Autofocus using spot measurements

Abstract

Some implementations of the present disclosure relate to an imaging system that includes one or more image sensors and a Z-stage. The imaging system is configured to perform operations including: capturing, using the one or more image sensors, a first image of a first pair of light spots projected at a first sample position of a sample; determining whether the first image of the first pair of light spots is valid; and when the first image is determined to be valid: obtaining a current spacing distance measurement of the first pair of light spots based on the first image; and controlling the Z stage to focus the imaging system at the first sample location based at least on the current spacing distance measurement.

Description

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 410,961, entitled “SPOTERROR HANDLING FOR FOCUS TRACKING,” filed on September 28, 2022, and U.S. Provisional Patent Application No. 63 / 424,771, entitled “SPOT ERROR HANDLING FOR FOCUSTRACKING,” filed on November 11, 2022. The entire texts of all of the above applications are incorporated herein by reference. Background Art

[0002] Increasing the numerical aperture (NA) of an optical imaging system improves optical imaging resolution. In sequencing applications, this reduces the spacing between sequencing clusters and increases cluster density, making sequencing cheaper. However, increasing the NA also reduces the depth of field (DoF) - the distance at which an imaged object (e.g., a cluster) remains in focus when the object is translated along the optical axis of the optical imaging system.

[0003] As optical imaging systems with higher NA continue to be used in imaging applications to reduce costs (e.g., to reduce sequencing costs), it becomes more difficult to ensure that the imaged sample will remain in focus as it is translated along the optical axis. Figure 1A As illustrated, some current sequencers are able to establish a best fit plane for the entire sample so that the entire sample remains within the DoF of the optical imaging system, thereby removing sample tilt. Figure 1B As illustrated, as the available DoF of the optical imaging system decreases, and even with global adjustment of sample tilt, local tilt within the sample itself may produce a DoF shift large enough to cause at least a portion of the sequencing image to be out of focus, resulting in poor data quality and data loss in the degraded portion of the image. Summary of the invention

[0004] Specific implementations of the present disclosure relate to systems and methods for dynamically tilting a sample holder in one or more directions based on the local topography of the sample to maintain sample focus during sample imaging. Additional specific implementations of the present invention relate to describing techniques for handling spot errors during focus tracking, which can achieve smoother movement of the Z stage and / or tilt stage during imaging of the sample.

[0005] In one embodiment, an imaging system includes: a sample holder including a surface for supporting a sample container having a plurality of sample positions; an objective lens; an assembly including one or more actuators physically coupled to the sample holder, the one or more actuators for tilting the sample holder relative to the objective lens during imaging of the plurality of sample positions to focus the imaging system on a current sample position; a first light source for projecting a first pair of light spots onto the sample container; and a controller for controlling the one or more actuators to tilt the sample holder along a first direction of the imaging or a second direction substantially perpendicular to the first direction based on sample tilt, the sample tilt being determined from a first separation measurement of the first pair of light spots from an image captured by an image sensor at one or more of the plurality of sample positions.

[0006] In some specific implementations, the first light source is used to project the first pair of light spots and the second pair of light spots onto the sample container; and the sample tilt is determined from the first spacing measurement result and the second spacing measurement result of the second pair of light spots from the image captured by the image sensor at the one or more sample positions.

[0007] In some implementations, the sample tilt is determined at least along the second direction substantially perpendicular to the first direction of imaging; and the controller is used to control the one or more actuators to tilt the sample holder along the second direction based on the sample tilt.

[0008] In some implementations, the sample tilt is determined along the first direction and the second direction; and the controller is configured to control the one or more actuators based on the sample tilt to tilt the sample holder along the first direction and the second direction.

[0009] In some specific implementations, the sample tilt along the second direction is determined by the imaging system by the following steps: determining a first sample height based at least on the first spacing measurement result; determining a second sample height based at least on the second spacing measurement result; and calculating the sample tilt along the second direction based at least on the first sample height and the second sample height.

[0010] In some implementations, the one or more actuators include a plurality of actuators; and the controller is configured to control the plurality of actuators to center the tilt axis of the sample holder along the first direction of imaging.

[0011] In some embodiments, the controller is configured to control the one or more actuators to tilt the sample holder along the first direction or the second direction based on a tilt map determined from a plurality of separation measurements of the first pair of spots from a plurality of images captured by the image sensor at a plurality of sample positions.

[0012] In some specific implementations, the controller is configured to control the one or more actuators to tilt the sample stage along the first direction and the second direction based on a tilt map determined from a plurality of first separation measurements of the first pair of spots and a plurality of second separation measurements of the second pair of spots from a plurality of images captured by the image sensor at a plurality of sample positions.

[0013] In some specific implementations, the imaging system further includes a second light source, wherein: the second light source includes a line scanner, the line scanner is used to scan the sample during the imaging of the sample container; and the direction of the imaging is the direction in which the line scanner scans the sample container.

[0014] In some implementations, the one or more actuators include a plurality of linear actuators; the sample holder is a movable platform mounted on the plurality of linear actuators; and the plurality of linear actuators are used to tilt the sample holder along the first direction and the second direction.

[0015] In some implementations, each of the linear actuators includes a carriage driven up and down by a screw of a tilt motor.

[0016] In some implementations, the assembly further comprises a stage; the movable platform is mounted on the stage; and the stage is used to move the sample holder laterally.

[0017] In one embodiment, a method includes: projecting a first pair of light spots and a second pair of light spots at a first sample position of a sample using one or more light sources of an imaging system; capturing images of the first pair of light spots and the second pair of light spots at the first sample position using an image sensor of the imaging system; determining a first spacing distance of the first pair of light spots and a second spacing distance of the second pair of light spots based at least on the image; determining a first sample height based at least on the first spacing distance; determining a second sample height based at least on the second spacing distance; and calculating a first tilt of the sample at the first sample position along a scanning direction of the imaging system or a direction substantially perpendicular to the scanning direction based at least on the first sample height and the second sample height.

[0018] In some implementations, the method further includes: based on the first tilt of the sample, using one or more actuators of the imaging system to tilt the sample in the direction substantially perpendicular to the scanning direction of the imaging system.

[0019] In some specific implementations, calculating the first tilt of the sample includes calculating the first tilt of the sample at the first sample position along the scanning direction of the imaging system and the direction substantially perpendicular to the scanning direction based at least on the first sample height and the second sample height.

[0020] In some implementations, the method further includes: based on the first tilt of the sample, tilting the sample in the scanning direction and in the direction substantially perpendicular to the scanning direction using one or more actuators of the imaging system.

[0021] In some specific implementations, the method also includes: generating a tilt map based on the first tilt of the sample at the first sample position and additional tilts of the sample determined at additional sample positions of the sample, the tilt map including multiple entries corresponding to multiple sample tilt positions, each entry indicating an amount by which the sample is tilted along the scanning direction or in a direction substantially perpendicular to the scanning direction for the corresponding sample tilt position.

[0022] In some implementations, the method further comprises: tilting the sample in the direction substantially perpendicular to the scanning direction at each of the plurality of sample tilt positions using one or more actuators of the imaging system based on the tilt map.

[0023] In one embodiment, an imaging system includes: an image sensor; a component including one or more actuators, the one or more actuators being used to tilt a sample holder during imaging of multiple sample positions to focus the imaging system on a current sample position; a light source, the light source being used to project a first pair of light spots and a second pair of light spots onto a sample container mounted on the sample holder; and a controller, the controller being used to control the one or more actuators to tilt the sample holder along a first direction of the imaging or a second direction substantially perpendicular to the first direction based on a sample tilt, the sample tilt being determined from images of the first pair of light spots and the second pair of light spots captured by the image sensor at one or more sample positions among the multiple sample positions.

[0024] In some specific implementations, the imaging system also includes: a row generating device, which includes: a second light source, which is used to emit a first light beam of a first wavelength; a third light source, which is used to emit a second light beam of a second wavelength; and one or more row forming optical devices, which are used to shape the first light beam into a first row and shape the second light beam into a second row.

[0025] In one embodiment, a non-transitory computer-readable medium has executable instructions stored thereon, which, when executed by a processor, cause an imaging system to perform operations including the following steps: using one or more image sensors of the imaging system to capture a first image of a first pair of light spots projected at a first sample position of a sample; determining whether the first image of the first pair of light spots is valid; and when the first image is determined to be valid: obtaining a current separation distance measurement result of the first pair of light spots based on the first image; and controlling components of the imaging system to focus the imaging system at the first sample position based on at least the current separation distance measurement result. For example, the Z stage of the imaging system can be controlled to focus the imaging system at the first sample position.

[0026] In some specific implementations, determining whether the first image of the first pair of light spots is valid includes: determining whether the first image of the first pair of light spots is valid based on an intensity distribution of pixels in the first image.

[0027] In some specific implementations, determining whether the first image of the first pair of light spots is valid based on the intensity distribution of the pixel in the first image includes determining: whether the intensity distribution of each of the first light spot and the second light spot in the image is a Gaussian distribution; whether there is a first peak intensity corresponding to the first light spot in the image and whether there is a second peak intensity corresponding to the second light spot in the image; whether the number of intensity peaks in the image matches the valid number; or whether one or more of the pixels are saturated.

[0028] In some implementations, determining whether the first image of the first pair of spots is valid includes: determining that the first image is invalid; and in response to determining that the first image is invalid, storing an identifier indicating a reason why the first image is invalid in a memory of the imaging system.

[0029] In some implementations, the operation further includes: before controlling the Z stage, determining that the current separation distance measurement result of the first pair of spots is within a threshold range, the threshold range being determined from a plurality of previous separation distance measurement results of the first pair of spots associated with other sample positions of the sample.

[0030] In some implementations, the threshold range is within a number of standard deviations above or below a mean of the plurality of prior interval distance measurements, the number being greater than zero and less than four.

[0031] In some implementations, the operations further include storing the current interval distance measurement result in a circular buffer including the plurality of previous interval distance measurements.

[0032] In some specific implementations, the operation further includes: when the first image is determined to be invalid, controlling the Z stage to focus on the sample at the first sample position based at least on one or more previous separation distance measurements of the first pair of spots associated with one or more other sample positions of the sample.

[0033] In some specific implementations, the one or more previous spacing distance measurements are a plurality of previous spacing distance measurements stored in a memory of the imaging system; and controlling the Z stage to focus on the sample at the first sample position based at least on the one or more previous spacing distance measurements includes: retrieving a most recent previous spacing distance measurement of the plurality of previous spacing distance measurements stored in the memory; and controlling the Z stage to focus on the sample at the first sample position based on the most recent previous spacing distance measurement of the plurality of previous spacing distance measurements.

[0034] In some specific implementations, the one or more previous separation distance measurements are multiple previous separation distance measurements stored in a memory of the imaging system; and controlling the Z stage to focus on the sample at the first sample position based at least on the one or more previous separation distance measurements includes: controlling the Z stage to focus on the sample at the first sample position based on a combination of two or more of the previous separation distance measurements.

[0035] In some specific implementations, the operation also includes: capturing, using the one or more image sensors of the imaging system, a second image of a second pair of light spots projected at the first sample position that is substantially parallel to the first pair of light spots; determining whether the second image of the second pair of light spots is valid; and when the second image is determined to be valid, obtaining a current separation distance measurement result of the second pair of light spots based on the second image.

[0036] In some specific implementations, the operation also includes: when the first image is determined to be invalid and the second image is determined to be valid, controlling the Z stage of the imaging system to focus the imaging system on the first sample position based at least on the current separation distance measurement result of the second pair of spots.

[0037] In some specific implementations, the operation also includes: when the first image is determined to be invalid and the second image is determined to be valid, controlling the Z stage of the imaging system to focus the imaging system at the first sample position based at least on the current separation distance measurement result of the second pair of spots and one or more previous separation distance measurement results of the first pair of spots associated with one or more other sample positions of the sample.

[0038] In some specific implementations, the operation also includes: when both the first image and the second image are determined to be valid, controlling the Z stage of the imaging system to focus the imaging system on the first sample position based at least on the current separation distance measurement result of the first pair of spots and the current separation distance measurement result of the second pair of spots.

[0039] In some specific implementations, controlling the Z-stage of the imaging system to focus the imaging system on the first sample position based at least on the current spacing distance measurement result of the first pair of spots and the current spacing distance measurement result of the second pair of spots includes: determining an average value of the current spacing distance measurement result of the first pair of spots and the current spacing distance measurement result of the second pair of spots; and controlling the Z-stage of the imaging system to focus the imaging system on the first sample position based at least on the average value.

[0040] In some implementations, the operation further includes: when both the first image and the second image are determined to be valid, determining a tilt measurement result based on a difference between the current separation distance measurement result of the first pair of spots and the current separation distance measurement result of the second pair of spots.

[0041] In some implementations, the operation also includes determining whether a current sample tilt measurement result is within a threshold range determined from a previous sample tilt measurement result.

[0042] In one embodiment, a system includes: one or more light sources to project a first pair of light spots at a first sample position of a sample; one or more image sensors to capture a first image of the first pair of light spots projected at the first sample position; and one or more processors to perform operations including the following steps: determining whether the first image of the first pair of light spots is valid; and when the first image is determined to be valid: obtaining a current separation distance measurement result of the first pair of light spots based on the first image; and controlling a Z stage to focus on the first sample position based on at least the current separation distance measurement result. In some specific implementations, other components other than or in addition to the Z stage can be controlled to focus on the first sample position.

[0043] In some specific implementations, determining whether the first image of the first pair of light spots is valid includes: determining whether the first image of the first pair of light spots is valid based on an intensity distribution of pixels in the first image.

[0044] In some specific implementations, the system also includes a memory for storing one or more previous spacing distance measurement results associated with one or more other sample positions of the sample, and the operation also includes: when the first image is determined to be invalid, controlling the Z stage to focus on the sample at the first sample position based at least on the one or more previous spacing distance measurement results.

[0045] In some specific implementations, the one or more light sources are used to project a second pair of light spots substantially parallel to the first pair of light spots at the first sample position; and the one or more image sensors are used to capture a second image of the second pair of light spots projected at the first sample position.

[0046] In some specific implementations, the one or more light sources include: a first light source, which is used to project the first pair of light spots at the first sample position; and a second light source, which is used to project the second pair of light spots at the first sample position; and the one or more image sensors include: a first image sensor, which is used to capture the first image; and a second image sensor, which is used to capture the second image.

[0047] In some implementations, the first image sensor and the second image sensor are substantially parallel linear sensors.

[0048] In some specific implementations, the operation further includes: determining whether the second image of the second pair of light spots is valid; and when the second image is determined to be valid, obtaining a current separation distance measurement result of the second pair of light spots based on the second image.

[0049] In some implementations, the operation further includes: when the first image is determined to be invalid and the second image is determined to be valid, controlling the Z stage to focus on the first sample position based at least on the current separation distance measurement result of the second pair of spots.

[0050] In some specific implementations, the system also includes a tilt assembly, which includes one or more actuators, which are used to tilt a sample holder of the sample during imaging of the sample; and the operation also includes: when both the first image and the second image are determined to be valid, determining a tilt measurement result based on a difference between the current separation distance measurement result of the first pair of spots and the current separation distance measurement result of the second pair of spots; and controlling the tilt assembly to tilt the sample holder to focus on the first sample position based on at least the tilt measurement result.

[0051] In some implementations, the system further includes the Z stage, and the Z stage includes an objective lens.

[0052] Other features and aspects of the disclosed technology will become apparent from the following detailed description in conjunction with the accompanying drawings, which illustrate features of specific implementations according to the disclosed technology by way of example. The Summary of the Invention is not intended to limit the scope of any invention described herein, which is defined by the claims and equivalents. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] The present disclosure according to one or more specific implementations is described in detail with reference to the following drawings. These drawings are provided for illustrative purposes only and only describe example implementations. In addition, it should be noted that for clarity and ease of description, the elements in the drawings are not necessarily drawn to scale.

