directing a first excitation beam having a first wavelength and a second excitation beam having a second wavelength different from the first wavelength, spatially separating the first excitation beam toward a first region of a sample and the second excitation beam toward a second region of the sample, the sample being on a sample stage moving at a sample stage velocity, the sample comprising a first fluorophore that generates a first fluorescent emission after interaction with the first excitation beam, a second fluorophore that generates a second fluorescent emission after interaction with the second excitation beam, a third fluorophore that generates a third fluorescent emission after interaction with the first excitation beam, and a fourth fluorophore that generates a fourth fluorescent emission after interaction with the second excitation beam, respectively, wherein the first and third fluorescent emissions generated by the first excitation beam and the second and fourth fluorescent emissions generated by the second excitation beam are spatially separated; b) directing the first fluorescent radiation to impinge on a first sensor array and generate a first charge that moves across the first sensor array at a first charge velocity; directing the second fluorescent radiation to impinge on a second sensor array and generate a second charge that moves across the second sensor array at a second charge velocity; directing the third fluorescent radiation to impinge on a third sensor array and generate a third charge that moves across the third sensor array at a third charge velocity; and directing the fourth fluorescent radiation to impinge on a fourth sensor array and generate a fourth charge that moves across the fourth sensor array at a fourth charge velocity, wherein at least one of the first charge velocity and the second charge velocity is synchronized with the sample stage velocity; c) scanning the sample in the scan dimension and repeating steps a) and b) to form an image of the sample.

22. 22. The method of claim 21, wherein the method comprises imaging the sample containing four different fluorophores simultaneously.

23. 22. The method of claim 21, wherein the first excitation beam and the second excitation beam comprise an excitation line.

24. 22. The method of claim 21 , wherein scanning the sample comprises moving the sample stage.

25. 22. The method of claim 21, wherein scanning the sample comprises moving the sample stage at a constant velocity.

26. 22. The method of claim 21, further comprising storing a data representation of the image of the sample in a computer readable memory.

27. 22. The method of claim 21, wherein the excitation beam comprises UV radiation, VIS radiation, or IR radiation.

28. 22. The method of claim 21 , wherein the excitation beam comprises an excitation beam having a wavelength of 405 nm, 470 nm, 488 nm, 514 nm, 520 nm, 532 nm, 561 nm, 633 nm, 639 nm, 640 nm, 800 nm, 808 nm, 912 nm, 1024 nm, or 1500 nm.

29. 22. The method of claim 21 , wherein the sample comprises modified nucleotides.

30. 22. The method of claim 21, wherein the sample comprises an array having a plurality of individual sites.

Images