Autofocus with spot measurement

Abstract

Some embodiments of the present disclosure relate to an imaging system including one or more image sensors and a Z-stage, wherein the imaging system is configured to perform operations including: capturing, using the one or more image sensors, a first image of a first spot pair projected onto a first sample position of a sample; determining whether the first image of the first spot pair is valid; if the first image is determined to be valid, obtaining a current separation distance measurement for the first spot pair based on the first image; and controlling the Z-stage to focus the imaging system on the first sample position based on at least the current separation distance measurement.

Description

[Technical Field]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 410,961, entitled "SPOT ERROR HANDLING FOR FOCUS TRACKING," filed September 28, 2022, and U.S. Provisional Patent Application No. 63 / 424,771, entitled "SPOT ERROR HANDLING FOR FOCUS TRACKING," filed November 11, 2022. All of the above applications are incorporated herein by reference in their entirety. [Background technology]

[0002] Increasing the numerical aperture (NA) of an optical imaging system improves optical imaging resolution. In sequencing applications, this reduces sequencing cluster pitch, increasing cluster density and enabling lower-cost sequencing. However, increasing the NA also reduces the depth of field (DoF), i.e., the distance over which an imaged object (e.g., a cluster) remains in focus as the object is translated along the optical axis of the optical imaging system.

[0003] As optical imaging systems with higher NAs continue to be used in imaging applications to reduce costs (e.g., to reduce sequencing costs), ensuring that the imaged sample remains in focus when translated along the optical axis becomes more challenging. For example, as illustrated by FIG. 1A, some current sequencers can tilt the sample by establishing a best-fit plane for the entire sample so that the entire sample remains within the DoF of the optical imaging system. However, as illustrated in FIG. 1B, with a reduction in the available DoF of the optical imaging system, and even with global adjustments to the sample tilt, local tilt within the sample itself can create a DoF excursion 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 for the degraded portion of the image. Summary of the Invention

[0004] Embodiments of the present disclosure relate to systems and methods for dynamically tilting a sample holder in one or more directions during sample imaging based on the local topography of the sample to keep the sample in focus. Further embodiments of the present disclosure describe techniques for handling spot errors during focus tracking that can enable smoother movement of the Z-stage and / or tilt stage during imaging of the sample.

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

[0006] In some embodiments, the first light source is for projecting a first pair of spots and a second pair of spots onto the sample container, and the sample tilt is determined from a first separation measurement from images taken by the image sensor at one or more sample positions and a second separation measurement of the second pair of spots.

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

[0008] In some embodiments, the sample tilt is determined along a first direction and a second direction, and the controller is for controlling the one or more actuators to tilt the sample holder along the first direction and the second direction based on the sample tilt.

[0009] In some embodiments, the sample tilt along the second direction is determined by the imaging system by determining a first sample height based on at least a first separation measurement, determining a second sample height based on at least a second separation measurement, and calculating the sample tilt along the second direction based on at least the first sample height and the second sample height.

[0010] In some embodiments, the one or more actuators comprise a plurality of actuators, and the controller is for controlling 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 for controlling 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 spot pair from a plurality of images taken by the image sensor at a plurality of sample locations.

[0012] In some embodiments, the controller is for controlling one or more actuators to tilt the sample stage along a first direction and a second direction based on a tilt map determined from a plurality of first separation measurements of a first spot pair and a plurality of second separation measurements of a second spot pair from a plurality of images taken by the image sensor at a plurality of sample positions.

[0013] In some embodiments, the imaging system further comprises a second light source, the second light source comprising a line scanner for scanning the sample during imaging of the sample container, and the imaging direction is the direction in which the line scanner scans the sample container.

[0014] In some embodiments, the one or more actuators comprise a plurality of linear actuators, the sample holder being a movable platform mounted on the plurality of linear actuators, and the plurality of linear actuators being for tilting the sample holder along the first direction and the second direction.

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

[0016] In some embodiments, the assembly further comprises a stage, the movable platform being mounted on the stage, the stage being for laterally moving the sample holder.

[0017] In one embodiment, a method includes projecting a first pair of spots and a second pair of spots at a first sample position on a sample using one or more light sources of an imaging system; capturing images of the first pair of spots and the second pair of spots at the first sample position using an image sensor of the imaging system; determining a first separation distance of the first pair of spots and a second separation distance of the second pair of spots based on at least the images; determining a first sample height based on at least the first separation distance; determining a second sample height based on at least the second separation 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 on at least the first sample height and the second sample height.

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

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

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

[0021] In some embodiments, the method further 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 a plurality of entries corresponding to a plurality of sample tilt positions, each entry indicating an amount to tilt the sample along the scan direction or a direction substantially perpendicular to the scan direction for a corresponding sample tilt position.

[0022] In some embodiments, the method further includes tilting the sample in a direction substantially perpendicular to the scanning direction at each of a plurality of sample tilt positions using one or more actuators of the imaging system according to the tilt map.

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

[0024] In some embodiments, the imaging system further comprises a line generating device, the line generating device including a second light source for emitting a first light beam at a first wavelength, a third light source for emitting a second light beam at a second wavelength, and one or more line forming optics for shaping the first light beam into a first line and shaping the second light beam into a second line.

[0025] In one embodiment, the non-transitory computer-readable medium, when executed by a processor, causes an imaging system to perform operations including: capturing, using one or more image sensors of the imaging system, a first image of a first spot pair projected onto a first sample position of a sample; determining whether the first image of the first spot pair is valid; if the first image is determined to be valid, obtaining a current separation distance measurement for the first spot pair based on the first image; and controlling components of the imaging system to focus the imaging system on the first sample position based on at least the current separation distance measurement. For example, a Z-stage of the imaging system may be controlled to focus the imaging system on the first sample position.