[0054] Some of the drawings included herein illustrate various implementations of the disclosed technology from different perspectives. Although the accompanying descriptive text may refer to such views as "top", "bottom" or "side", such references are merely descriptive and do not imply or require that the disclosed technology be implemented or used in a specific spatial orientation unless otherwise explicitly stated.

[0055] Figure 1A De-tilting of a sample by establishing a best-fit plane for the entire sample is illustrated.

[0056] Figure 1B Illustrated is a local tilt within a sample which produces a depth of field shift causing a portion of the sample image to be out of focus.

[0057] Figure 2 A block diagram of an optical imaging system according to some implementations of the present disclosure is illustrated.

[0058] Figure 3 According to some specific implementations of the present disclosure, Figure 2 A perspective view of optical components used in conjunction with the optical system shown.

[0059] Figure 4 yes Figure 3 A plan view of the optical assembly is shown.

[0060] Figure 5 Illustrated when Figure 3 Optical components shown with incident and reflected light beams focused relative to an object.

[0061] Figure 6 The detector surface is shown as Figure 5 The beam spot provided by the reflected beam is shown.

[0062] Figure 7 Illustrated when Figure 3 Incident and reflected beams with the optical components shown below focus.

[0063] Figure 8 The detector surface is shown as Figure 7 The beam spot provided by the reflected beam is shown.

[0064] Fig. 9 Illustrated when Figure 3 Incident and reflected beams when the optical components are shown in focus.

[0065] Fig.10 The detector surface is shown as Fig. 9 The beam spot provided by the reflected beam is shown.

[0066] Fig.11 A series of graphs illustrating surface profiles of flow cell samples scanned using a line scanning system are shown according to some implementations of the present disclosure.

[0067] Fig.12 A coordinate system that may be used when designing an assembly that dynamically moves a sample in lateral and / or angular directions according to some implementations of the present disclosure is shown.

[0068] Fig.13 A sample stage assembly configured to mount a movable platform that accommodates a sample container is shown according to some implementations of the present disclosure.

[0069] Fig.14 A movable platform mounted on an actuator of a dump tilt assembly according to an implementation of the present disclosure is depicted.

[0070] Fig.15 Depicted is a movable platform on a sample stage assembly according to some implementations of the present disclosure.

[0071] Fig.16A An exploded view of an actuator according to some implementations of the present disclosure is shown.

[0072] Fig. 16B Shows Fig.16A Perspective view of the actuator.

[0073] Fig. 16C Shows Fig.16A Side view of the actuator.

[0074] Fig.16D Shows Fig.16A Another perspective view of the actuator.

[0075] Fig.17 Some specific implementations of the present disclosure are shown. Fig.14 The underside of the movable platform.

[0076] Fig.18Some specific implementations of the present disclosure are shown. Fig.14 A perspective view of a movable platform.

[0077] Fig.19 A movable platform is shown interfacing and coupled to an actuator according to some implementations of the present disclosure.

[0078] Fig. 20 A process of using two pairs of projected spot beams to determine the tilt of a sample along its row scanning direction according to some specific implementations of the present disclosure is described.

[0079] Fig.21A is an operational flow chart illustrating an example method of dynamically tilting a sample based on a predetermined tilt trajectory according to some implementations of the present disclosure.

[0080] Fig. 21B Tilt angles at different sample positions of a tilt map according to some implementations of the present disclosure are illustrated.

[0081] Fig. 21C The smooth fitting of tilted trajectories in the z direction based on different tilt angles according to some specific implementations of the present disclosure is conceptually illustrated.

[0082] Fig. 22 is a block diagram illustrating an example mechanism for tilting a sample holder according to some implementations of the present disclosure.

[0083] Fig.23A is an operational flow chart illustrating an example method of determining a tilt map along a scan direction according to some specific implementations of the present disclosure.

[0084] Fig. 23B Depicted in Fig.23A A focus tracking module projects a pair of spot beams on the surface of the sample at different sample positions in the scanning direction during the process.

[0085] Fig.23C A smooth tilt trajectory generated by interpolating between five entries corresponding to spot beam measurements according to some implementations of the present disclosure is illustrated.

[0086] Fig.24A is an operational flow chart illustrating an example method of determining a tilt map along a scan direction and a direction orthogonal to the scan direction according to some specific implementations of the present disclosure.

[0087] Fig. 24B Depicted in Fig.24A A focus tracking module projects two pairs of spot beams on the surface of the sample at different sample positions in the scanning direction during the process.

[0088] Fig.24CA process of using two pairs of projected spot beams to determine the tilt of a sample in a scanning direction and a direction orthogonal to the scanning direction according to some specific implementations of the present disclosure is described.

[0089] Fig.25A Two pairs of projection beam spots and a relative orientation of two scan lines in a dual-channel, line-scan imaging system according to some implementations of the present disclosure are illustrated.

[0090] Fig.25B Another relative orientation of two pairs of projection beam spots and two scan lines in a dual-channel, line-scan imaging system according to some specific implementations of the present disclosure is illustrated.

[0091] Fig.25C Another relative orientation of two pairs of projection beam spots and two scan lines in a dual-channel, line-scan imaging system according to some specific implementations of the present disclosure is illustrated.

[0092] Fig.25D Another relative orientation of two pairs of projection beam spots and two scan lines in a dual-channel, line-scan imaging system according to some specific implementations of the present disclosure is illustrated.

[0093] Fig.25E Another relative orientation of two pairs of projection beam spots and two scan lines in a dual-channel, line-scan imaging system according to some specific implementations of the present disclosure is illustrated.

[0094] Fig.25F Another relative orientation of two pairs of projection beam spots and two scan lines in a dual-channel, line-scan imaging system according to some specific implementations of the present disclosure is illustrated.

[0095] Fig.26 The advantages of using multiple tilt actuators according to some implementations of the present disclosure are conceptually illustrated.

[0096] Fig. 27 is a block diagram illustrating some components of an example focus control system for focus tracking according to some implementations of the present disclosure.

[0097] Fig.28 is a diagram illustrating an example architecture of a Z-stage controller according to some specific implementations of the present disclosure.

[0098] Fig.29 Depicted are examples of the design and operation of a focus tracking system utilizing two pairs of focus tracking spots per sample surface in accordance with some implementations of the present disclosure.

[0099] Fig. 30A Depicts the light incident on the spot pair as Fig.29 The pixel intensity of a sensor as a function of the sensor pixel number.

[0100] Fig. 30B Depicts the light incident on the spot pair as Fig.29 Another sensor's pixel intensity as a function of its pixel number.

[0101] Fig.31 Describes the use of reference Fig.29 One example implementation of a focus tracking system is described in both the object plane and the sensor plane during scanning of the flow cell surface.

[0102] Fig.32 is a graph showing the Z step size (in nm) as a function of time for a Z stage used with a focus tracking system that does not implement spot error handling.

[0103] Fig.33A An image of a portion of a flow cell with a large bubble is depicted.

[0104] Fig.33B A graph is depicted showing the difference in the light spot error when the spot error processing is turned off. Fig.33A Focus tracking performance (including summary of out-of-focus errors) when performing focus tracking on a flow cell.

[0105] Fig.33C A graph is depicted showing that when the spot error processing is turned on, Fig.33A Focus tracking performance (including summary of out-of-focus errors) when performing focus tracking on a flow cell.

[0106] Fig.34A Depicted is an image of a portion of a flow cell having a defect that causes the focus tracking spot of the sensor to saturate over approximately eight frames.

[0107] Fig.34B A graph is depicted showing the difference in the light spot error when the spot error processing is turned off. Fig.34A Focus tracking is performed on the flow cell with spot error processing turned on. Fig.34A Focus tracking performance (including summary of out-of-focus errors) when performing focus tracking on a flow cell for simulation.

[0108] Fig.35A Depicted is an image of a portion of a flow cell with a large bubble that causes poor measurements of the sensor's focus tracking spot over approximately fifty frames.

[0109] Fig.35B A graph is depicted showing the difference in the light spot error when the spot error processing is turned off. Fig.35A Focus tracking is performed on the flow cell with spot error processing turned on. Fig.35AFocus tracking performance (including summary of out-of-focus errors) when performing focus tracking on a flow cell for simulation.

[0110] Fig.36A Depicted is an image of a portion of a flow cell having debris (appearing as black spots) on the surface, which causes poor measurement of the focus tracking spot.

[0111] Fig.36B A graph is depicted showing the difference in the light spot error when the spot error processing is turned off. Fig.36A Focus tracking performance (including summary of out-of-focus errors) when performing focus tracking on a flow cell.

[0112] Fig.36C A graph is depicted showing that when the spot error processing is turned on, Fig.36A Focus tracking performance (including summary of out-of-focus errors) when performing focus tracking on a flow cell.

[0113] Fig.37A Depicted is an image of a portion of a flow cell having residue (appearing as a black pattern) on the surface, which results in poor measurement of the focus tracking spot.

[0114] Fig.37B A graph is depicted showing the difference in the light spot error when the spot error processing is turned off. Fig.37A Focus tracking performance (including summary of out-of-focus errors) when performing focus tracking on a flow cell.

[0115] Fig.37C A graph is depicted showing that when the spot error processing is turned on, Fig.37A Focus tracking performance (including summary of out-of-focus errors) when performing focus tracking on a flow cell.

[0116] Fig.38A Depicted is a graph showing focus tracking performed on a flow cell using a four-beam, dual-sensor focus tracking system without spot error processing, where there are frequent Z-step spikes due to short-duration pulse interference with the measurement results of the focus tracking spot on one or both detectors.

[0117] Fig.38B Depicted is a diagram showing the use of a four-beam, dual-sensor focus tracking system with spot error handling in Fig.38A Figure 2 is a graph of focus tracking performed on the same flow cell, where the Z-step spikes caused by short pulses of the focus tracking spot interfering with the measurement have been eliminated.

[0118] Fig.39 is a flow chart illustrating an example method of implementing spot error processing according to some specific implementations of the present disclosure.

[0119] Fig.40 is a flow chart depicting a method of spot error processing in a system for performing focus tracking using at least two pairs of substantially parallel spots according to some implementations of the present disclosure.

[0120] Fig.41 is a flow chart depicting a method of detecting tilt spikes in a system for performing focus tracking using at least two pairs of substantially parallel light spots projected onto at least two locations on a sample according to some implementations of the present disclosure.

[0121] The drawings are not exhaustive and do not limit the disclosure to the precise forms disclosed. DETAILED DESCRIPTION

[0122] As used herein to refer to a sample, the term "feature" is intended to mean a point or region in a pattern that is distinguishable from other points or regions based on relative position. A single feature may include one or more molecules of a particular type. For example, a feature may contain a single target nucleic acid molecule having a particular sequence, or a feature may contain several nucleic acid molecules having the same sequence (and / or its complement).

[0123] As used herein, the term "scanning swath" is intended to mean a rectangular portion of an object. A scanning swath may be an elongated strip that is scanned by relative movement between the object and the detector in a direction parallel to the longest dimension of the strip. Generally, the width of the rectangular portion or strip will be constant along its entire length. Multiple scanning swaths of an object may be parallel to each other. Multiple scanning swaths of an object may be adjacent to each other, overlap each other, abut each other, or be separated from each other by gap regions.

[0124] As used herein, the term "tile" generally refers to one or more images of the same area of ​​a sample, wherein each of the one or more images may represent a corresponding color channel. A tile may form a subset of imaging data of an imaging data set of one imaging cycle.

[0125] As used herein, the term "xy coordinates" is intended to mean information that specifies a position, size, shape, and / or orientation in an xy plane. The information may be, for example, numerical coordinates in a Cartesian system. The coordinates may be provided relative to one or both of the x-axis and the y-axis, or may be provided relative to another position in the xy plane. For example, the coordinates of a feature of an object may specify the position of the feature relative to a reference or other feature of the object.

[0126] As used herein, the term "xy plane" is intended to refer to a 2-dimensional region defined by linear axes x and y. When used with reference to a detector and an object observed by the detector, the region may be further specified as being orthogonal to the viewing direction between the detector and the object being detected. When used herein to refer to a line scanner, the term "y direction" refers to the scanning direction.

[0127] As used herein, the term "z-coordinate" is intended to mean information specifying the position of a point, line, or area along an axis orthogonal to the xy plane. In a particular implementation, the z-axis is orthogonal to the area of ​​the object observed by the detector. For example, the focus direction of an optical imaging system may be specified along the z-axis.

[0128] As used herein, the term "scan" is intended to mean a 2-dimensional cross-section of the detection object in the xy plane, which cross-section is rectangular or oblong. For example, in the case of fluorescence imaging, a region of the object having a rectangular or oblong shape can be specifically excited (to the exclusion of other regions) and / or the emission from this region can be specifically acquired (to the exclusion of other regions) at a given time point in the scan.

[0129] As mentioned above, there is a growing need to implement dynamic tilting of samples in optical imaging systems that utilize higher NA to resolve finer optical features at the expense of DoF. In such systems, even a small amount of twisting of the sample that defocuses a portion of the sample within the field of view can result in significant errors. A small amount of tilt in the scan direction or in a direction perpendicular to the scan direction can blur or defocus the region of interest being scanned. Therefore, dynamic, multi-axis tilting of the sample is needed.

[0130] Various embodiments of the present disclosure relate to systems and methods for dynamically adjusting one or more components of an imaging system based on the local topography of the sample to maintain sample focus during sample scanning. One set of embodiments describes a sample stage assembly including a tip-tilt assembly to achieve multi-axis tilt of a sample holder during scanning. Another set of embodiments describes techniques for determining local sample tilt along a scan direction and / or in a direction orthogonal to the scan direction using a focus tracking system. Another set of embodiments describes techniques for generating a tilt map that is utilized by a controller to guide the operation of a component that dynamically tilts a sample holder during sample scanning. Another set of embodiments describes techniques for handling spot errors during focus tracking that can achieve smoother movement of a Z stage and / or a tilt stage during imaging of a sample.

[0131] Figure 2A block diagram of an optical imaging system 100 according to some specific implementations of the present disclosure is illustrated. The optical imaging system 100 images a sample of interest (object 110) for analysis. For example, in a specific implementation in which the optical imaging system 100 is used as a sequencer, the imaged sample can be sequenced. In some specific implementations, the optical imaging system 100 can be used as a profiler for determining the surface profile (e.g., topography) of the imaged object. In addition, various other types of optical imaging systems can use the mechanisms and systems described herein. In the illustrated embodiment, the optical imaging system 100 includes an optical assembly 106, an object holder 102 for supporting an object 110 near a focal plane FP of the optical assembly 106, and a stage controller 115 configured to move the object holder 102 in a lateral direction (along an X-axis and / or Y-axis extending into a page), in a vertical / elevational direction along the Z-axis, and / or in an angular direction along the X-axis (tilt), the Y-axis (tilt), and / or the Z-axis (twist). The optical imaging system 100 may also include a system controller or computing system 120 operably coupled to the optical assembly 106 , the stage controller 115 , and / or the object holder 102 .

[0132] In some implementations, object 110 is a sample container including a biological sample imaged using one or more fluorescent dyes. For example, in a specific implementation, the sample container may be implemented as a patterned flow cell, which includes a translucent cover, a substrate or a liquid sandwiched therebetween, and the biological sample may be located at the inner surface of the translucent cover or the inner surface of the substrate or the flow cell. The flow cell may include a large number (e.g., thousands, millions, or billions) of holes or regions that are patterned on the substrate to define an array (e.g., a hexagonal array, a rectangular array, etc.). Each region may form a cluster (e.g., a monoclonal cluster) of a biological sample (such as DNA, RNA, or another genomic material) that may be sequenced, for example, using sequencing by synthesis. The flow cell may be divided into a plurality of physically separated lanes (e.g., eight lanes), each of which includes an array of clusters. During each sequencing cycle, each surface (e.g., upper and lower) of each lane may be imaged in separate scan bands (e.g., three), and any number of images or tiles may be collected for each scan band.