[0026] In some embodiments, determining whether the first image of the first spot pair is valid includes determining whether the first image of the first spot pair is valid based on an intensity distribution of pixels in the first image.

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

[0028] In some embodiments, determining whether the first image of the first spot pair is valid includes determining that the first image is not valid, and in response to determining that the first image is not valid, storing an identifier in a memory of the imaging system indicating why the first image is not valid.

[0029] In some embodiments, the operations further include, before controlling the Z stage, determining that the current separation distance measurement of the first spot pair is within a threshold range determined from multiple previous separation distance measurements of the first spot pair associated with other sample positions of the sample.

[0030] In some embodiments, the threshold range is within a number of standard deviations above or below the mean of a number of previous separation distance measurements, the number being greater than 0 and less than 4.

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

[0032] In some embodiments, the operations further include, if 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 spot pair associated with one or more other sample positions of the sample.

[0033] In some embodiments, 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 on at least the one or more previous separation distance measurements includes retrieving a most recent separation distance measurement from the plurality of previous separation 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 separation distance measurement from the plurality of previous separation distance measurements.

[0034] In some embodiments, 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 on at least 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 embodiments, the operations further include capturing, using one or more image sensors of the imaging system, a second image of a second pair of spots projected substantially parallel to the first pair of spots at the first sample position; determining whether the second image of the second pair of spots is valid; and, if the second image is determined to be valid, obtaining a current separation distance measurement of the second pair of spots based on the second image.

[0036] In some embodiments, the operation further includes, if the first image is determined to be invalid and the second image is determined to be valid, controlling a Z stage of the imaging system to focus the imaging system on the first sample position based on the current separation distance measurement of at least the second spot pair.

[0037] In some embodiments, if the first image is determined to be invalid and the second image is determined to be valid, the operations further include controlling a Z stage of the imaging system to focus the imaging system on the first sample position based on at least a current separation distance measurement of the second spot pair and one or more previous separation distance measurements of the first spot pair associated with one or more other sample positions of the sample.

[0038] In some embodiments, the operation further includes, if both the first image and the second image are determined to be valid, controlling a Z stage of the imaging system to focus the imaging system on the first sample position based on at least the current separation distance measurement of the first spot pair and the current separation distance measurement of the second spot pair.

[0039] In some embodiments, controlling the Z stage of the imaging system to focus the imaging system on the first sample position based on at least the current separation distance measurement of the first pair of spots and the current separation distance measurement of the second pair of spots includes determining an average of the current separation distance measurement of the first pair of spots and the current separation distance measurement 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 on at least the average.

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

[0041] In some embodiments, the operations further include determining whether the current specimen tilt measurement is within a threshold range determined from the previous specimen tilt measurement.

[0042] In one embodiment, a system includes one or more light sources for projecting a first pair of spots onto a first sample position on a sample, one or more image sensors for capturing a first image of the first pair of spots projected onto the first sample position, and one or more processors for performing operations including: determining whether the first image of the first pair of spots is valid; if the first image is determined to be valid, obtaining a current separation distance measurement for the first pair of 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. In some implementations, other components other than or in addition to the Z-stage may be controlled to focus on the first sample position.

[0043] In some embodiments, determining whether the first image of the first spot pair is valid includes determining whether the first image of the first spot pair is valid based on an intensity distribution of pixels in the first image.

[0044] In some embodiments, the system further comprises a memory for storing one or more previous separation distance measurements associated with one or more other sample positions of the sample, and the operations further include, if the first image is determined to be invalid, controlling the Z stage to focus on the sample at the first sample position based on at least the one or more previous separation distance measurements.

[0045] In some embodiments, the one or more light sources are for projecting a second pair of spots at the first sample position substantially parallel to the first pair of spots, and the one or more image sensors capture a second image of the second pair of spots projected at the first sample position.

[0046] In some embodiments, the one or more light sources include a first light source for projecting a first pair of spots onto the first sample location and a second light source for projecting a second pair of spots onto the first sample location, and the one or more image sensors include a first image sensor for capturing the first image and a second image sensor for capturing the second image.

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

[0048] In some embodiments, the operation further includes determining whether a second image of the second spot pair is valid, and if the second image is determined to be valid, obtaining a current separation distance measurement of the second spot pair based on the second image.

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

[0050] In some embodiments, the system further comprises a tilt assembly including one or more actuators for tilting a sample holder of the sample during imaging of the sample, and the operation further comprises, when both the first image and the second image are determined to be valid, determining a tilt measurement based on a difference between a current separation distance measurement of the first spot pair and a current separation distance measurement of the second spot pair, and controlling the tilt assembly to tilt the sample holder to focus at the first sample position based at least on the tilt measurement.

[0051] In some embodiments, the system further comprises a Z-stage, the Z-stage comprising an objective lens.

[0052] Other features and aspects of the disclosed technology will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, features according to embodiments of the disclosed technology. The Summary is not intended to limit the scope of any inventions described herein, which are defined by the claims and equivalents. [Brief explanation of the drawings]

[0053] The present disclosure, in accordance with one or more embodiments, will now be described in detail with reference to the following figures. The figures are provided for illustrative purposes only and merely depict exemplary embodiments. Furthermore, it should be noted that for clarity and ease of illustration, elements in the figures have not necessarily been drawn to scale.

[0054] Some of the figures included herein illustrate various embodiments of the disclosed technology from different viewing angles. Although the accompanying description may refer to such figures as "top," "bottom," or "side" views, such references are merely illustrative and do not imply or require that the disclosed technology be implemented or used in a particular spatial orientation unless expressly stated otherwise. [Figure 1A] 1 illustrates de-tilting a sample by establishing a best fit plane for the entire sample. [Figure 1B] Illustrates a local tilt within the sample that creates a depth of field excursion that causes parts of the sample image to be out of focus. [Figure 2] 1 illustrates a block diagram of an optical imaging system according to some embodiments of the present disclosure. [Figure 3] 3 is a perspective view of an optical assembly that can be used with the optical system shown in FIG. 2 according to some embodiments of the present disclosure. [Figure 4] FIG. 4 is a plan view of the optical assembly shown in FIG. 3. [Figure 5] 4 illustrates incident and reflected light beams when the optical assembly shown in FIG. 3 is focused on an object. [Figure 6] 6 illustrates the beam spot on the detector surface provided by the reflected light beam shown in FIG. 5. [Figure 7] 4 illustrates the incident and reflected light beams when the optical assembly shown in FIG. 3 is below focus. [Figure 8] 8 illustrates the beam spot on the detector surface provided by the reflected light beam shown in FIG. 7; [Figure 9] 4 illustrates the incident and reflected light beams when the optical assembly shown in FIG. 3 is above focus. [Figure 10] 10 illustrates the beam spot on the detector surface provided by the reflected light beam shown in FIG. 9. [Figure 11]1 shows a series of plots illustrating the surface profile of a flow cell sample scanned using a line scanning system, according to some embodiments of the present disclosure. [Figure 12] 1 illustrates a coordinate system that may be used when designing an assembly for dynamically moving a sample laterally and / or angularly, according to some embodiments of the present disclosure. [Figure 13] 1 illustrates a sample stage assembly configured to mount a movable platform containing a sample container, according to some embodiments of the present disclosure. [Figure 14] 1 depicts a movable platform mounted on an actuator of a tilt-tilt assembly, according to some embodiments of the present disclosure. [Figure 15] 1 depicts a movable platform on a sample stage assembly according to some embodiments of the present disclosure. [Figure 16A] 1 illustrates an exploded view of an actuator according to some embodiments of the present disclosure. [Figure 16B] 16B shows a perspective view of the actuator of FIG. 16A. [Figure 16C] 16B shows a side view of the actuator of FIG. 16A. [Figure 16D] 16B shows another perspective view of the actuator of FIG. 16A. [Figure 17] 15 illustrates the underside of the movable platform of FIG. 14 according to some embodiments of the present disclosure. [Figure 18] 15 shows a perspective view of the movable platform of FIG. 14 according to some embodiments of the present disclosure. [Figure 19] 1 illustrates a movable platform interfacing and coupling with an actuator according to some embodiments of the present disclosure. [Figure 20] 1 depicts a process for determining the tilt of a specimen along a line scan direction using two pairs of projected spot beams, according to some embodiments of the present disclosure. [Figure 21A]FIG. 1 is an operational flow diagram illustrating an exemplary method for dynamically tilting a specimen based on a predetermined tilt trajectory, according to some embodiments of the present disclosure. [Figure 21B] 10 illustrates tilt angles at different sample positions for a tilt map according to some embodiments of the present disclosure. [Figure 21C] 10 conceptually illustrates the smooth adaptation of tilt trajectories in the z direction based on different tilt angles, according to some embodiments of the present disclosure. [Figure 22] FIG. 1 is a block diagram illustrating an exemplary mechanism for tilting a sample holder, according to some embodiments of the present disclosure. [Figure 23A] 1 is an operational flow diagram illustrating an example method for determining a gradient map along a scan direction, according to some embodiments of the present disclosure. [Figure 23B] FIG. 23A depicts a focus tracking module projecting one spot beam pair onto the surface of the sample across different sample positions in the scan direction during the process. [Figure 23C] 10 illustrates a smooth tilt trajectory generated by interpolating between five entries corresponding to spot beam measurements, according to some embodiments of the present disclosure. [Figure 24A] 10 is an operational flow diagram illustrating an example method for determining a gradient map along a scan direction and a direction orthogonal to the scan direction, according to some embodiments of the present disclosure. [Figure 24B] FIG. 24A depicts a focus tracking module projecting two spot beam pairs onto the surface of the sample across different sample positions in the scan direction during the process. [Figure 24C] 1 depicts a process for determining the tilt of a specimen in the scan direction and in a direction orthogonal to the scan direction using two pairs of projected spot beams, according to some embodiments of the present disclosure. [Figure 25A] 1 illustrates two projected beam spot pairs and the relative orientation of one of the two scan lines in a two-channel line-scan imaging system, according to some embodiments of the present disclosure. [Figure 25B]10 illustrates two projected beam spot pairs and different relative orientations of two scan lines in a two-channel line-scan imaging system, according to some embodiments of the present disclosure. [Figure 25C] 10 illustrates two projected beam spot pairs and different relative orientations of two scan lines in a two-channel line-scan imaging system, according to some embodiments of the present disclosure. [Figure 25D] 10 illustrates two projected beam spot pairs and different relative orientations of two scan lines in a two-channel line-scan