[0133] Although not shown, the optical imaging system 100 may include one or more subsystems or devices for performing various assay protocols. For example, in the case where the sample includes a flow cell with a flow channel, the optical imaging system 100 may include a fluid control system, which includes a reservoir coupled to the flow channel through a fluid network. The fluid control system may direct the flow of reagents (e.g., fluorescently labeled nucleotides, buffers, enzymes, cleavage reagents, etc.) to (and through) a sample container and a waste valve. Another subsystem that may be included is a temperature control system, which may have a heater / cooler configured to adjust the temperature of the sample and / or the fluid flowing through the sample. The temperature control system may include a sensor for detecting the temperature of the fluid.

[0134] As shown, the optical assembly 106 is configured to direct input light to the object 110, and receive output light and direct the output light to one or more detectors. The output light may be input light that is at least one of reflected and refracted by the object 110, and / or the output light may be light emitted from the object 110. To direct the input light, the optical assembly 106 may include at least one reference light source 112 and at least one excitation light source 114 that directs light (such as a light beam having a predetermined wavelength) through one or more optical components of the optical assembly 106. The optical assembly 106 may include various optical components, including a conjugate lens 118 for directing the input light to the object 110 and directing the output light to the detector.

[0135] The reference light source 112 may be used by a distance measurement system and / or a focus control system (or a focus mechanism) of the optical imaging system 100, and when the object 110 includes a biological or chemical sample, the excitation light source 114 may be used to excite the biological or chemical substances of the object 110. The excitation light source 114 may be arranged to illuminate the bottom surface of the object 110, such as in TIRF imaging, or may be arranged to illuminate the top surface of the object 110, such as in epifluorescence imaging. Figure 2 As shown, the conjugate lens 118 directs the input light to a focal region 122 located within the focal plane FP. The lens 118 has an optical axis 124 and is positioned at a working distance WD1 from the object 110 measured along the optical axis 124. The stage controller 115 can move the object 110 in the Z direction to adjust the working distance WD1 so that, for example, a portion of the object 110 is located within the focal region 122. In addition, the stage controller 115 can actively orient the region of interest of the object 100 within the FP by rotating the object holder 102 around the X-axis, the Y-axis, and / or the Z-axis.

[0136] In order to determine whether the object 110 is in focus (i.e., substantially within the focus region 122 or the focal plane FP), the optical assembly 106 is configured to direct at least a pair of light beams to the focus region 122 where the object 110 is substantially located. The object 110 reflects the light beams. More specifically, the outer surface of the object 110 or the interface within the object 110 reflects the light beams. The reflected light beams are then returned and propagated through the lens 118. As shown, each light beam has an optical path including a portion that has not been reflected by the object 110 and a portion that has been reflected by the object 110. The portions of the light path before reflection are designated as incident light beams 130A and 132A and are indicated by arrows pointing to the object 110. The portions of the light path that have been reflected by the object 110 are designated as reflected light beams 130B and 132B and are indicated by arrows pointing away from the object 110. For illustrative purposes, the light beams 130A, 130B, 132A, and 132B are shown as having different optical paths within the lens 118 and near the object 110. However, in this embodiment, light beams 130A and 132B propagate in opposite directions and are configured to have the same or substantially overlapping optical paths within lens 118 and near object 110, and light beams 130B and 132A propagate in opposite directions and are configured to have the same or substantially overlapping optical paths within lens 118 and near object 110.

[0137] exist Figure 2 In the illustrated embodiment, light beams 130A, 130B, 132A, and 132B pass through the same lens used for imaging. In an alternative embodiment, light beams used for distance measurement or focus determination may pass through different lenses that are not used for imaging. In this alternative embodiment, lens 118 is dedicated to passing light beams 130A, 130B, 132A, and 132B for distance measurement or focus determination, and a separate lens (not shown) is used to image object 110. Similarly, it should be understood that the systems and methods for focus determination and distance measurement described herein can occur using a common objective lens shared with the imaging optics, or alternatively, the objective lens listed herein can be dedicated to focus determination or distance measurement.

[0138] The reflected light beams 130B and 132B propagate through the lens 118 and may optionally be further guided by other optical components of the optical assembly 106. As shown, the reflected light beams 130B and 132B are detected by at least one focus detector 144. In the illustrated embodiment, both the reflected light beams 130B and 132B are detected by a single focus detector 144. The reflected light beams can be used to determine the relative spacing RS1. For example, the relative spacing RS1 can be determined by the distance (i.e., the spacing distance) separating the beam spots that strike the reflected light beams 130B and 132B relative to the focus detector 144. The relative spacing RS1 can be used to determine the degree of focus of the optical imaging system 100 relative to the object 110. However, in an alternative embodiment, each reflected light beam 130B and 132B can be detected by a separate corresponding focus detector 144, and the relative spacing RS1 can be determined based on the position of the beam spot on the corresponding focus detector 144.

[0139] If the object 110 is not within a sufficient degree of focus, the computing system 120 may operate the stage controller 115 to move the object holder 102 to a desired position. As an alternative or in addition to moving the object holder 102, the optical assembly 106 may be moved in the Z direction and / or along the XY plane. For example, if the object 110 is above the focal plane FP (or the focal region 122), the object 110 may be relatively moved toward the focal plane FP by a distance ΔZ1, or if the object 110 is below the focal plane FP (or the focal region 122), the object 110 may be relatively moved toward the focal plane FP by a distance ΔZ2. In some embodiments, the optical imaging system 100 may replace the lens 118 with another lens 118 or other optical component to move the focal region 122 of the optical assembly 106.

[0140] The above and related aspects have been given with respect to a system for controlling focus or for determining a degree of focus. Figure 2 The system can also be used to determine (e.g., based on the relative spacing of the reflected light beams) a working distance WD1 between the object 110 and the lens 118. In such embodiments, the focus detector 144 can be used as a working distance detector, and the distance separating the beam spots on the working distance detector can be used to determine the working distance between the object 110 and the lens 118.

[0141] In addition, as further described below, the system can be used to determine the surface profile of the object 110 along one or more dimensions of the object. For example, by determining the change in the relative spacing of the reflected light beams at different locations along the object, the change in the working distance between the object 110 and the lens 118 along the imaging direction can be determined, and this can be mapped to the object height along the imaging direction (i.e., in the z-direction). In a specific implementation further described below, the optical assembly 106 is configured to direct multiple pairs (e.g., at least two pairs) of light beams along different locations of the surface of the scanned object. Based on the relative spacing of each pair of light beams in the multiple pairs of light beams and the distances between different pairs of light beams, the surface profile of the object can be determined in one or more dimensions. With the surface profile of the object known, the optical imaging system 100 can actively orient the region of interest of the object 100 within the FP by rotating the object holder 102 around the X-axis, the Y-axis, and / or the Z-axis via the stage controller 115.

[0142] Thus, the systems and methods described herein may be used to control focus or determine the degree of focus, determine the working distance between an object and a lens, determine the surface profile of an object, and / or linearly or rotationally orient a holder that holds an imaging object to keep the object in focus.

[0143] In one embodiment, during operation, the excitation light source 114 directs input light (not shown) onto the object 110 to excite the fluorescently labeled biological or chemical substance. The marker of the biological or chemical substance provides a light signal 140 (also referred to as light emission) having a predetermined wavelength. The light signal 140 is received by the lens 118 and then directed to at least one object detector 142 by other optical components of the optical assembly 106. Although the illustrated embodiment shows only one object detector 142, the object detector 142 may include multiple detectors. For example, the object detector 142 may include: a first detector configured to detect light of one or more wavelengths; and a second detector configured to detect light of one or more different wavelengths. The optical assembly 106 may include a lens / filter assembly that directs different light signals along different optical paths to corresponding object detectors.

[0144] The object detector 142 transmits object data associated with the detected light signal 140 to the computing system 120. The computing system 120 may then record, process, analyze, and / or transmit the data to other users or computing systems (including remote computing systems) via a communication line (e.g., the Internet). As an example, the object data may include imaging data that is processed to generate images of the object 110. These images may then be analyzed by the computing system and / or a user of the optical imaging system 100. In other embodiments, the object data may include not only light emissions from biological or chemical substances, but also light that is at least one of reflected and refracted by an optical substrate or other component. For example, the light signal 140 may include light that has been reflected by an encoded particle (such as a holographically encoded optical identification element).

[0145] In some embodiments, a single detector can provide both functions as described above with respect to object detector 142 and focus detector 144. For example, a single detector can detect a pair of reflected light beams (e.g., reflected light beams 130B and 132B) as well as an optical signal (e.g., optical signal 140).

[0146] The optical imaging system 100 may include a user interface 125 for interacting with a user through the computing system 120. For example, the user interface 125 may include: a display (not shown) that displays and requests information from the user; and a user input device (not shown) that is used to receive user input.

[0147] The computing system 120 may include an object analysis module 150 and a focus control module 152, among other things. The focus control module 152 is configured to receive focus data obtained by the focus detector 144. The focus data may include a signal representing a beam spot incident on the focus detector 144. The data may be processed to determine a relative spacing (e.g., a spacing distance between the beam spots). The degree of focus of the optical imaging system 100 relative to the object 110 may then be determined based on the relative spacing. In a particular embodiment, a working distance WD1 between the object 110 and the lens 118 may be determined. Similarly, the object analysis module 150 may receive object data obtained by the object detector 142. The object analysis module may process or analyze the object data to generate an image of the object.

[0148] In addition, the computing system 120 may include any processor-based or microprocessor-based system, including systems using microcontrollers, reduced instruction set computers (RISC), application specific integrated circuits (ASICs), field programmable gate arrays (FPGAs), logic circuits, and any other circuits or processors capable of performing the functions described herein. The above examples are exemplary only and are therefore not intended to limit the definition and / or meaning of the term system controller in any way. In one embodiment, the computing system 120 executes a set of instructions stored in one or more storage elements, memories, or modules to obtain at least one of the object data and the analysis object data. The storage element can be in the form of an information source or physical memory element within the optical imaging system 100.

[0149] The set of instructions may include various commands that direct the optical imaging system 100 to perform a particular protocol. For example, the set of instructions may include various commands for performing an assay and imaging the object 110, for moving the object holder 102 linearly or rotationally, or for determining a surface profile of the object 110. The set of instructions may be in the form of a software program.

[0150] As described above, the excitation light source 114 generates excitation light that is directed onto the object 110. The excitation light source 114 may generate one or more laser beams at one or more predetermined excitation wavelengths. The light may be moved in a raster pattern over portions of the object 110, such as groups of columns and rows of the object 110. Alternatively, the excitation light may illuminate one or more complete areas of the object 110 at once and successively stop through these areas in a "step-by-step illumination" scanning pattern.

[0151] In some implementations, the excitation light source 114 utilizes line scanning to image the sample. For example, the excitation light source 114 can be implemented as part of a line generation module, which includes one or more light sources operating at one or more wavelengths and a beam shaping optical device aligned at a predetermined angle with each light source. The beam shaping optical device can be used to provide uniform line illumination at a desired aspect ratio. In a specific implementation, the line generation module is implemented as part of a dual-channel imaging system, which includes a first light source operating at a first wavelength and a second light source operating at a second wavelength. For example, the first wavelength can be a "green light" wavelength (e.g., from about 520nm to 565nm), and the second wavelength can be a "red light" wavelength (e.g., from about 625nm to 740nm). Such a line scanning system can be used in conjunction with a TDI sensor.

[0152] Object 110 generates light signals 140, which may include light emissions generated in response to illumination of markers in object 110, and / or light that has been reflected or refracted by an optical substrate of object 110. Alternatively, light signals 140 may be generated without illumination based solely on emission properties of materials within object 110 (e.g., radioactive or chemiluminescent components in the object).

[0153] The object detector 142 and the focus detector 144 can be, for example, a photodiode or a camera. In some embodiments herein, the detectors 142 and 144 can include a charge coupled device (CCD) camera (e.g., a time delay integration (TDI) CCD camera) that can interact with various filters. The camera is not limited to a CCD camera, and other camera and image sensor technologies can be used. In a specific embodiment, the camera sensor can have a pixel size of about 1 μm to about 15 μm.

[0154] Figure 3 and Figure 4 202 is formed according to one embodiment. The optical assembly 202 can be used with the optical imaging system 100 ( Figure 2 ) or other optical imaging systems. As shown, the optical assembly 202 includes an optical system 240 of optical components 241-245 that guide light beams 230 and 232 along an optical track or route between an object of interest (not shown) and a focus detector 250. In some embodiments, the focus detector may also be referred to as a distance detector. The series of optical components 241-245 of the optical system 240 include a dual beam generator 241, a beam splitter 242, a conjugate lens 243, a beam combiner 244, and a folding mirror 245.

[0155] The optical assembly 202 includes a reference light source 212 that provides a light beam 228 to a dual beam generator 241. The reference light source 212 may emit light having a wavelength between about 620 nm and 700 nm. For example, the reference light source may be a 660 nm laser. The dual beam generator 241 provides a pair of parallel incident light beams 230A and 232A and directs the incident light beams 230A and 232A to a beam splitter 242. In the illustrated embodiment, the dual beam generator 241 includes a beam having relatively parallel surfaces 260 and 262 ( Figure 4 ). The first surface 260 reflects a portion of the light beam 228 that forms the incident light beam 230A and refracts a portion of the light beam 228. The refracted portion of the light beam 228 is reflected toward the first surface 260 by the opposing second surface 262, which forms the incident light beam 232A.

[0156] The dual beam generator 241 directs parallel incident light beams 230A and 232A toward the beam splitter 242. The beam splitter 242 reflects the incident light beams 230A and 232A toward the conjugate lens 243. In this example, the beam splitter 242 includes a pair of reflectors (e.g., aluminum-plated tabs) that are positioned to reflect the incident light beams 230A and 232A and the reflected light beams 230B and 232B. The beam splitter 242 is positioned to reflect the incident light beams 230A and 232A so that the incident light beams 230A and 232A propagate parallel to the optical axis 252 of the lens 243. The optical axis 252 extends through the center of the lens 243 and intersects the focal region 256. The lens 243 can be a near-infinity conjugate objective lens. Alternatively, the incident light beams 230A and 232A may propagate in a non-parallel manner relative to the optical axis 252. As also shown in FIG. Figure 4 As shown, when the incident light beams 230A and 232A propagate through the lens 243, the incident light beams 230A and 232A may be equally spaced apart from the optical axis 252.

[0157] As described above with respect to optical imaging system 100, incident light beams 230A and 232A may converge toward focal region 256 and be reflected by object 268 located near focal region 256 (at Figure 5 230B and 232B and propagate through lens 243. Reflected light beams 230B and 232B may propagate along substantially equal or overlapping optical paths relative to incident light beams 232A and 230A, respectively, through lens 243 and toward dual beam generator 241. More specifically, reflected light beam 230B propagates along substantially the same optical path as incident light beam 232A in an opposite direction, and reflected light beam 232B propagates along substantially the same optical path as incident light beam 230A in an opposite direction. Reflected light beams 230B and 232B leave lens 243 separated by a path spacing PS2 that is substantially equal to the path spacing separating incident light beams 230A and 232A (as shown in FIG. 230B ). Figure 4 Path spacing PS1 as shown).

[0158] like Figure 3 and Figure 4As shown, the reflected light beams 230B and 232B are incident on the dual beam generator 241 and are directed by the dual beam generator through the range limiter 254 toward the beam combiner 244. In the illustrated embodiment, the beam combiner 244 is configured to modify the path spacing PS of the separated reflected light beams 230B and 232B. The path spacing PS at the beam combiner 244 can be rescaled to be substantially equal to the separation distance SD1 of the reflected light beams 230B and 232B detected by the focus detector 250. The separation distance SD1 is the distance measured between the reflected light beams at a predetermined portion of the optical track (such as at the focus detector 250). In some embodiments, the separation distance SD1 at the focus detector 250 is less than the path spacing PS at the beam combiner 244, so that only a single focus detector 250 can detect both the reflected light beams 230B and 232B. In addition, the beam combiner 244 can make the optical path lengths of the reflected light beams 230B and 232B substantially equal.