imaging system, according to some embodiments of the present disclosure. [Figure 25E] 10 illustrates two projected beam spot pairs and different relative orientations of two scan lines in a two-channel line-scan imaging system, according to some embodiments of the present disclosure. [Figure 25F] 10 illustrates two projected beam spot pairs and different relative orientations of two scan lines in a two-channel line-scan imaging system, according to some embodiments of the present disclosure. [Figure 26] 10 conceptually illustrates the advantages of using multiple tilt actuators, according to some embodiments of the present disclosure. [Figure 27] FIG. 1 is a block diagram illustrating some components of an exemplary focus control system for focus tracking, according to some embodiments of the present disclosure. [Figure 28] FIG. 2 illustrates an example architecture of a Z stage controller, according to some embodiments of the present disclosure. [Figure 29] 1 depicts an example of the design and operation of a focus tracking system utilizing two focus tracking spot pairs per sample surface, according to some embodiments of the present disclosure. [Figure 30A] 29 depicts pixel intensity as a function of pixel number for one of the sensors of FIG. 29 after incidence of light corresponding to a spot pair. [Figure 30B] 29 depicts pixel intensity as a function of pixel number for the other sensors of FIG. 29 after incidence of light corresponding to a spot pair. [Figure 31] 29 depicts both the object plane and the sensor plane during scanning of the flow cell surface in one exemplary embodiment utilizing the focus tracking system described with reference to FIG. [Figure 32] 10 is a plot showing Z steps (in nm) as a function of time for a Z stage used with a focus tracking system that does not implement spot error processing. [Figure 33A] 1 depicts an image of a portion of a flow cell with a large air bubble. [Figure 33B] 33B depicts plots showing focus tracking performance, including a summary of defocus error, when focus tracking is performed on the flow cell of FIG. 33A with spot error processing turned off. [Figure 33C] 33B depicts a plot showing focus tracking performance, including a summary of defocus error, when focus tracking is performed on the flow cell of FIG. 33A with spot error processing turned on. [Figure 34A] 1 depicts images of a portion of a flow cell having a defect that causes saturation of the focal tracking spot of the sensor over approximately 8 frames. [Figure 34B] 3A depicts a plot showing focus tracking performance, including a summary of defocus error, when focus tracking is performed on the flow cell of FIG. 34A with spot error processing turned off, and a simulation of performing focus tracking on the flow cell of FIG. 34A with spot error processing turned on. [Figure 35A] 1 depicts images of a portion of a flow cell with a large air bubble causing poor measurement of the sensor's focal tracking spot over approximately 50 frames. [Figure 35B] 35A depicts a plot showing focus tracking performance, including a summary of defocus error, when focus tracking is performed on the flow cell of FIG. 35A with spot error processing turned off, and a simulation of performing focus tracking on the flow cell of FIG. 35A with spot error processing turned on. [Figure 36A] 1 depicts an image of a portion of a flow cell that has debris on the surface (appears as dark dots) causing poor measurement of the focal tracking spot. [Figure 36B] 36B depicts plots showing focus tracking performance, including a summary of defocus error, when focus tracking is performed on the flow cell of FIG. 36A with spot error processing turned off. [Figure 36C] 36B depicts a plot showing focus tracking performance, including a summary of defocus error, when focus tracking is performed on the flow cell of FIG. 36A with spot error processing turned on. [Figure 37A] Depicts an image of a portion of a flow cell that has residues on the surface (appearing as dark patterns) that cause poor measurement of the focal tracking spot. [Figure 37B] 37B depicts plots showing focus tracking performance, including a summary of defocus error, when focus tracking is performed on the flow cell of FIG. 37A with spot error processing turned off. [Figure 37C] 37B depicts a plot showing focus tracking performance, including a summary of defocus error, when focus tracking is performed on the flow cell of FIG. 37A with spot error processing turned on. [Figure 38A-1] 10 depicts a plot showing focus tracking performed on a flow cell using a four-beam, two-sensor focus tracking system without spot error processing, with frequent Z-step spikes present due to glitches in the measurement of the focus tracking spot on one or both detectors. [Figure 38A-2] 10 depicts a plot showing focus tracking performed on a flow cell using a four-beam, two-sensor focus tracking system without spot error processing, with frequent Z-step spikes present due to glitches in the measurement of the focus tracking spot on one or both detectors. [Figure 38B-1] FIG. 38B depicts a plot showing focus tracking performed on the same flow cell as FIG. 38A using a four-beam, two-sensor focus tracking system with spot error processing, eliminating Z-step spikes due to glitched measurements of the focus tracking spot. [Figure 38B-2]FIG. 38B depicts a plot showing focus tracking performed on the same flow cell as FIG. 38A using a four-beam, two-sensor focus tracking system with spot error processing, eliminating Z-step spikes due to glitched measurements of the focus tracking spot. [Figure 39] FIG. 1 is a flow diagram illustrating an example method for implementing spot error processing according to some embodiments of the present disclosure. [Figure 40] FIG. 1 is a flow diagram depicting a method of spot error handling in a system that performs focus tracking using at least two pairs of substantially parallel spots, according to some embodiments of the present disclosure. [Figure 41] FIG. 1 is a flow diagram depicting a method for detecting gradient spikes in a system that performs focus tracking using at least two pairs of substantially parallel spots projected onto at least two locations on a sample, according to some embodiments of the present disclosure.

[0055] The figures are not exhaustive and are not intended to limit the disclosure to the precise form disclosed. DETAILED DESCRIPTION OF THE INVENTION

[0056] As used herein to refer to a sample, the term "feature" is intended to mean a point or area in a pattern that can be distinguished from other points or areas according to their relative position. An individual feature may contain 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 complementary sequence).

[0057] As used herein, the term "swath" is intended to mean a rectangular portion of an object. A 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 is constant along its entire length. Multiple swaths of an object may be parallel to one another. Multiple swaths of an object may overlap one another, be adjacent to one another, or be separated from one another by interstitial areas.

[0058] As used herein, the term "tile" generally refers to one or more images of the same region of a sample, where each of the one or more images may represent a respective color channel. A tile may form an imaging data subset of the imaging dataset of one imaging cycle.

[0059] As used herein, the term "x-y coordinates" is intended to mean information that specifies a position, size, shape, and / or orientation within an x-y plane. The information may be, for example, numerical coordinates in a Cartesian coordinate system. The coordinates may be provided relative to one or both of the x-axis and y-axis, or may be provided relative to another location within the x-y plane. For example, the coordinates of a feature of an object may specify the location of the feature relative to the location of a reference, or other features of the object.

[0060] As used herein, the term "xy plane" is intended to mean the two-dimensional area defined by linear axes x and y. When used in reference to a detector and an object observed by the detector, the area may be further specified as being orthogonal to the direction of observation between the detector and the object being detected. When used herein to refer to a line scanner, the term "y direction" refers to the direction of scanning.

[0061] As used herein, the term "z-coordinate" refers to information specifying the location of a point, line, or area along an axis perpendicular to the x-y plane. In certain embodiments, the z-axis is perpendicular to the area of ​​an object observed by a detector. For example, the direction of focus of an optical imaging system may be specified along the z-axis.

[0062] As used herein, the term "scanning" is intended to mean detecting a two-dimensional cross-section in the xy plane of an object, where the cross-section is rectangular or elliptical. For example, in the case of fluorescence imaging, an area of ​​an object having a rectangular or elliptical shape can be specifically excited (to the exclusion of other areas) and / or emission from that area can be specifically acquired (to the exclusion of other areas) at a given time point in the scan.

[0063] As mentioned above, there is an increasing need to enable dynamic sample tilt in optical imaging systems that utilize larger NAs and sacrifice DoF to resolve finer optical features. In such systems, even small amounts of sample twist that defocus a portion of the sample within the field of view can cause significant errors. Small amounts of tilt in the scan direction or perpendicular to the scan direction can blur or defocus the region of interest being scanned. Therefore, there is a need for dynamic multi-axis sample tilt.

[0064] 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 focus of the sample during a sample scan. One set of embodiments describes a sample stage assembly including a tilt-tilt assembly for enabling multi-axis tilt of the sample holder during a scan. Another set of embodiments describes techniques for utilizing a focus tracking system to determine local sample tilt along the scan direction and / or a direction orthogonal to the scan direction. A further set of embodiments describes techniques for generating a tilt map utilized by a controller to direct the operation of an assembly that dynamically tilts the sample holder during a sample scan. A further set of embodiments describes techniques for handling spot errors during focus tracking, which can enable smoother movement of the Z-stage and / or tilt stage during imaging of the sample.

[0065] FIG. 2 illustrates a block diagram of an optical imaging system 100 according to some embodiments of the present disclosure. The optical imaging system 100 images a sample of interest (object 110) for analysis. For example, in embodiments in which the optical imaging system 100 functions as a sequencer, the imaged sample can be subjected to the sequencer. In some embodiments, the optical imaging system 100 can function as a profilometer to determine the surface profile (e.g., topography) of the imaged object. Additionally, 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 the object 110 near a focal plane FP of the optical assembly 106, and a stage controller 115 configured to move the object holder 102 laterally (along an X-axis and / or a Y-axis extending into the page), vertically / elevationally along a Z-axis, and / or angularly along the X-axis (tilt), Y-axis (tilt), and / or Z-axis (torsion). 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 .

[0066] In some embodiments, object 110 is a sample container containing a biological sample to be imaged using one or more fluorescent dyes. For example, in certain embodiments, the sample container may be implemented as a patterned flow cell including a translucent cover plate, a substrate, and a liquid sandwiched therebetween, and the biological sample may be located on the inner surface of the translucent cover plate or the inner surface of the substrate. The flow cell may include a large number (e.g., thousands, millions, or billions) of wells or regions within the substrate that are patterned into a defined array (e.g., a hexagonal array, a rectangular array, etc.). Each region may form a cluster (e.g., a monoclonal cluster) of biological sample, such as DNA, RNA, or another genomic material, that can be sequenced using sequencing-by-synthesis. The flow cell may be divided into several physically separated lanes (e.g., eight lanes), each containing an array of clusters. During each cycle of sequencing, each surface (e.g., top and bottom) of each lane can be imaged in separate swaths (e.g., three), and any number of images or tiles can be collected for each swath.

[0067] Although not shown, the optical imaging system 100 may include one or more subsystems or devices for performing various test protocols. For example, if the sample includes a flow cell with a flow channel, the optical imaging system 100 may include a fluid control system including a liquid reservoir fluidly 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) sample containers and waste valves. Another subsystem that may be included is a temperature control system, which may have heaters / coolers configured to adjust the temperature of the sample and / or fluids flowing through the sample. The temperature control system may include a sensor to detect the temperature of the fluid.

[0068] As shown in the figure, optical assembly 106 is configured to direct input light toward object 110 and to receive and direct output light toward one or more detectors. The output light may be input light that has been at least one of reflected and refracted by object 110 and / or the output light may be light emitted from object 110. To direct the input light, optical assembly 106 may include at least one reference light source 112 and at least one excitation light source 114 that direct light, such as a light beam having a predetermined wavelength, through one or more optical components of optical assembly 106. Optical assembly 106 may include various optical components, including a conjugate lens 118, for directing the input light toward object 110 and the output light toward detector(s).