[0159] Reflected light beams 230B and 232B propagate substantially parallel to each other between optical components after leaving lens 243. In the illustrated embodiment, reflected light beams 230B and 232B propagate substantially parallel to each other along the optical track between lens 243 and focus detector 250. As used herein, two light beams propagate "substantially parallel" if they are substantially coplanar, and if allowed to propagate infinitely, the two light beams will not intersect each other or converge / diverge relative to each other at a slow rate. For example, if the intersection angle is less than 20°, or more specifically less than 10°, or even more specifically less than 1°, the two light beams are substantially parallel. For example, reflected light beams 230B and 232B may propagate substantially parallel to each other between beam splitter 242 and dual beam generator 241, between dual beam generator 241 and beam combiner 244, between beam combiner 244 and folding mirror 245, and between folding mirror 245 and focus detector 250.

[0160] The optical system 240 can be configured to maintain a projection relationship between the reflected light beams 230B and 232B throughout the optical track so that the degree of focus can be determined. As an example, if the optical assembly 202 is focused with the object, the reflected light beams 230B and 232B will propagate parallel to each other between each optical component in the optical system 240. If the optical assembly 202 is not focused with the object, the reflected light beams 230B and 232B are coplanar, but propagate at a slight angle relative to each other. For example, when the reflected light beams 230B and 232B travel along the optical track to the focus detector 250, the reflected light beams 230B and 232B may diverge relative to each other or converge toward each other.

[0161] To this end, each optical component 241-245 may have one or more surfaces that are shaped and oriented to reflect and refract at least one of the reflected light beams 230B and 232B so that the reflected light beams 230B and 232B maintain the projection relationship between the reflected light beams 230B and 232B. For example, the optical components 242 and 245 have flat surfaces that reflect both the incident light beams 230B and 232B. The optical components 241 and 244 may also have parallel surfaces, each of which reflects one of the incident light beams 230B and 232B. Therefore, if the reflected light beams 230B and 232B are parallel, the reflected light beams 230B and 232B will remain parallel to each other after leaving each optical component. If the reflected light beams 230B and 232B diverge or converge toward each other at a certain rate, the reflected light beams 230B and 232B will converge or diverge toward each other at the same rate after leaving each optical component. Thus, the optical component along the optical track may include a flat surface that reflects at least one of the reflected light beams, or a pair of parallel surfaces, where each surface reflects a corresponding one of the reflected light beams.

[0162] The optical imaging system may include one or more optical components for determining working distance or focus as discussed above. For example, the optical imaging system may include Figure 3 and Figure 4 Two optical assemblies of the type shown may be provided to allow focus to be determined at two different locations on an object or to provide determination of a working distance between an optical imaging system and an object at two different locations. For embodiments in which more than one optical assembly is present, the optical assemblies may be discrete and separate, or the optical assemblies may share optical components. The optical assemblies may share optical components, such as a reference light source 212, a focus detector 250, a fold mirror 245, a beam combiner 244, a dual beam generator 241, a beam splitter 242, an epi-fluorescence (EPI) input reflector 280, and a range limiter 254. Optical components may be shared by placing a beam splitter upstream of the shared components in the optical system. Although the optical components are shown in FIG. Figure 3 and Figure 4 The optical components shown are examples, but one or more versions of other optical components listed herein may be present in a particular optical imaging system. In addition, a particular optical imaging system may include various combinations of the optical components described herein.

[0163] like Figure 4As shown, the reflected light beams 230B and 232B are finally incident on the detector surface 264 of the focus detector 250 at corresponding beam spots. The beam spots are spaced apart by a spacing distance SD1. The spacing distance SD1 can indicate whether the optical assembly 202 has a sufficient degree of focus relative to the object. The spacing distance SD1 on the detector surface 264 can also indicate the working distance between the lens 243 and the object being imaged.

[0164] In other embodiments, the optical components 241-245 can be replaced with alternative optical components that perform substantially the same functions as described above. For example, the beam splitter 242 can be replaced with a prism that directs the incident light beams 230A and 232A through the lens 243 parallel to the optical axis 252. The beam combiner 244 can be omitted, or it can be replaced with an optical plane that does not affect the path spacing of the reflected light beams. In addition, the optical components 241-245 can have different sizes and shapes and be arranged in different configurations or orientations as needed. For example, the optical system 240 of the optical assembly 202 can be configured for a compact design.

[0165] In addition, in an alternative embodiment, parallel beams may be provided without the dual beam generator 241. For example, the reference light source 212 may include a pair of light sources configured to provide parallel incident beams. In an alternative embodiment, the focus detector 250 may include two focus detectors arranged side by side in a fixed, known position relative to each other. Each focus detector may detect a separate reflected beam. The relative spacing between the reflected beams may be determined based on the position of the beam spot with the corresponding focus detector and the relative position of the focus detectors relative to each other.

[0166] Although in Figure 3 and Figure 4 268. Although not shown in the drawings, the optical assembly 202 may also be configured to facilitate the collection of output light projected from the object 268. For example, the optical assembly 202 may include an epifluorescence (EPI) input reflector 280 positioned to reflect incident light provided by an excitation light source (not shown). The light may be directed to a beam splitter 242, which reflects at least a portion of the excitation light and directs the light along an optical axis 252 through a lens 243. The lens 243 directs the light onto the object 268, which may provide output light. The lens 243 then receives the output light (e.g., light emission) from the object 268 and directs the output light back to the beam splitter 242. The beam splitter 242 may allow a portion of the output light to propagate through it along the optical axis. The output light may then be detected by an object detector (not shown).

[0167] like Figure 4As shown, the EPI input reflector 280 includes two passages 282 and 284 that allow the light beams 230 and 232 to propagate therein without being affected by the input reflector 280. Thus, the beam splitter 242 can reflect the incident and reflected light beams 230A, 230B, 232A, and 232B, and can also reflect the excitation light.

[0168] Figures 5 to 8 Different projection relationships between reflected light beams 230B and 232B and corresponding beam spots 270 and 272 on the detector surface 264 are shown. As discussed above, the projection relationship between the reflected light beams is based on the position of the object relative to the focal region. When the object moves relative to the focal region, the projection relationship between the reflected light beams changes, and thus the relative spacing between the reflected light beams also changes. Figures 5 to 8 exemplifies how the separation distance SD measured between the beam spots can change as the projection relationship between the reflected beams changes. However, the separation distance SD is only one way to determine the relative spacing between the reflected beams. Therefore, those skilled in the art understand that Figures 5 to 8 Only one way of determining the relative spacing is illustrated, and other ways of determining the relative spacing or projection relationship are possible.

[0169] Figures 5 and 6 It is shown that when the optical component 202 ( Figure 3 ) is focused relative to the object 268. As shown, the incident light beams 230A and 232A propagate through the lens 243 in parallel with each other and are spaced apart by a path spacing PS1. In the illustrated embodiment, the incident light beams 230A and 232A propagate parallel to the optical axis 252 of the lens 243 and are equidistant from the optical axis 252. In an alternative embodiment, the incident light beams 230A and 232A may propagate in a non-parallel manner relative to the optical axis 252 and have a different spacing therefrom. In a specific alternative embodiment, one of the incident light beams 230A or 232A coincides with the optical axis 252 of the lens 243, and the other is spaced apart from the optical axis 252.

[0170] Incident light beams 230A and 232A are directed by lens 243 to converge toward focal region 256. In such embodiments where the incident light beams are not parallel to the optical axis, the focal region may have a Figure 5 The positions shown are different. Incident light beams 230A and 232A are reflected by object 268 and form reflected light beams 230B and 232B. Reflected light beams 230B and 232B return and propagate through lens 243 and are parallel to optical axis 252. Reflected light beams 230B and 232B leave lens 243 parallel to each other and are spaced apart by path spacing PS2. When optical assembly 202 is focused, path spacings PS1 and PS2 are equal.

[0171] Thus, when the optical assembly 202 is focused, the projection relationship of the reflected light beams 230B and 232B leaving the lens 243 includes two parallel light beams. The optical system 240 is configured to maintain the parallel projection relationship. For example, when the optical assembly 202 is focused, the reflected light beams 230B and 232B are parallel to each other when leaving the dual beam generator 241, when leaving the beam combiner 244, and when reflected by the folding mirror 245. Although the projection relationship is maintained, the path spacing PS2 can be rescaled by the beam combiner.

[0172] like Figure 6 As shown, Figure 5 The reflected light beams 230B and 232B are incident on the detector surface 264 and form beam spots 270 and 272. When the optical assembly 202 is focused, the beam spots 270 and 272 have a separation distance SD 2 . Spacing distance SD 2 The separation distance SD may be based on the size of the beam combiner 244 and the angle of incidence (or a function thereof) relative to the beam combiner 244 and the parallel surfaces impinging the reflected beams 230B and 232B. 2 It is also based on the projection relationship of the reflected light beams 230B and 232B leaving the lens 243. Figure 6 As shown, the detector surface 264 has a center point or area 266. If all optical components 241-245 ( Figure 3 ) are in respective desired positions, the beam spots 270 and 272 may be equally spaced from the central region 266 along the X-axis and vertically centered within the detector surface 264. It is also shown that the beam spots 270 and 272 may have a selected topography associated with the focused optical component 202. For example, the beam spots 270 and 272 may have an Airy radius associated with the focused optical component 202.

[0173] Figures 7 and 8 It is shown that when the optical component 202 ( Figure 3 ) is below the focal point. As described above, the incident light beams 230A and 232A propagate through the lens 243 in parallel with each other and are spaced apart by the path spacing PS1. The incident light beams 230A and 232A intersect each other at the focal region 256 and are then reflected by the object 268 to form the reflected light beams 230B and 232B. However, as Figure 7 As shown, when reflected light beams 230B and 232B exit lens 243, reflected light beams 230B and 232B are slightly oriented toward optical axis 252 and converge with each other. It is also shown that path spacing PS2 is greater than path spacing PS1.

[0174] Thus, when the object 268 is located below the focal region 256, the projection relationship of the reflected light beams 230B and 232B includes two beams converging toward each other. Similar to the above, the optical system 240 is configured to maintain a converging projection relationship. For example, the reflected light beams 230B and 232B converge toward each other when leaving the dual beam generator 241, when leaving the beam combiner 244, and when reflected by the fold mirror 245.

[0175] like Figure 8 As shown, when the object 268 is located below the focal region 256, the beam spots 270 and 272 have a spacing less than the spacing distance SD2 ( Figure 6 ). The separation distance SD3 is small because the reflected light beams 130B and 132B converge toward each other over the entire optical track between the lens 243 and the focus detector 250. Figure 8 As shown, the beam spots 270 and 272 may have selected topologies associated with the beam spots 270 and 272. When the object 268 is below the focal region 256, the topologies of the beam spots 270 and 272 are different from the topologies of the beam spots 270 and 272 when the object 268 is in focus. The beam spots 270 and 272 may have different Airy radii associated with the object being below the focal region 256.

[0176] Figures 9 and 10 It is shown that when the optical component 202 ( Figure 3 ) is above the focal point. As described above, the incident light beams 230A and 232A propagate through the lens 243 in parallel with each other and are separated by the path spacing PS1. Before the incident light beams 230A and 232A reach the focal area 256, the incident light beams 230A and 232A are reflected by the object 268 to form the reflected light beams 230B and 232B. However, as Fig. 9 As shown, reflected light beams 230B and 232B diverge away from optical axis 252 and away from each other when they exit lens 243. Also shown is that path spacing PS2 is less than path spacing PS1.

[0177] Thus, when object 268 is located above focal region 256, the projection relationship of reflected light beams 230B and 232B includes two beams that diverge away from each other. Optical system 240 is configured to maintain a divergent projection relationship. For example, reflected light beams 230B and 232B diverge away from each other when leaving dual beam generator 241, when leaving beam combiner 244, and when reflected by fold mirror 245.

[0178] like Fig.10As shown, when the object 268 is located above the focus region 256, the beam spots 270 and 272 have a separation distance SD4 that is greater than the separation distance SD2. The separation distance SD4 is greater because the reflected light beams 130B and 132B diverge relative to each other over the entire optical track between the lens 243 and the focus detector 250. Fig.10 As shown, the beam spots 270 and 272 can have selected topologies associated with the beam spots 270 and 272. The topologies of the beam spots 270 and 272 when the object 268 is above the focal region 256 are different from the topologies of the beam spots 270 and 272 when the object 268 is in focus or below the focal region 256. Likewise, the beam spots 270 and 272 can have different Airy radii associated with the object being below the focal region 256.

[0179] As described above, if the object 268 is below the focus area 256, the separation distance SD3 is less than the separation distance SD2 when the object 268 is within the focus area 256. If the object 268 is above the focus area 256, the separation distance SD4 is greater than the separation distance SD2. Therefore, the optical assembly 202 not only determines that the object 268 is not within the focus area 256, but also determines the direction in which the object 268 is moved relative to the lens 243. In addition, the value of the separation distance SD3 can be used to determine how far the object 268 is moved relative to the lens 243.

[0180] like Figures 6 to 10 In addition to determining that an object is in the focus region, the measurement of the separation distance on the detector can also be used to determine the working distance between the lens and the object being detected through the lens, as illustrated in the example of . In addition, the separation distance on the detector can be used to determine the contour of the object surface.

[0181] As illustrated in the foregoing examples, the relative spacing (e.g., spacing distance) can be a function of the projection relationship (i.e., at what rate the reflected light beams 230B and 232B diverge or converge) and the length of the optical track measured from the lens 243 to the focus detector 250. When the length of the optical track between the lens 243 and the focus detector 250 increases, if the object is not focused, the spacing distance may decrease or increase. Therefore, the length of the optical track can be configured to facilitate distinguishing the spacing distances SD3 and SD4. For example, the optical track can be configured so that the converging reflected light beams do not intersect each other, and / or so that the diverging light beams do not exceed a predetermined relative spacing from each other. To this end, the optical track between the optical components of the optical system 240 can be extended or shortened as needed.

[0182] As the previous examples also illustrate, the working distance between the lens and the object being imaged (e.g., Figure 5 WD 2 , Figure 7 WD3 and Fig. 9 WD 4 ) can be determined based on the measurement of the separation distance on the detector. In addition, this can be used to determine the contour of the object surface. For example, the optical system can record the relative separation of a series of data points and associate each data point with a position along the surface. When the working distance decreases (i.e., when the height of the object surface increases), the relative separation can increase, such as Fig.10 As the working distance increases (ie, as the height of the object surface decreases), the relative spacing may decrease, as shown in FIG. Figure 8 Thus, the surface contour or topography of an object can be determined based on the relative spacing of the reflected light beams.

[0183] As discussed above, a sample may have many variations in its topography along the imaging direction (e.g., scanning direction) that cannot be accounted for by performing a single global tilt of the sample prior to imaging. Fig.11 A series of graphs illustrating the surface profile of a flow cell sample scanned using a line scanning system is shown. As depicted, the surface profile of the flow cell can vary significantly in the scan direction (eg, from about -10 μm to 10 μm).

[0184] As also discussed above, to account for local variations in sample topography, the optical imaging system may include a controller configured to dynamically move the sample holder in a lateral direction (along the X and / or Y axes extending into the page), in a vertical / elevational direction along the Z axis, and / or in an angular direction along the X axis (tilt), Y axis (tilt), and / or Z axis (twist) during imaging. To this end, it is helpful to consider the coordinate systems that can be used when designing components that dynamically move the sample in lateral and / or angular directions. Fig.12 One such example coordinate system is depicted. In this example, the z-axis is aligned with the optical axis of the objective lens and may be generally pointing upward toward the sky. The y-axis is the axis along which the sample may actively travel during imaging (e.g., during sequencing using a line scanner). For example, the sample may be a flow cell having flow cell lanes co-aligned with the Y-axis. Any movement of the sample holder (and by extension, the sample) in an angular direction may be represented as twisting the sample holder about the z-axis (e.g., a change in θz), tilting the sample holder about the y-axis (e.g., a change in θy), or tipping / tilting the sample holder about the x-axis (e.g., a change in θx).