[0069] The reference light source 112 may be used by a distance measurement system and / or a focus control system (or focusing mechanism) of the optical imaging system 100, and the excitation light source 114 may be used to excite biological or chemical substances in the object 110 if the object 110 includes a biological or chemical sample. The excitation light source 114 may be positioned to illuminate the bottom surface of the object 110, such as in TIRF imaging, or to illuminate the top surface of the object 110, such as in epifluorescence imaging. As shown in FIG. 2 , the conjugate lens 118 directs input light to a focal zone 122 located within a 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 may move the object 110 in the Z direction to adjust the working distance WD1, for example, so that a portion of the object 110 is within the focal zone 122. Additionally, the stage controller 115 can actively orient the area of ​​interest of the object 100 within the FP by rotating the object holder 102 about the X-, Y-, and / or Z-axes.

[0070] To determine whether the object 110 is in focus (i.e., sufficiently within the focal region 122 or focal plane FP), the optical assembly 106 is configured to direct at least one pair of light beams to the focal region 122 where the object 110 is approximately located. The object 110 reflects the light beams. More specifically, an outer surface of the object 110 or an interface within the object 110 reflects the light beams. The reflected light beams then return to and propagate through the lens 118. As shown in the figure, each light beam has an optical path that includes a portion that has not yet been reflected by the object 110 and a portion that has been reflected by the object 110. The portions of the optical path before reflection are designated as incident light beams 130A and 132A and are shown with arrows pointing toward the object 110. The portions of the optical path that are reflected by the object 110 are designated as reflected light beams 130B and 132B and are shown with arrows pointing away from the object 110. For purposes of illustration, light beams 130A, 130B, 132A, and 132B are shown as having different optical paths within lens 118 and near object 110. However, in this embodiment, light beams 130A and 132B are configured to propagate in counter directions and have the same or substantially overlapping optical paths within lens 118 and near object 110, and light beams 130B and 132A are configured to propagate in counter directions and have the same or substantially overlapping optical paths within lens 118 and near object 110.

[0071] 2, 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 can pass through a different lens not used for imaging. In this alternative embodiment, lens 118 is dedicated to passing 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 will be understood that the systems and methods described herein for focus determination and distance measurement can be performed using a common objective lens shared with imaging optics, or alternatively, the objective lens illustrated herein can be dedicated to focus determination or distance measurement.

[0072] The reflected light beams 130B and 132B propagate through the lens 118 and may optionally be further directed 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 reflected light beams 130B and 132B are detected by a single focus detector 144. The reflected light beams may be used to determine a relative separation RS1. For example, the relative separation RS1 may be determined by the distance separating the beam spots (i.e., the separation distance) from the reflected light beams 130B and 132B incident on the focus detector 144. The relative separation RS1 may be used to determine the degree of focus of the optical imaging system 100 with respect to the object 110. However, in an alternative embodiment, each reflected light beam 130B and 132B may be detected by a separate corresponding focus detector 144, and the relative separation RS1 may be determined based on the position of the beam spot on the corresponding focus detector 144.

[0073] If the object 110 is not within a sufficient degree of focus, the computing system 120 can operate the stage controller 115 to move the object holder 102 to a desired position. Alternatively or in addition to moving the object holder 102, the optical assembly 106 can be moved in the Z direction and / or along the XY plane. For example, if the object 110 is positioned above the focal plane FP (or focal zone 122), the object 110 can be relatively moved a distance ΔZ1 toward the focal plane FP, or if the object 110 is positioned below the focal plane FP (or focal zone 122), the object 110 can be relatively moved a distance ΔZ2 toward the focal plane FP. In some embodiments, the optical imaging system 100 can replace the lens 118 with another lens 118 or other optical component to move the focal zone 122 of the optical assembly 106.

[0074] The example described above and in Figure 2 is presented for a system for controlling focus or determining the degree of focus. The system is also useful for determining a working distance WD1 between the object 110 and the lens 118 (e.g., based on the relative separation of the reflected light beams). In such an embodiment, the focus detector 144 can function 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.

[0075] Additionally, as described further below, the system can be useful for determining the surface profile of the object 110 along one or more dimensions of the object. For example, by determining the variation in the relative separation of the reflected light beams along different positions on the object, the variation in the working distance between the object 110 and the lens 118 along the imaging direction can be determined, which can be mapped to the object height along the imaging direction (i.e., in the z-direction). In certain embodiments described further below, the optical assembly 106 is configured to direct multiple light beam pairs (e.g., at least two pairs) along different positions on the scanned object surface. Based on the relative separation of each of the light beam pairs and the distance between different light beam pairs, the surface profile of the object can be determined in one or more dimensions. Given knowledge of the object's surface profile, the optical imaging system 100 can actively orient an area of ​​interest of the object 100 within the FP by rotating the object holder 102 about the X-axis, Y-axis, and / or Z-axis via the stage controller 115.

[0076] Thus, the systems and methods described herein can 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 imaged object to keep the object in focus.

[0077] In one embodiment, during operation, excitation light source 114 directs input light (not shown) toward object 110 to excite fluorescently labeled biological or chemical substances. The biological or chemical substance labels provide optical signals 140 (also referred to as optical emissions) having predetermined wavelength(s). Optical signals 140 are received by lens 118 and then directed by other optical components of optical assembly 106 to at least one object detector 142. While the illustrated embodiment shows only one object detector 142, object detector 142 may comprise multiple detectors. For example, object detector 142 may include a first detector configured to detect one or more wavelengths of light and a second detector configured to detect one or more different wavelengths of light. Optical assembly 106 may include lens / filter assemblies that direct different optical signals along different optical paths toward corresponding object detectors.

[0078] The object detector 142 communicates object data associated with the detected optical signal 140 to the computing system 120. The computing system 120 may then record, process, analyze, and / or communicate the data to other users or computing systems, including remote computing systems, via communications lines (e.g., the Internet). By way of example, the object data may include imaging data that is processed to generate image(s) of the object 110. The 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 light emitted from biological or chemical substances, as well as light reflected and / or refracted by an optical substrate or other component. For example, the optical signal 140 may include light reflected by an encoded particulate, such as a holographically encoded optical identification element.

[0079] In some embodiments, a single detector may provide both functions as described above for object detector 142 and focus detector 144. For example, a single detector may also detect a reflected light beam pair (e.g., reflected light beams 130B and 132B) as well as an optical signal (e.g., optical signal 140).

[0080] Optical imaging system 100 may include a user interface 125 for interacting with a user through computing system 120. For example, user interface 125 may include a display (not shown) for presenting and requesting information from the user and a user input device (not shown) for receiving user input.

[0081] Computing system 120 may include, among other things, object analysis module 150 and focus control module 152. Focus control module 152 is configured to receive focus data acquired by focus detector 144. The focus data may include signals representative of beam spots incident on focus detector 144. The data may be processed to determine a relative separation (e.g., a separation distance between the beam spots). The degree of focus of optical imaging system 100 with respect to object 110 may then be determined based on the relative separation. In certain embodiments, a working distance WD1 between object 110 and lens 118 may be determined. Similarly, object analysis module 150 may receive object data acquired by object detector 142. The object analysis module may process or analyze the object data to generate an image of the object.

[0082] Furthermore, computing system 120 may include any processor- or microprocessor-based system, including systems using microcontrollers, reduced instruction set computers (RISC), application specific integrated circuits (ASIC), field programmable gate arrays (FPGA), logic circuits, and any other circuits or processors capable of performing the functions described herein. The above examples are illustrative only and, thus, are not intended to limit the definition and / or meaning of the term system controller. In one embodiment, computing system 120 executes instruction sets stored in one or more storage elements, memories, or modules to acquire and analyze at least one of the object data. The storage elements may be in the form of information sources or physical memory elements within optical imaging system 100.

[0083] The set of instructions may include various commands that instruct the optical imaging system 100 to perform a particular protocol. For example, the set of instructions may include various commands to perform tests and image the object 110, to move the object holder 102 linearly or rotationally, or to determine the surface profile of the object 110. The set of instructions may be in the form of a software program.

[0084] As described above, the excitation light source 114 generates excitation light that is directed toward 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 across a portion of the object 110, such as a group of columns and rows of the object 110. Alternatively, the excitation light may illuminate one or more entire regions of the object 110 at a time and stop sequentially through the regions in a "step-and-shoot" scanning pattern.

[0085] In some embodiments, the excitation light source 114 utilizes line scanning to image the sample. For example, the excitation light source 114 may be implemented as part of a line-generating module that includes one or more light sources operating at one or more wavelengths and beam-shaping optics aligned at a predetermined angle relative to each light source. The beam-shaping optics may be used to provide uniform line illumination with a desired aspect ratio. In certain embodiments, the line-generating module is implemented as part of a two-channel imaging system that 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 may be a "green" wavelength (e.g., approximately 520-565 nm) and the second wavelength may be a "red" wavelength (e.g., approximately 625-740 nm). Such a line-scanning system may be utilized in conjunction with a TDI sensor.

[0086] Object 110 generates optical signal 140, which may include optical emissions generated in response to illumination of indicia within object 110 and / or light reflected or refracted by the optical substrate of object 110. Alternatively, optical signal 140 may be generated without illumination based entirely on the emission characteristics of materials within object 110 (e.g., radioactive or chemiluminescent components within the object).

[0087] The object detector 142 and the focus detector 144 may be, for example, photodiodes or cameras. In some embodiments herein, the detectors 142 and 144 may comprise charge-coupled device (CCD) cameras (e.g., time delay integration (TDI) CCD cameras) that may interact with various filters. The cameras are not limited to CCD cameras; other camera and image sensor technologies may be used. In certain embodiments, the camera sensors may have a pixel size of about 1 to about 15 μm.

[0088] 3 and 4 illustrate perspective and / or top views of an optical assembly 202 formed in accordance with one embodiment. The optical assembly 202 may be used with the optical imaging system 100 (FIG. 2) or other optical imaging systems. As shown, the optical assembly 202 includes an optical train 240 of optical components 241-245 that directs light beams 230 and 232 along an optical track or path 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 range detector. The series of optical components 241-245 in the optical train 240 includes a dual beam generator 241, a beam splitter 242, a conjugate lens 243, a beam combiner 244, and a folding mirror 245.

[0089] 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 approximately 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 toward a beam splitter 242. In the illustrated embodiment, the dual beam generator 241 comprises a single body having opposing parallel surfaces 260 and 262 ( FIG. 4 ). The first surface 260 reflects a portion of the light beam 228, forming the incident light beam 230A, and refracts a portion of the light beam 228. The refracted portion of the light beam 228 is reflected by an opposing second surface 262 back toward the first surface 260, forming the incident light beam 232A.