[0185] Figures 13 to 15 Depicted are example components that can enable dynamic adjustment of the lateral and angular positions of a sample according to some implementations of the present disclosure. Fig.13 A device configured to accommodate an imaging sample ( Fig.14) of a sample stage assembly 300 of a movable platform. As illustrated in the figure, the sample stage assembly 300 includes an XY hanger 301, an XY stage 302, a controller 303 and a tilt-tilt assembly (TTA) including a plurality of actuators 304. Fig.14 An example movable platform 400 is depicted mounted on the actuator 304 of the TTA. Fig.15 A movable platform 400 is depicted mounted on the sample stage assembly 300. The movable platform 400 includes a sample container 405, which in this example is a flow cell.

[0186] In this example, the XY stage 302 is configured to move the sample holder laterally along the X-axis and the Y-axis. The TTA is configured to control the angular alignment of the sample holder to position the sample surface within the focus range of the optics of the imaging system. Fig.11 As depicted, TTA can affect all three axes of rotation. In some implementations, TTA can actively control rotation about all three axes during imaging. In other implementations, TTA can actively control rotation about only some axes during imaging. For example, rotation about only the X-axis and Y-axis can be actively controlled during the imaging process, and rotation about the Z-axis can be manually adjusted as needed.

[0187] The controller 303 may be configured to apply parameters for one or more drive signals that are applied to one or more actuators to linearly move the XY stage 302 or angularly move the movable platform 400 for each imaging operation. Generally speaking, for larger linear or rotational translations, a larger control output (e.g., one or more parameters such as a larger drive current, a larger voltage, and a larger duty cycle) will be specified. Similarly, for smaller translations, a smaller control output (e.g., a smaller drive current, a lower voltage, and a smaller duty cycle) will be specified. For example, the control output may be adjusted by adjusting the current or voltage applied to one or more actuators. In addition, in some examples, the time at which the drive signal is applied to one or more actuators may be adjusted based on the amount of translation required to change the focus. For example, in the case where the required translation is larger, the drive signal may be applied earlier. However, in other examples, the drive signal is applied as soon as possible after imaging is completed at the current sample position, regardless of the difference in focus settings. The parameters of the drive signal and the time at which the drive signal is applied may be determined based on the actuator type (e.g., piezoelectric coil vs. voice coil) and the drive requirements. Thus, drive signals may be provided to one or more actuators at different output levels to linearly move, tilt, tip, or otherwise position the sample during imaging.

[0188] In this example, TTA accomplishes θX and θY alignment through active manipulation of three linear actuators 304, whereby the sample holder is located on a movable platform 400 that is kinematically mounted to the three linear actuators 304. The actuators 304 can be sufficiently spaced apart so that relatively large displacements of these actuators can achieve small changes in the tilt of the platform. In some implementations, the 3-point kinematic mount can utilize a "3V coupling," also known as a "Maxwell coupling." In other implementations, a 3-2-1 coupling can be utilized. Although angular alignment control via the use of a 3-point kinematic mount is shown in this example, it should be understood that other types or numbers of actuators or other configurations of actuators can be utilized to achieve angular control to position the sample in a focused manner.

[0189] FIG. 16A to FIG. 16D An exploded view, a perspective view, a side view, and another perspective view of a specific actuator 304 that can be used in some specific implementations of the present disclosure are shown respectively. As depicted, the actuator 304 may include a tilt motor 304-1, an encoder 304-2, a tilt plate 304-3, a lead screw 304-4, a proximity sensor reflector 304-5, an encoder scale 304-6, a linear bearing 304-7, a bracket 304-8, a lead screw nut 304-9, and a ball 304-10. In this specific implementation, the actuator 304 acts as a linear stage with a bracket 304-8 driven up and down by a lead screw 304-4, which is integrated into the tilt motor 304-1 (e.g., a stepper motor). The linear bearing 304-7 guides the bracket 304-8 up and down, while the encoder 304-2 monitors its relative position. The encoder 304-2 can be light reflective. The scale 304-6 is located on the movable carriage, while the encoder sensor is located on the tilt plate 304-3. A separate proximity sensor can be found on the tilt plate 304-3. This proximity sensor can help the software verify the absolute position and orientation.

[0190] like FIG. 16B to FIG. 16C 304, homing of the actuator 304 may be accomplished by driving the carriage 304-8 all the way down until it contacts the motor support structure at 304-11. Alternatively, global positioning may be determined solely by relying on proximity sensors. The actuator 304 may be zeroed at the bottom of carriage travel during instrument initialization, and all actuator commands may be relative to this fully bottomed position.

[0191] like Fig.16DAs depicted, the actuator 304 may include an upper hard stop to prevent the carriage 304-8 from leaving the end of the lead screw 304-4. The hard stop may be implemented by a hard mounted machine screw 304-13 that protrudes through a slot 304-12 in the carriage 304-8. The length of the slot 304-12 may be greater than the range of normal travel of the carriage 304-8 during use of the instrument.

[0192] Fig.17 The underside of the movable platform 400 is depicted according to some implementations of the present disclosure. The underside includes three V-shaped grooves 415 configured to couple to the ball 304-10 of the actuator 304.

[0193] Fig.18 Another perspective view of the movable platform 400 is depicted. To ensure adequate stability of the system, including under external loads (such as wire and pipe disturbances that may disturb the kinematic balance of gravity), the movable platform 400 may include tension springs 425 to further increase the contact force at each coupling interface between the actuator 304 and the movable platform 400 and resist unintentional lift-off.

[0194] Fig.19 The movable platform 400 is depicted interfacing and coupled with the actuator 304. As shown, the threaded hole 425 interfaces with the compression spring fastener and the ball 304-10 interfaces with the V-groove 415.

[0195] In order to achieve dynamic tilting of the sample holder to keep the sample in focus during image scanning, there are different strategies that can potentially be adopted. In some embodiments, a feedback removal tilt mechanism can be used, whereby the tilt is measured in real time during image scanning, and the tilt measurement results are directly fed into one or more tilt motor drivers corresponding to one or more tilt actuators (e.g., one or more tilt actuators 304). As described above, the spot beam interval of the projection spot pair of the focus tracking module can be mapped to the sample height position. A light source with a wavelength between about 620nm and 700nm can be used to generate the projection spot pair. By projecting two different pairs of spots along two different scanning positions (i.e., two different Y positions), the sample height at two different positions in the scanning direction can be measured and mapped to the change in sample tilt between the two scanning positions. For example, as Fig. 20 Depicted is a diagram showing scanning of a sample 500 using a line scan 510 with a spot beam spacing Δx 1 can be mapped to the first sample height h1, and the spot beam spacing Δx 2 Can be mapped to the second sample height h2. Based on the interval L between the two pairs of spot beams yand the corresponding heights h1 and h2, the tilt angle (θy) of the sample between the two scan points can be estimated (e.g., by calculating the slope between the two points). The system controller can use the determined tilt angle to cause one or more actuators to tilt the sample holder. In some implementations of this embodiment, to ensure that the tilt is measured before imaging of the sample cross section, the system can use a forward-looking focus tracking beam that projects at least one pair of spots before reaching the line scan of the sample cross section.

[0196] While feedback-based tilting as described above can provide real-time tilt adjustment of the sample, any real-time feedback loop may be limited by: i) the maximum speed at which the sample stage can be tilted, and ii) the time delay in transmitting the real-time measurement to the tilt controller. If the local tilt changes faster than the combined time delay of the maximum tilt speed and the time delay in transmitting the real-time measurement to the tilt controller, any real-time feedback mechanism may experience a time delay that makes such an approach insufficient.

[0197] Therefore, in some embodiments, in order to adjust for this time delay problem, it may be preferable to use a technique that generates a "tilted trajectory" before the sample is tilted during the image scan. FIG. 21A to FIG. 21C Example. Specifically, Fig.21A 1 is an operational flow chart showing an exemplary method for dynamically tilting a sample based on a predetermined tilt trajectory. The method may be implemented by an optical imaging system (e.g., optical imaging system 100) that includes a suitable mechanism for dynamically tilting a stage carrying a sample (e.g., sample stage assembly 300).

[0198] Operation 610 includes determining a tilt map of the sample, the tilt map including a plurality of entries corresponding to a plurality of sample positions, each entry indicating, for a corresponding sample position, an amount by which the sample is tilted at least along a scanning direction. The tilt map may be in the form of a table, a one-dimensional array, a two-dimensional (2D) array, or other suitable data structure. For example, Fig. 21B As depicted, the tilt map may specify five tilt angles θy1 for five different sample locations. In practice, tilt maps may be generated for more than five locations, and may be constructed by estimating a smooth fit between multiple sample tilt measurements. Fig. 21C The smooth fit of the tilted trajectory in the z direction based on different tilt angles is conceptually illustrated. An initial tilt map is generated before scanning the sample. As discussed further below, the tilt map can be generated by performing a "pre-scan" at several positions along the scan direction (e.g., several Y positions along the Y direction) to determine the sample height at various positions. In the case of a sample scanned in multiple strips (e.g., a flow cell scanned in a scanning strip), the sample height can be determined at multiple Y positions in each strip.

[0199] Operation 620 includes scanning the sample during a first imaging cycle, and adjusting the tilt of the sample holder at each of the sample positions during the first imaging cycle by causing one or more actuators to tilt the sample holder at least along the scanning direction according to the tilt map. The image of the sample can be collected by moving the sample holder at a constant speed in a direction perpendicular to the length dimension of the image sensor array (e.g., TDI sensor) using a motorized stage (e.g., XY stage 302). In an embodiment in which the sample is imaged in strips (e.g., flow cell), after each strip is imaged, the motorized stage can move the sample in the X direction by a distance corresponding to the strip width. In such embodiments, a tilt map can be generated and used for each sample strip (e.g., operations 610 to 640 can be applied to each sample strip).

[0200] Operation 630 includes updating the tilt map for the next imaging cycle. Over time, the topography of the sample may change due to thermal expansion (e.g., due to excitation or other light sources that increase the sample temperature) and / or due to other changes in the sample. Therefore, in order to account for potential changes in the sample topography, the tilt map may be updated for each imaging cycle. In an alternative embodiment, the tilt map may be updated after a predetermined number of imaging cycles. In order to update the tilt map before the next imaging cycle, a tilt map measurement for the next imaging cycle may be performed during the current imaging cycle. The optical imaging system may utilize the same mechanism used to generate the original tilt map in order to generate an updated tilt map.

[0201] Operation 640 includes scanning the sample on the sample holder during a next imaging cycle, and adjusting the tilt of the sample at each of the sample positions by causing one or more actuators to tilt the sample holder at least along the scanning direction according to the updated tilt map during the next imaging cycle. Operations 630 to 640 may be repeated until all imaging cycles (e.g., sequencing cycles) are completed. In the case of a sequencer, each of the above imaging cycles may correspond to a sequencing cycle.

[0202] To illustrate one particular implementation of a system that generates a "tilt trajectory" prior to tilting a sample during an image scan, it is helpful to consider an example system that utilizes a controller of assembly 300 to perform dynamic corrections during the duration of each scan of a flow cell. For example, consider an optical imaging system that images a flow cell and has the following parameters: a scan rate of 1 Hz, a dynamic de-tilt servo update rate of about 10 Hz, a tile θY capture rate of about 100 Hz, 99 tiles per scan band, 2 surfaces per flow cell (top and bottom), 2 flow cells per instrument, 8 lanes per flow cell surface, and 4 scans per flow cell lane. In this embodiment, the system can perform 128 scans per sequencing cycle.

[0203] In the aforementioned example, it is assumed that at least 10 measurements per scan are required to characterize the required tilt correction (approximately 1 correction per 10 tiles). Therefore, for the entire sequencing cycle, a set of correction tables with 10 entries per table and 1 table per scan can be created, requiring a total of 128 10-entry tables per imaging cycle. Each correction entry can be based on the centroid calculation performed at each of the 10 measurement points along each scan (e.g., using a dual projection beam as described above). These tables can be stored in the controller of the TTA. In this example, in order to minimize the energy input into the instrument structure and maximize the quality of the tilt correction removal, a smooth tilt removal trajectory can be determined. Smooth curve fitting can be used instead of performing a piecewise linear correction between subsequent entries in the table to create a tilt removal trajectory. For example, if the curve fit interpolates 9 points between each entry (plus 5 points at the beginning of the scan, and 5 points at the end of the scan), the total number of corrections per scan will be 100, requiring a dumping / tilt update rate of 100Hz for a scan rate of 1Hz. To generate a smooth de-sloped trajectory, a mathematical operation (such as a cubic Hermitian fit) may be applied to all the data collected, which has the beneficial effect of specifying both the position and slope at all target trajectory points.

[0204] In this example, a first set of correction tables for the first sequencing cycle can be created by scanning the flow cell before sequencing begins. Subsequent sequencing cycles can use correction tables updated based on the centroid calculations performed during the scans of each previous cycle. An advantage of this exemplary method is that it avoids the need for a low-latency link between the centroid calculations and the controller of the TTA. During the inter-cycle time frame, the controller may have enough time to update 128 tables for the next sequencing cycle, or, as it completes each scan in the current sequencing cycle, it may update each table for the next sequencing cycle in the background.

[0205] Fig. 22 is a block diagram illustrating an example mechanism for tilting the sample holder 720. In this example, only the high-resolution tilt actuator 721-1 is configured to tilt the sample holder 720 (e.g., by moving the holder up and down) during a sample imaging cycle. The standard-resolution tilt actuator 721-2 can adjust the global tilt of the sample holder 720 between imaging cycles. For example, where the actuator corresponds to actuator 304 of assembly 300, Fig.13 The actuator 304 shown on the right side of the image can be fixed during the image scan, and Fig.13 The actuator 304 shown on the left side of FIG. 1 can dynamically tilt the sample up and down during an image scan.

[0206] FIG. 23A to FIG. 23C An example implementation for generating a tilt map according to some implementations of the present disclosure is illustrated. Specifically, Fig.23A 6 is an operational flow chart illustrating an example method of determining a tilt map along a scan direction. The method can be implemented, for example, to generate an initial tilt map before starting imaging of a sample (e.g., operation 610), and to generate a tilt map for a subsequent imaging cycle during each imaging cycle (e.g., operation 630). In this example, the tilt map is determined using an optical assembly (e.g., optical assembly 202) that projects a dual focused beam onto the surface of the sample.

[0207] Operation 810 includes scanning the sample in a scanning direction along a plurality of sample locations of the sample by projecting a pair of beam spots onto a surface of each sample location. Operation 820 includes, for each sample location in the plurality of sample locations, estimating a height of the sample location based at least on a separation distance of the pair of beam spots. For example, Fig. 23B As depicted, a focus tracking module or other suitable module may project a pair of spot beams on the surface of the sample at different sample positions in the scanning direction, and an image sensor of the optical imaging system may be used to capture the image. The sensor used to capture the image of the projected spot beam pair may be the same as the image sensor used to capture the sample image, or may be a different image sensor. As discussed above, the spacing distance of the imaging spot beams may be mapped to the distance between the objective lens and the sample surface. Therefore, the spot beam spacing may be mapped to the sample height at a given sample position.

[0208] Operation 830 includes calculating the tilt of the sample along the scanning direction based on the heights of the plurality of sample positions. For example, based on the estimated heights of two adjacent sample positions and the separation distance of the two sample positions, the tilt slope and angle between adjacent positions may be determined.

[0209] Operation 840 includes generating a tilt map based on the tilt of the sample along the scanning direction, the tilt map including a plurality of entries corresponding to a plurality of sample tilt positions, each entry indicating an amount by which the sample is tilted along the scanning direction for a corresponding sample tilt position. In some implementations, the tilt map entries correspond to each sample position of a pair of sample positions on which a beam spot is projected. For example, each entry may indicate a tilt angle, a tilt slope, or a tilt height for each sample position of a pair of sample positions on which a beam spot is projected. In some implementations, in order to minimize the energy input into the instrument structure and maximize the quality of the de-tilt correction, a tilt map may be created to provide a smooth de-tilt trajectory. In such implementations, a smooth curve fit may be performed between entries corresponding to the initial spot beam measurement results. As an example, Fig.23CA smooth tilt trajectory generated by interpolating between five initial entries is illustrated. In this illustration, the black dots may correspond to the five initial entries generated from the spot beam spacing measurements. The gray dots may represent additional entries added by interpolation. During sample imaging, each tilt map entry may then be read by the controller of the TTA to cause the actuator to tilt the sample holder.

[0210] Although the foregoing examples have been described primarily in the context of determining sample tilt along the scanning direction (Y direction), it is also important to account for sample tilt in a direction orthogonal to the scanning direction (X direction). FIG. 24A to FIG. 24C By way of further example, the optical imaging system may utilize a focusing system that generates at least two pairs of beam spots to account for tilt in two directions.

[0211] Specifically, Fig.24A 6 is an operational flow chart illustrating an example method of determining a tilt map along a scan direction and a direction orthogonal to the scan direction. The method can be implemented, for example, to generate an initial tilt map before starting imaging of a sample (e.g., operation 610), and to generate a tilt map for a subsequent imaging cycle during each imaging cycle (e.g., operation 630). In this example, the tilt map is determined using an optical assembly (e.g., optical assembly 202) that projects at least two pairs of focused beams onto a surface of the sample.

[0212] Operation 910 includes scanning the sample in a scanning direction along a plurality of sample positions of the sample by projecting two pairs of beam spots onto a surface at each sample position. Operation 920 includes estimating, for each sample position in the plurality of sample positions, a height of the sample position and a tilt of the sample position in a second direction substantially orthogonal to the scanning direction based on at least a separation distance of each pair of beam spots in the two pairs of beam spots.

[0213] like FIG. 24B to FIG. 24C As depicted, the focus tracking module or other suitable module can project two pairs of spot beams on the surface of the sample at different sample positions in the scanning direction. Compared with the previous embodiment in which each pair of spot beams is spaced along the X-axis, in this embodiment, each pair of spot beams is rotated 90 degrees and spaced along the scanning axis (Y-axis). For example, referring to Fig.24C, the first pair of spot beams are spaced Δy1 along the Y axis, and the second pair of spot beams are spaced Δy2 along the Y axis. In this example, the height of the sample at the center of the field of view of the sample position can be estimated based on the beam spot spacing Δy1 and the beam spot spacing Δy2 as follows. The first beam spot spacing can be mapped to a first sample height, and the second beam spot spacing can be mapped to a second sample height. The average value between the first sample height and the second sample height can be determined. Alternatively, one of the two sample heights can be selected as the sample height at the position. In addition to determining the sample height, the difference between the intervals (Δy1, Δy2) of the two beam spot pairs and the distance (L x ) can be used to determine the tilt along the X direction.

[0214] Operation 930 includes calculating the tilt of the sample along the scanning direction and the tilt of the sample along the second direction based on the height of the plurality of sample positions. Operation 940 includes generating a tilt map based on the tilt of the sample along the scanning direction and the tilt of the sample along the second direction. As previously described, the tilt map may include a plurality of entries corresponding to a plurality of sample tilt positions. In this case, each entry may indicate the amount by which the sample is tilted in both the scanning direction and the direction substantially orthogonal to the scanning direction for the corresponding sample tilt position. Such a tilt map may also be referred to as a "dump tilt map". During sample imaging, each tilt-dump map entry may then be read by a controller of the TTA to cause one or more actuators to tilt and dump the sample holder along both the X and Y axes as desired.

[0215] It should be appreciated that while the foregoing examples have been described in the context of generating tilt maps along a scan direction or both along the scan direction and a direction orthogonal to the scan direction, the above techniques may be applied to generate tilt maps only for a direction orthogonal to the scan direction.

[0216] Although the foregoing examples for enabling dynamic tilting of a sample have been primarily described in the context of an imaging system utilizing a tilt map that may be updated after each imaging cycle or after some multiple imaging cycles, it should be appreciated that the foregoing examples may also be used in embodiments utilizing a feedback removal tilt mechanism that does not rely on a tilt map generated prior to scanning in a region. For example, Fig.23A or Fig.24A The method can be adapted so that the tilt measurement results (e.g., as determined for each sample position) are provided directly to the tilt controller, which adjusts one or more tilt actuators in response to receiving the measurement results. In such specific implementations, the frequency of sample holder tilting can be the same as, greater than, or less than the frequency of scanning.

[0217] In the foregoing examples, the relative positioning between the projected beam spots (e.g., two pairs of beam spots) used to capture the tilt measurement results and the projected excitation light (e.g., scan lines) used to capture the sample image can vary depending on the system requirements. Fig.24C In the example, two pairs of beam spots are projected before the scanning line in the scanning direction. However, in some specific implementations, one pair of beam spots in the two pairs of beam spots may be projected before the scanning line, and another pair of beam spots in the plurality of pairs of beam spots may be projected after the scanning line. In other specific implementations, both pairs of beam spots may be projected after the scanning line. In other specific implementations, one pair of beam spots in the plurality of pairs of beam spots may be projected on the scanning line.

[0218] Likewise, in a multi-channel, line-scanning imaging system that uses multiple excitation light sources to image a sample, the relative orientation between the multiple scan lines and the projection beam spot pairs can vary. FIG. 25A to FIG. 25F The relative orientations of two pairs of projection beam spots and two scanning lines (e.g., laser lines) in a dual-channel, line-scanning imaging system in different configurations are illustrated. Fig.25A In , both pairs of beam spots are projected before two scanning lines. Fig.25B In , one pair of beam spots is projected before two scanning lines, and one pair of beam spots is projected after two scanning lines. Fig.25C In , both pairs of beam spots are projected after two scanning lines. Fig.25D In , both pairs of beam spots are projected before one scan line and after the other scan line. FIG. 25E to FIG. 25F In the example, one pair of beam spots is projected before a scan line, and another pair of beam spots is projected after the same scan line.

[0219] Fig.26 The advantages of using multiple tilt actuators according to some specific implementations of the present disclosure are conceptually illustrated. As depicted in the above figure, when a single tilt actuator is used, the tilt / rotation axis can be offset from the center of the sample surface being scanned. Depending on the lever arm L, dynamically changing the tilt may result in delay-induced errors due to the z displacement, and the x position of the sample feature may shift as the sample holder is tilted up and down. To overcome this problem, in some specific implementations, multiple tilt actuators can be used to change the position of the tilt axis so that it is closer to the sample area being scanned or centered on the sample area being scanned. For example, as depicted in the following figure, the rotation axis x can be changed by moving z1 and z2 simultaneously, where In one embodiment, using multiple tilt actuators, the tilt axis can be selected to be centered for each swath of the flow cell being scanned. In one embodiment, three tilt actuators are utilized to ensure that the tilt axis is centered.

[0220] While some of the foregoing examples have been described in the context of using spot-beam spacing measurements to enable dynamic tilting of a sample holder to keep a sample in focus during image scanning, it should be appreciated that these measurements may also be used to move a Z stage to provide movement of the objective relative to the sample container to keep the sample in focus. For example, one or more actuators may be configured to move the objective and / or sample container in the z direction while keeping the sample within a focus region of the focal plane of the imaging system. For illustration, Fig. 27 2734 is a block diagram illustrating some components of an example focus control system for focus tracking according to some implementations of the present disclosure. The example focus control system includes a focus tracking circuit system 2732 configured to determine a focus setting for generating a drive signal that drives a focus tracking feedback loop in a Z stage 2734. Fig. 27 In an example of , commands 2752 based on the focus setting difference are fed to the Z stage 2734. In this example, the Z stage 2734 is configured to move the objective lens 2746. In other specific implementations, the Z stage can move the stage where the sample holder is placed. The actuator 2744 moves the optical table, and in particular the objective lens 2746, in response to a drive signal provided by the Z stage amplifier 2738. The actuator 2744 may include a piezoelectric actuator, a voice coil actuator, a motor or other similar actuator. The encoder 2742 may provide information about the actuator position and its movement. The encoder information 2754 can be fed back to the focus tracking circuit system 2732 through the Z stage controller 2736 and can be used to determine an error signal.

[0221] Fig.282870 is a diagram illustrating an example architecture of a Z-stage controller according to some specific implementations of the present disclosure. The example includes feedback control and feedforward control. In operation, the position of the Z-stage is controlled using a target focus setting (e.g., a target z position 2870 as determined using spot spacing measurements). The target z position 2870 is provided to a controller 2888, which determines the drive signal required to command an actuator 2890 to position the Z-stage. The controller 2888 may also include a drive circuit system that generates a drive signal. The drive signal determination may be performed using the magnitude of the difference between the target focus setting (e.g., the target z position 2870 as determined by the most recent spot spacing measurements) and the current focus setting (e.g., the current z position 2872 as determined by the past spot spacing measurements), which may be provided, for example, by the actuator 2890. In this example, the drive signal used to drive the actuator is adjusted by a signal from a feedforward control path 2876, and a measured focus correction signal 2878 is generated by a focus tracking circuit system 2892. For example, the correction information may be determined using look-ahead predictive focus tracking or predictive focus tracking based on focus history data or using other predictive focus tracking techniques. For example, the focus settings may be electronically stored in a history file in memory so that these focus settings may be recalled for later use during a scanning operation. The history file or the information therein may be tagged to be identified as containing focus information for a particular sample container. The correction information may be added to the commanded stage position to adjust the drive signal according to the slope of the change in focus setting for the scanning operation.

[0222] More generally, the techniques described herein may be implemented by creating a relative tilt between a sample and an image sensor based on sample tilt measurements that images the sample by adjusting any component of an optical imaging system along an imaging light path from the sample to the sample image sensor. Thus, based on the sample tilt measurements, the system may instead be configured to tilt an image sensor that images the sample, a camera that carries the sample image sensor, and / or a sample holder. Other optical components along the sample imaging / light path from the sample to the sample image sensor may be tilted and / or otherwise adjusted to create a relative tilt between the sample and the image sensor that images the sample. Such optical components may, for example, include an objective lens or one or more reflectors that receive light corresponding to an image of the sample.

[0223] Adjustments to imaging system components other than the sample holder (e.g., adjustments to one or more mirrors and / or image sensors) to account for sample tilt may be made based on real-time tilt measurements transmitted in real time to one or more controllers or based on a sample tilt trajectory calculated prior to removing the tilt during an image scan, as described above with reference to FIG. FIG. 21A to FIG. 25FFor example, this can be achieved by creating a tilt map including entries corresponding to a plurality of sample positions. Fig.21A , Fig.23A , Fig.24A Embodiments of the invention, wherein each entry indicates an amount to tilt the image sensor or otherwise adjust some optical component of the imaging system to de-tilt the sample. Thus, de-tilting the sample may be achieved by tilting or otherwise adjusting one or more components of the imaging system along an imaging path from the sample to the sample image sensor based on one or more sample tilt measurements. One or more system controllers may be used to control the tilting or adjustment of one or more optical components.

[0224] In implementations where the sample container is a flow cell, tilt adjustment may occur after each scan swath, multiple times per scan swath, after each tile, or some other sub-portion of the sample container. Such adjustments may account for sample tilt along one axis (e.g., tilt along the scan direction or a direction substantially orthogonal to the scan direction), or sample tilt along multiple axes (e.g., tilt along both the scan direction and a direction substantially orthogonal to the scan direction). Such embodiments may be implemented with or without a tiltable sample holder.

[0225] As described above, the optical sequencing system described herein may map the spot beam spacing of one or more focus tracking modules to a sample height position. This mapping may be performed when operating the sequencing system in a focus model generation mode, a sequencing mode, or both. During focus tracking model generation, the system may determine and store information about the focus setting for a given sample container to control the focus of the system during real-time imaging. For example, using one or more focus tracking modules, a sample container may be scanned at each of a plurality of sample positions on the sample container, and a focus setting may be measured and determined for each of the plurality of sample positions.

[0226] Fig.29 and FIG. 30A to FIG. 30B Specific examples of the design and operation of a focus tracking system utilizing two pairs of focus tracking spots per sample surface according to some implementations of the present disclosure are illustrated. The focus tracking system utilizes a beam architecture that projects two pairs of spots onto each of a first surface and a second surface (e.g., a top surface and a bottom surface) of a sample container (e.g., a flow cell lane). In alternative designs, the two pairs of spots may be projected onto a single sample surface or onto more than two sample surfaces.

[0227] like Fig.29As depicted, two reflected light beams corresponding to each pair of light spots are incident on the surface of the corresponding sensors 2911, 2912 to form beam spots (e.g., light spot pairs 2901, 2902 corresponding to light reflected from the top surface of the sample and light spot pairs 2903, 2904 corresponding to light reflected from the bottom surface of the sample). For simplicity, the discussion will focus on the light spot pairs 2901 and 2902 projected on one surface. The light spot pairs 2901, 2902 have corresponding light spot intervals Δx detected using the corresponding sensors 2911, 2912. 1 , Δx 2 . In this example, L is the "lever arm" and the ROI refers to a region of interest that can be dynamically determined and set near the peak maximum. For example, the ROI can be set near the peak maximum with a width of twice the full width at half maximum (FWHM). The two sensors can be used as a parallel linear sensor array for spot detection. In some specific implementations, the corresponding focus tracking module can be associated with the corresponding light source that projects the two spots and the sensor that detects the two spots. For example, the four-beam system described herein can be associated with two separate focus tracking modules.

[0228] Fig. 30A 2901 and 2903 after the light corresponding to the spot pair 2901 and 2903 is illustrated as a function of the pixel number of the sensor 2911. As depicted, by determining the pixel corresponding to the peak intensity of each detected spot and determining the difference, the spot spacing Δx corresponding to the left and right spots of the spot pair 2901 on the sensor 2911 can be calculated. 1 . Fig. 30B 2904 as a function of the pixel number of the sensor 2912. As depicted, by determining the pixel corresponding to the peak intensity of each detected spot and determining the difference, the spot spacing Δx corresponding to the left and right spots of the spot pair 2902 on the sensor 2912 can be calculated. 2 .

[0229] In some implementations, the following parameters may be defined for the four-beam focus tracking system described above. The average value Δx of the spot spacing between two pairs of spots in the sensor plane may be defined by equation (1):

[0230]

[0231] Where Δx = Δx at the best focus 0 The change Δz of the position of the sample container (e.g., flow cell) relative to the z-stage can be defined by equation (2):

[0232]

[0233] Where DSG (pix / μm) is the differential spot gain from best focus. The difference dx in the spot spacing between two pairs of spots in the sensor plane can be defined by equation (3):

[0234] dx=Δx 1 -Δx 2 (3)

[0235] where dx = dx at zero image tilt 0 The differential tilt gain (DTG) in pixels / μrad can be defined by equation (4):

[0236]

[0237] Where L is the spot spacing on the sample in μm. The variation of the tilt angle around the y-axis can be defined by equation (5):

[0238]

[0239] Fig.31 Describes the use of reference Fig.29 One example implementation of the described focus tracking system is in both the object plane and the sensor plane during scanning of the flow cell surface. In this case, Δy 1 and Δy 2 refers to the spot spacing at the surface of the sample, and L refers to the distance between each pair of spots at the surface of the flow cell. The focus tracking module spots are shown relative to two scanning laser lines. For illustration purposes, sensors 2911 and 2912 with incident beams are placed on the figure. In a specific embodiment, Δy 1 and Δy 2 At best focus is about 60 μm, and L is about 900 μm.

[0240] Although the imaging systems described herein may utilize an estimate of the focus tracking spot spacing to maintain sample focus (e.g., by moving the z-stage in the z-direction and / or by tilting the stage on which the sample holder is placed), in some cases, the spot spacing may not be accurately estimated due to a problem with the sample or sample container (e.g., bubbles, defects, debris on the surface), a problem with the sensor (e.g., a short pulse interference in the sensor reading), or some other problem that affects the readout of one or both spots on the sensor of the focus tracking module during imaging. Such problems may cause the detected spots to become saturated or non-Gaussian in nature, which leads to inaccurate estimates of the spot spacing. These inaccurate estimates of the spot spacing may result in large Z step spikes during focus tracking, which may interact with the sensitivity of the Z-stage control loop and introduce large errors in the movement along the Z-direction. This problem is caused by Fig.32By way of example, the figure is a graph showing the Z step size (in nm) as a function of time for a Z stage used with a focus tracking system that does not implement "spot error" handling. Inaccurate estimates of these spot spacings can also introduce errors in the movement of the stage that tilts the sample holder.

[0241] To address potential issues with spot errors, some implementations of the present disclosure involve correcting spot errors based at least on previous focus tracking measurements of spot spacing, including potentially inaccurate measurements of spot spacing. According to some implementations further described below, statistics of previous spot spacing measurements, statistics of previous sample tilt measurements, and / or error codes for tracking current conditions of the measured focus tracking spot can be used to determine which Z stage and / or tilt adjustment to apply to a given frame, including which spot spacing measurements to use for a given frame. By implementing the techniques described herein, significant improvements in focus tracking performance can be achieved. In addition, the techniques described herein can be implemented to address spot errors and / or spot spacing glitches in real time (e.g., at an update rate of about 3.3 kHz).

[0242] Although the spot error processing techniques described herein will be primarily described in the context of an imaging system utilizing two pairs of substantially parallel focus tracking spots detected using two substantially parallel sensors, it should be understood that the techniques described herein can be applied with: a focus tracking system utilizing a single pair of focus tracking spots detected using one or more sensors, a focus tracking system utilizing two pairs of substantially parallel focus tracking spots detected using one or more sensors, or a focus tracking system utilizing more than two pairs of substantially parallel focus tracking spots detected using one or more sensors.

[0243] Before describing specific implementations of the spot error processing techniques described herein, it is helpful to consider examples of different issues with samples / sample holders and / or sensors that may cause spot errors, focus tracking performance when spot errors are not accounted for, and improvements in focus tracking performance when spot errors are accounted for, according to some implementations of the present disclosure. To this end, FIG. 33A to FIG. 37C It is illustrative.

[0244] Fig.33AAn image of a portion of a flow cell 3300 with a large bubble 3301 is depicted. Sensor 3321 and sensor 3322 are overlaid on the image. Sensor 3321 is configured to detect reflections of a first (right) pair of focused tracking beams, and sensor 3322 is configured to detect reflections of a second (left) pair of focused tracking beams. Defect 3301 results in a higher reflection of the right pair of focused tracking beams. This higher reflection may result in saturation of the signal readout of the two reflected beams incident on sensor 3321, resulting in a noisy sensor signal that does not have clear peak intensity spikes (e.g., corresponding to the intensity of the light beams as shown in FIG. 1 ) that enable spot spacing measurements. FIG. 30A to FIG. 30B The center of one or two spots illustrated in FIG.

[0245] FIG. 33B to FIG. 33C A graph is depicted which shows that when the spot error processing is turned off ( Fig.33B ) and spot error processing is turned on ( Fig.33C ) when performing focus tracking on a flow cell 3300 with defect 3301. In these graphs, the x-axis refers to the current focus tracking frame. The y-axis of the bottom graph measures the distance (in sensor pixels) from the best z focus position for each sensor ("Sensor 1" and "Sensor 2"). As depicted, the defect occurs between focus tracking frames 38690 and 38700 for sensor 3321 (shown as "Sensor 1" in the graph). By accounting for the spot error caused by the defect, aggressive Z stage movements can be avoided when the focus tracking spot of sensor 1 is over defect 3301. As measured, focus tracking performance was improved by about 20% on average for this flow cell 3300 with defect 3301. Performance on the worst scan band was improved by about 50%.

[0246] Fig.34A An image of a portion of a flow cell 3400 having a defect 3401 that causes the focus tracking spot of sensor 1 to saturate over approximately 8 frames is depicted. Fig.34B Depicted are graphs showing focus tracking performance (including a summary of through-focus errors) when focus tracking is performed on a flow cell 3400 with spot error processing turned off and when focus tracking is simulated with spot error processing turned on. In this example, the graph with spot error processing turned off and the simulated graph with spot error processing turned on are overlaid. As depicted with spot error processing turned on, the curves are smoothed out and no Z step spikes are commanded to the Z stage when a flow cell defect is encountered.

[0247] Fig.35AAn image of a portion of a flow cell 3500 with a large bubble 3501 is depicted which causes poor measurements of the focus tracking spot of sensor 2 over approximately 50 frames. Fig.35B Depicted are graphs showing the focus tracking performance (including a summary of through-focus errors) when focus tracking is performed on the flow cell 3500 with spot error processing turned off and when focus tracking is simulated with spot error processing turned on. In this example, the graph with spot error processing turned off and the simulated graph with spot error processing turned on are overlaid. As depicted with spot error processing turned on, the curves are smoothed out and no Z step spikes are commanded to the Z stage when a flow cell bubble is encountered.

[0248] Fig.36A An image of a portion of a flow cell 3600 is depicted with debris 3601 (appearing as black spots) on the surface, which causes poor measurements of the focus tracking spot. For example, the debris blocks some light from reaching the portion of the flow cell that is obscured by it. FIG. 36B to FIG. 36C A graph is depicted which shows that when the spot error processing is turned off ( Fig.36B ) and spot error processing is turned on ( Fig.36C ) when performing focus tracking on the flow cell 3600. As depicted with spot error processing turned on, no Z step spikes are commanded to the Z stage when debris is encountered.

[0249] 37 depicts an image of a portion of a flow cell 3700 having residue 3701 (appearing as a black pattern) on the surface that causes poor measurement results of the focus tracking spot. For example, the residue 3701 causes unpredictable reflections of the incident focus tracking light, which skews the measured spot. FIG. 37B to FIG. 37C A graph is depicted which shows that when the spot error processing is turned off ( Fig.37B ) and spot error processing is turned on ( Fig.37C ) when performing focus tracking on the flow cell 3700 (including summary of through-focus errors). As depicted with spot error processing turned on, no Z step spikes are commanded to the Z stage when residue is encountered.

[0250] In some cases, glitches in the system electronics (eg, focus tracking sensor) and / or during data collection result in missed and / or incorrect measurements. Fig.38ADepicted are graphs showing focus tracking performed on a flow cell using a four-beam, dual-sensor focus tracking system without spot error processing. In this example, a spot interval glitch occurred on one sensor and was not accounted for. The fourth graph shows an error code for the current condition of the focus tracking spot measured without using tracking. Fig.38B Depicted is a diagram showing the use of a four-beam, dual-sensor focus tracking system with spot error handling in Fig.38A Graph of focus tracking performed on a flow cell. Fig.38A The example shown, in contrast, commands a smoother Z movement of the Z stage despite the presence of abnormal spot spacing glitches on one or both sensors.The fourth graph shows the error code using the current condition of the focus tracking spot measured by tracking.

[0251] Fig.39 A flow chart illustrating an example method 3900 for implementing spot error processing according to some implementations of the present disclosure is depicted.

[0252] Operation 3901 includes capturing an image of a pair of light spots at a sample location. For example, using one or more light sources of an imaging system, a first pair of light spots may be projected at a sample location in a sample container, and an image sensor of the imaging system may capture an image of the first pair of light spots. The image may be captured using a focus tracking system as described above.

[0253] At decision 3902, a determination is made as to whether the image of the pair of spots is valid. In some embodiments, this determination may be made based on the intensity distribution of pixels in the first image. For example, as described above with reference to FIG. 30A to FIG. 30B As discussed, when detecting a pair of light spots on an image sensor, it is expected that a valid image will produce two intensity peaks from a sample with one surface, or four intensity peaks from a sample with two surfaces, each intensity peak having a substantially Gaussian distribution. When this is not the case (e.g., there are no intensity peaks, there are one intensity peak, there are three intensity peaks, and / or a non-Gaussian distribution of intensities around the peaks, and / or an asymmetric pixel position of the peaks), this may indicate that a light spot error has occurred. Therefore, in some specific implementations, one or more of the following parameters may be checked by the system to determine whether the image is valid: the intensity distribution of the first light spot, the intensity distribution of the second light spot, the intensity distribution of the third light spot, the intensity distribution of the fourth light spot, the pixel position of the peak intensity, the number of intensity peaks, whether there are any saturated pixels, etc.

[0254] In some implementations, when an image is determined to be invalid, an identifier (such as an error code) may be stored in the system memory, indicating the reason why the image is invalid. In some implementations, the identifier may be a number or other alphanumeric indication associated with the type of error / problem. For example, the system may associate different identifiers with problems such as one or more spots having saturated pixels, non-Gaussian spots, incorrect number of valid peaks, incorrect symmetry, other errors, or unidentifiable errors. In some implementations, the system may also associate an identifier with an image determined to be valid. Thus, an identifier indicating whether an image is valid or invalid (and optionally, the reason why it is invalid) may be stored for each frame. By implementing and storing the identifier in the memory, the system may maintain an error history over different frames, which may be used to implement spot error processing. In some cases, the identifier may also be used to troubleshoot an imaging system (e.g., a sensor of a focus tracking system that collects images) or a sample container during a sample pre-scan, during a sample scan, and / or after a sample scan. For example, an identifier for saturated pixels may identify the presence of bubbles. As another example, an identifier for a non-Gaussian spot may identify the presence of debris or scratches on a sample container. In some implementations, the identifier may be stored in a circular buffer (e.g., during a current swath scan or other sample scan) that maintains a history of recent identifiers. In implementations where multiple spot pairs are detected using multiple corresponding sensors, the system may store a history of identifiers associated with each sensor.

[0255] When the image is determined to be valid, at operation 3903, a current separation distance measurement result of the pair of light spots is obtained based on the image (eg, Δx as described above). 1 ). As described herein, the spacing distance measurement result can be FIG. 30A to FIG. 30B The depiction is obtained in pixels.

[0256] At decision 3904, a determination is made as to whether the current interval distance measurement is within a threshold range determined from previously interval distance measurements. For example, to prevent large Z step sizes that may indicate measurement errors and / or be detrimental to imaging, the system may maintain a recent history of previously interval distance measurements (e.g., in a circular buffer or other fixed-size data structure). The threshold range may be based on an average / moving average of recent measurements, or a change between adjacent frames from recent measurements, or a frequency content of recent measurements. For example, the threshold range may be set to a number of standard deviations (e.g., 1, 1.5, 2, 2.5, 3, 3.5, 4, etc.) above / below the average past valid measurements, or above / below the average change from the last valid measurement. If the most recent measurement is outside the threshold range, it may indicate an abnormally large Z step size that should not be performed. In some implementations, the threshold range can be set so that the most recent measurements in the fixed data structure are weighted more (e.g., an average of the measurements or an average of the variation of the measurements between adjacent frames weights the most recent measurements in the data structure more than the last measurement in the data store). In some implementations, the threshold range can be set based on the most recent valid interval distance measurement used during Z stage adjustment.

[0257] When the separation distance measurement is within the threshold range, the Z stage may be controlled to focus the imaging system at the sample location based on at least the current separation distance measurement at operation 3905. When the image of the pair of spots is determined to be invalid (decision 3902) or the current separation distance measurement is not within the threshold range (decision 304), the Z stage may be controlled to focus the imaging system at the sample location based on more previous separation distance measurements at operation 3906. For example, the Z stage may be controlled based on the most recent one of the previous separation distance measurements determined to be valid or a combination of the previous separation distance measurements (e.g., a moving average weighted toward more recent measurements).

[0258] During operation, the Z stage may be controlled based on a Z step size that is commanded based on one or more standoff distance measurements. The Z step size may cause an objective lens of an imaging system and / or a stage on which a sample container is placed to move up or down in the Z direction. As discussed above, the Z step size may be calculated as a function of one or more standoff distance measurements (e.g., a current standoff distance measurement) and a gain value associated with the optics of the focus tracking module. For example, the Z step size may be calculated by taking the current standoff distance measurement in pixels and multiplying it by a gain value in nm / pixel.

[0259] In an alternative implementation, method 3900 may be implemented without decision 3904 (eg, after an image is determined to be valid and a current separation distance measurement is obtained, the current separation distance measurement is used to control the Z stage).

[0260] Although method 3900 has been described in the context of spot error processing for an imaging / focus tracking system that generates a pair of imaged spots, it should be understood that method 3900 can be extended to an imaging / focus tracking system that generates multiple pairs of spots imaged using one or more sensors (e.g., two pairs of substantially parallel spots imaged by two sensors). In such a system, operations 3901 to 3904 as described above can be implemented for each pair of detected spots (e.g., for each sensor of the system).

[0261] to this end, Fig.40 4 is a flow chart depicting a method 4000 of spot error processing in a system using at least two pairs of substantially parallel spots for performing focus tracking according to some specific implementations of the present disclosure. Operation 4001 includes capturing a first image of a first pair of spots and a second image of a second pair of spots at a sample position. The two pairs of spots may be substantially parallel and captured by one sensor or multiple sensors (e.g., two substantially parallel linear sensors as described above).

[0262] Operation 4002 includes determining that a first image of a first pair of spots is invalid or that a current separation distance measurement of the first pair of spots obtained from the first image (eg, Δx 1 ) is not within the threshold range. For example, operation 4002 may be performed as described above with reference to operations 3902 to 3904 of method 3900.

[0263] At decision 4003, a determination is made as to whether the second image of the second pair of spots is valid. For example, this determination may be made based on the intensity distribution of pixels in the second image following the techniques described above with reference to decision 3902 of method 3900. When the second image is determined to be valid, at operation 4004, a current separation distance measurement of the second pair of spots is obtained based on the second image (e.g., Δx as described above). 2 ).

[0264] At decision 4005, a determination is made as to whether the current separation distance measurement of the second pair of spots is within a threshold range determined from a previous separation distance measurement of the second pair of spots. In alternative implementations, the comparison of the obtained separation distance measurement to the threshold value may be skipped. In such implementations, operation 4005 may be skipped, and operation 4002 merely includes determining that the first image of the first pair of spots is invalid.

[0265] In the event that the first image is invalid but the second image is valid, operation 4006 may be performed, which includes controlling the Z stage to focus the imaging system at the sample position based on the current separation distance measurement of the second pair of spots and optionally one or more of the previous separation distance measurements of the first pair of spots. Thus, by using two pairs of spots, even if there is a detected error at one of the pairs of spots, the separation distance measurement of the other pair of spots may be used to determine the Z step size. In some implementations, the current separation distance measurement of the second pair of spots may be averaged with a separation distance measurement determined from one or more of the previous separation distance measurements of the first pair of spots (e.g., the most recently valid one or some type of average of the previous measurements). This average may then be used to command the Z stage.

[0266] In the event that both the first image and the second image are invalid, operation 4007 may be performed, which includes controlling the Z stage to focus the imaging system at the sample position based on one or more previous separation distance measurements of the first pair of spots and / or one or more previous separation distance measurements of the second pair of spots. For example, an average of the most recent valid separation distance measurement of the first pair of spots and the most recent valid separation distance measurement of the second pair of spots may be used to determine the Z step size.

[0267] Although the foregoing examples have been described in the context of spot error processing to detect / prevent Z spikes, the spot error processing techniques described herein may also be implemented to detect / prevent tilt spikes. For example, the techniques described herein may be implemented to prevent any tilt spikes of a tilt stage that is configured to tilt a sample container in a direction orthogonal to a scan direction.

[0268] to this end, Fig.41 41 is a flow chart depicting a method 4100 for detecting tilt spikes in a system that performs focus tracking using at least two pairs of substantially parallel light spots projected onto at least two locations on a sample according to some implementations of the present disclosure. Prior to performing the method 4100, two separation distance measurements of the two pairs of light spots may be obtained (e.g., by following the techniques described above).

[0269] Operation 4101 includes determining a distance measurement based on the current separation distance measurement result (eg, Δx) of the first pair of light spots. 1 ) and the current separation distance measurement result of the second pair of spots (e.g., Δx 2 ) to obtain the current sample tilt measurement result. For example, the measurement result can be obtained based on the difference between two interval distance measurement results (eg, the above equation (3)).

[0270] At decision 4102, a determination is made as to whether the current sample tilt measurement is within a threshold range determined from previous sample tilt measurements. For example, to prevent large tilt steps, the system may maintain a recent history of previous sample tilt measurements (e.g., in a circular buffer or other fixed-size data structure). As discussed above, each tilt measurement may be obtained by taking the difference between two spaced-apart distance measurements of two pairs of substantially parallel light spots. The threshold range may be based on an average / moving average of recent measurements, or a change between adjacent frames from recent measurements, or a frequency content of recent measurements. For example, the threshold range may be set to a number of standard deviations (e.g., 1, 1.5, 2, 2.5, 3, 3.5, 4, etc.) above / below the average past valid measurements, or above / below the average change from the last valid measurement. If the most recent tilt measurement is outside the threshold range, it may indicate a tilt spike that should be avoided. In some implementations, the threshold range can be set so that the most recent measurement in the fixed data structure is weighted more (e.g., the average of the measurements weights the most recent measurement in the data structure more than the last measurement in the data store). In some implementations, the threshold range can be set based on the most recent valid sample tilt measurement used during tilt stage adjustment.

[0271] When the current sample tilt measurement result is within the threshold range, operation 4103 includes controlling the tilt stage to focus the sample at a desired position based on the current sample tilt measurement result. When the current sample tilt measurement result is not within the threshold range, optional operation 4104 includes identifying a sensor with a spot spacing short-time pulse interference. For example, in a specific implementation using two parallel sensors to detect corresponding two pairs of spots, a determination can be made as to whether the current spacing distance measurement result of one sensor has deviated significantly or whether the current spacing distance measurement results of two sensors have deviated significantly. In some specific implementations, when it is detected that the current sample tilt measurement result is not within the threshold range, the system can store an error code.

[0272] In this document, the terms "machine-readable medium", "computer-readable medium" and similar terms are generally used to refer to volatile or non-volatile, non-transitory media that store data and / or instructions that cause a machine to operate in a specific manner. Common forms of machine-readable media include, for example, hard disks, solid-state drives, magnetic tapes or any other magnetic data storage media, optical disks or any other optical data storage media, any physical media with a pattern of holes, RAM, PROM, EPROM, FLASH-EPROM, NVRAM, any other memory chip or cartridge, and networked versions thereof.

[0273] These and other various forms of computer-readable media may involve carrying one or more sequences of one or more instructions to a processing device for execution. Such instructions embodied on the medium are generally referred to as "instructions" or "codes". Instructions may be grouped in the form of computer programs or other groupings. When executed, such instructions may enable a processing device to perform features or functions of the present application as discussed herein.

[0274] In this document, a "processing device" may be implemented as a single processor that performs processing operations or a combination of dedicated and / or general-purpose processors that perform processing operations. A processing device may include a CPU, GPU, APU, DSP, FPGA, ASIC, SOC, and / or other processing circuit systems.

[0275] The terms "substantially" and "approximately" used throughout this disclosure (including claims) are used to describe and illustrate that there are small fluctuations, such as those caused by variations in processing. For example, they may refer to less than or equal to ±5%, such as less than or equal to ±2%, such as less than or equal to ±1%, such as less than or equal to ±0.5%, such as less than or equal to ±0.2%, such as less than or equal to ±0.1%, such as less than or equal to ±0.05%.

[0276] To the extent applicable, the terms "first", "second", "third", etc. herein are used merely to show the respective objects described by these terms as separate entities and are not meant to imply a time sequence unless otherwise expressly stated herein.

[0277] Unless expressly stated otherwise, the terms and phrases used in this document and variations thereof should be interpreted as open-ended and not limiting. As an example of the foregoing: the term "including" should be understood to mean "including but not limited to," etc.; the term "example" is used to provide an illustrative example of the items discussed, rather than an exhaustive or limiting list thereof; the term "one" or "an" should be understood to mean "at least one," "one or more," etc.; and adjectives such as "conventional," "traditional," "normal," "standard," "known," and terms of similar meaning should not be interpreted as limiting the items described to a given time period or to items available within a given time period, but should be understood to include conventional, traditional, normal, or standard technologies available or known at any time now or in the future. Similarly, where this document refers to technologies that are obvious or known to a person of ordinary skill in the art, such technologies include those technologies that are obvious or known to a person of ordinary skill in the art at any time now or in the future.

[0278] In some cases, the appearance of broadening words and phrases such as "one or more," "at least," "but not limited to," or other similar phrases should not be understood to mean that a narrower case is intended or required where such broadening phrases may not be present.

[0279] In addition, the various embodiments described herein are described in terms of exemplary block diagrams, flow charts, and other illustrations. As will be apparent to one of ordinary skill in the art after reading this document, the illustrated embodiments and their various alternatives may be implemented without limitation to the examples shown. For example, the block diagrams and their accompanying descriptions should not be construed as mandatory requirements for a particular architecture or configuration.

[0280] The terms "substantially" and "approximately" used throughout this disclosure (including claims) are used to describe and illustrate that there are small fluctuations, such as those caused by variations in processing. For example, they may refer to less than or equal to ±5%, such as less than or equal to ±2%, such as less than or equal to ±1%, such as less than or equal to ±0.5%, such as less than or equal to ±0.2%, such as less than or equal to ±0.1%, such as less than or equal to ±0.05%.

[0281] To the extent applicable, the terms "first", "second", "third", etc. herein are used merely to show the respective objects described by these terms as separate entities and are not meant to imply a time sequence unless otherwise expressly stated herein.

[0282] Although various embodiments of the present disclosure have been described above, it should be understood that they are presented only by way of example and not limitation. Similarly, each figure may depict an exemplary architecture or other configuration of the present disclosure, which is done to help understand the features and functions that may be included in the present disclosure. The present disclosure is not limited to the exemplary architecture or configuration shown, but various alternative architectures and configurations may be used to achieve the desired features. In fact, it is obvious to those skilled in the art how to implement alternative functions, logical or physical partitions and configurations to achieve the desired features of the present disclosure. In addition, many different component module names other than those described herein may be applied to various partitions. In addition, with respect to flow charts, operational descriptions, and method claims, unless the context otherwise dictates, the order of the actions presented herein should not enforce that the various embodiments perform the functions in the same order.

[0283] Although the present disclosure is described above in terms of various exemplary embodiments and implementations, it should be understood that the various features, aspects, and functions described in one or more separate embodiments in separate implementations are not limited in their applicability to the specific embodiments in which they are described, but may be applied to one or more other embodiments in other embodiments of the present disclosure, either alone or in various combinations, regardless of whether such embodiments are described and whether such features are presented as part of the described embodiments. Therefore, the breadth and scope of the present disclosure should not be limited by any of the exemplary embodiments described above.

[0284] It should be understood that all combinations of the aforementioned concepts (assuming such concepts are not mutually contradictory) are contemplated as being part of the inventive subject matter disclosed herein. Specifically, all combinations of the claimed subject matter appearing in this disclosure are contemplated as being part of the inventive subject matter disclosed herein.

Claims

1. A non-transitory computer readable medium having executable instructions stored thereon, which when executed by a processor cause an imaging system to perform operations comprising: capturing, using one or more image sensors of the imaging system, a first image of a first pair of light spots projected at a first sample location on the sample; determining whether the first image of the first pair of light spots is valid; and When the first image is determined to be valid: obtaining a current separation distance measurement result of the first pair of light spots based on the first image; and A Z stage of the imaging system is controlled based at least on the current separation distance measurement to focus the imaging system at the first sample position.

2. The non-transitory computer readable medium of claim 1 , wherein determining whether the first image of the first pair of spots is valid comprises: Whether the first image of the first pair of light spots is valid is determined based on an intensity distribution of pixels in the first image.

3. The non-transitory computer readable medium of claim 2, wherein determining whether the first image of the first pair of spots is valid based on the intensity distribution of the pixels in the first image comprises determining: whether the intensity distribution of each of the first light spot and the second light spot in the image is a Gaussian distribution; whether there is a first peak intensity corresponding to the first light spot in the image and whether there is a second peak intensity corresponding to the second light spot in the image; whether the number of intensity peaks in the image matches a valid number; or Whether one or more of the pixels are saturated.

4. The non-transitory computer readable medium of claim 1, wherein: Determining whether the first image of the first pair of light spots is valid includes: determining that the first image is invalid; and In response to determining that the first image is invalid, an identifier indicating why the first image is invalid is stored in a memory of the imaging system.

5. The non-transitory computer readable medium of claim 1 , wherein the operations further comprise: Prior to controlling the Z stage, it is determined that the current separation distance measurement of the first pair of spots is within a threshold range determined from a plurality of previous separation distance measurements of the first pair of spots associated with other sample positions of the sample. 6 . The non-transitory computer readable medium of claim 5 , wherein the threshold range is within a number of standard deviations above or below a mean of the plurality of previously spaced distance measurements, the number being greater than 0 and less than 4. 7 .

7. The non-transitory computer readable medium of claim 5, wherein the operations further comprise: The current separation distance measurement is stored in a circular buffer including the plurality of previous separation distance measurements.

8. The non-transitory computer readable medium of claim 1, wherein the operations further comprise: When the first image is determined to be invalid, the Z stage is controlled to focus on the sample at the first sample position based at least on one or more previous separation distance measurements of the first pair of spots associated with one or more other sample positions of the sample.

9. The non-transitory computer readable medium of claim 8, wherein: The one or more previous separation distance measurements are a plurality of previous separation distance measurements stored in a memory of the imaging system; and Controlling the Z stage to focus on the sample at the first sample position based at least on the one or more previous separation distance measurements comprises: retrieving a most recent previous separation distance measurement among the plurality of previous separation distance measurements stored in the memory; as well as The Z stage is controlled to focus on the sample at the first sample position based on the most recent previous separation distance measurement of the plurality of previous separation distance measurements.

10. The non-transitory computer readable medium of claim 8, wherein: The one or more previous separation distance measurements are a plurality of previous separation distance measurements stored in a memory of the imaging system; and Controlling the Z stage to focus on the sample at the first sample position based at least on the one or more previous separation distance measurements includes controlling the Z stage to focus on the sample at the first sample position based on a combination of two or more of the previous separation distance measurements.

11. The non-transitory computer readable storage medium of claim 1 , wherein the operations further comprise: capturing, using the one or more image sensors of the imaging system, a second image of a second pair of spots projected at the first sample location substantially parallel to the first pair of spots; determining whether the second image of the second pair of light spots is valid; and When the second image is determined to be valid, a current separation distance measurement result of the second pair of light spots is obtained based on the second image.

12. The non-transitory computer readable storage medium of claim 11, wherein the operations further comprise: When the first image is determined to be invalid and the second image is determined to be valid, the Z stage of the imaging system is controlled to focus the imaging system at the first sample position based at least on the current separation distance measurement of the second pair of spots.

13. The non-transitory computer readable storage medium of claim 11, wherein the operations further comprise: When the first image is determined to be invalid and the second image is determined to be valid, the Z stage of the imaging system is controlled to focus the imaging system at the first sample position based at least on the current separation distance measurement result of the second pair of spots and one or more previous separation distance measurements of the first pair of spots associated with one or more other sample positions of the sample.

14. The non-transitory computer readable storage medium of claim 11, wherein the operations further comprise: When both the first image and the second image are determined to be valid, the Z stage of the imaging system is controlled to focus the imaging system at the first sample position based at least on the current separation distance measurement of the first pair of spots and the current separation distance measurement of the second pair of spots.

15. The non-transitory computer readable storage medium of claim 14, wherein controlling the Z stage of the imaging system to focus the imaging system at the first sample position based at least on the current separation distance measurement of the first pair of spots and the current separation distance measurement of the second pair of spots comprises: Determine an average value of the current separation distance measurement result of the first pair of light spots and the current separation distance measurement result of the second pair of light spots; as well as The Z stage of the imaging system is controlled based at least on the average value to focus the imaging system at the first sample position.

16. The non-transitory computer readable storage medium of claim 11, wherein the operations further comprise: When both the first image and the second image are determined to be valid, a tilt measurement is determined based on a difference between the current separation distance measurement of the first pair of spots and the current separation distance measurement of the second pair of spots.

17. The non-transitory computer readable storage medium of claim 16, wherein the operations further comprise: A determination is made as to whether the current sample tilt measurement is within a threshold range determined from a previous sample tilt measurement.

18. A system, comprising: one or more light sources configured to project a first pair of light spots onto the sample at a first sample location; one or more image sensors to capture a first image of the first pair of light spots projected at the first sample location; and One or more processors, the one or more processors performing operations comprising the steps of: determining whether the first image of the first pair of light spots is valid; and When the first image is determined to be valid: obtaining a current separation distance measurement result of the first pair of light spots based on the first image; as well as The Z stage is controlled to focus at the first sample position based at least on the current separation distance measurement.

19. The system of claim 18, wherein determining whether the first image of the first pair of spots is valid comprises: Whether the first image of the first pair of light spots is valid is determined based on an intensity distribution of pixels in the first image.

20. The system of claim 18, further comprising a memory to store one or more previous separation distance measurements associated with one or more other sample positions of the sample, wherein the operations further comprise: When the first image is determined to be invalid, the Z stage is controlled to focus on the sample at the first sample position based at least on the one or more previous separation distance measurements.

21. The system of claim 18, wherein: The one or more light sources are configured to project a second pair of light spots substantially parallel to the first pair of light spots at the first sample location; and The one or more image sensors are used to capture a second image of the second pair of light spots projected at the first sample position.

22. The system of claim 21, wherein: The one or more light sources include: a first light source for projecting the first pair of light spots at the first sample position; and a second light source for projecting the second pair of light spots at the first sample position; and The one or more image sensors include: a first image sensor, the first image sensor is used to capture the first image; and a second image sensor, the second image sensor is used to capture the second image.

23. The system of claim 22, wherein the first image sensor and the second image sensor are substantially parallel linear sensors.

24. The system of claim 21, wherein the operations further comprise: determining whether the second image of the second pair of light spots is valid; as well as When the second image is determined to be valid, a current separation distance measurement result of the second pair of light spots is obtained based on the second image.

25. The system of claim 24, wherein the operations further comprise: When the first image is determined to be invalid and the second image is determined to be valid, the Z stage is controlled to focus at the first sample position based at least on the current separation distance measurement of the second pair of spots.

26. The system of claim 24, wherein: The system further includes a tilt assembly including one or more actuators for tilting a sample holder of the sample during imaging of the sample; and The operations also include: When both the first image and the second image are determined to be valid, determining a tilt measurement based on a difference between the current separation distance measurement of the first pair of spots and the current separation distance measurement of the second pair of spots; and The tilt assembly is controlled to tilt the sample holder to focus at the first sample position based at least on the tilt measurement.

27. The system of claim 18, further comprising the Z-stage, wherein the Z-stage comprises an objective lens.

Images