[0090] Dual beam generator 241 directs parallel incident light beams 230A and 232A toward beam splitter 242. Beam splitter 242 reflects incident light beams 230A and 232A toward conjugate lens 243. In this example, beam splitter 242 includes a pair of reflectors (e.g., aluminum tabs) positioned to reflect incident light beams 230A and 232A and reflected light beams 230B and 232B. Beam splitter 242 is positioned to reflect incident light beams 230A and 232A so that the incident light beams 230A and 232A propagate parallel to an optical axis 252 of lens 243. Optical axis 252 extends through the center of lens 243 and intersects focal zone 256. Lens 243 may be a near-infinity conjugate objective lens. Alternatively, the incident light beams 230A and 232A may propagate in a manner that is not parallel to the optical axis 252. As also shown in FIG. 4, the incident light beams 230A and 232A may be equally spaced from the optical axis 252 as the incident light beams 230A and 232A propagate through the lens 243.

[0091] As described above with respect to optical imaging system 100, incident light beams 230A and 232A may converge toward focal zone 256, be reflected by object 268 (shown in FIG. 5 ) positioned proximate focal zone 256, and return to and propagate through lens 243 as reflected light beams 230B and 232B. 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 in the opposite direction along substantially the same optical path as incident light beam 232A, and reflected light beam 232B propagates in the opposite direction along substantially the same optical path as incident light beam 230A. Reflected light beams 230B and 232B exit lens 243 separated by a path spacing PS2 that is substantially equal to the path spacing PS1 that separates incident light beams 230A and 232A (shown in FIG. 4).

[0092] As shown in FIGS. 3 and 4 , the reflected light beams 230B and 232B enter the dual beam generator 241, which directs them through a range limiter 254 to the beam combiner 244. In the illustrated embodiment, the beam combiner 244 is configured to modify the path spacing PS separating the 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 the focus detector 250. In some embodiments, the separation distance SD1 at the focus detector 250 is smaller than the path spacing PS at the beam combiner 244, such that only a single focus detector 250 can detect both the reflected light beams 230B and 232B. Additionally, beam combiner 244 may substantially equalize the optical path lengths of reflected light beams 230B and 232B.

[0093] After exiting lens 243, reflected light beams 230B and 232B propagate substantially parallel to each other between the optical components. 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" to each other if they are substantially coplanar, and if allowed to propagate infinitely, they do not intersect each other or converge / diverge with respect to each other at a slow rate. For example, two light beams are substantially parallel if the crossing angle is less than 20°, or more specifically, less than 10°, or even more specifically, less than 1°. For example, the reflected light beams 230B and 232B may propagate substantially parallel to each other between the beam splitter 242 and the dual beam generator 241, between the dual beam generator 241 and the beam combiner 244, between the beam combiner 244 and the folding mirror 245, and between the folding mirror 245 and the focus detector 250.

[0094] Optical train 240 may be configured to maintain a projective relationship between reflected light beams 230B and 232B throughout the optical track, so that the degree of focus may be determined. By way of example, when optical assembly 202 is focused on an object, reflected light beams 230B and 232B will propagate parallel to each other between each optical component in optical train 240. When optical assembly 202 is not focused on an object, reflected light beams 230B and 232B are coplanar but propagate at a slight angle relative to each other. For example, reflected light beams 230B and 232B may diverge from each other or converge toward each other as they travel along the optical track to focus detector 250.

[0095] To this end, each optical component 241-245 may have one or more surfaces that are shaped and oriented to at least one of reflect and refract the reflected light beams 230B and 232B so that the reflected light beams 230B and 232B maintain a projective relationship between the reflected light beams 230B and 232B. For example, optical components 242 and 245 may have a flat surface that reflects both incident light beams 230B and 232B. Optical components 241 and 244 may also each have parallel surfaces that reflect one of the incident light beams 230B and 232B. Thus, if the reflected light beams 230B and 232B are parallel, the reflected light beams 230B and 232B will remain parallel to one another after exiting each optical component. If reflected light beams 230B and 232B are converging or diverging toward each other at a particular speed, then reflected light beams 230B and 232B will be converging or diverging toward each other at the same speed after exiting each optical component. Thus, optical components 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, each surface reflecting a corresponding one of the reflected light beams.

[0096] The optical imaging system can include one or more optical assemblies, such as those discussed above, for determining working distance or focus. For example, the optical imaging system can include two optical assemblies of the type shown in FIGS. 3 and 4 to allow focus to be determined at two different locations on the object or to provide a determination of the working distance between the optical imaging system and the object at two different locations. In embodiments where more than one optical assembly is present, the optical assemblies can be discrete and separate, or the optical assemblies can share optical components. The optical assemblies can share optical components such as the reference light source 212, focus detector 250, folding mirror 245, beam combiner 244, dual beam generator 241, beam splitter 242, epi-fluorescent input (EPI) reflector 280, and range limiter 254. Optical components can be shared by placing a beam splitter in the optical train upstream of the shared components. 3 and 4, one or more versions of the other optical assemblies illustrated herein may be present in a particular optical imaging system. Additionally, a particular optical imaging system may include various combinations of the optical assemblies described herein.

[0097] 4, reflected light beams 230B and 232B ultimately impinge on detector surface 264 of focus detector 250 at corresponding beam spots. The beam spots are spaced apart by a separation distance SD1. The separation distance SD1 may indicate whether optical assembly 202 has a sufficient degree of focus on the object. The separation distance SD1 on detector surface 264 may also indicate the working distance between lens 243 and the object being imaged.

[0098] In other embodiments, optical components 241-245 may be replaced with alternative optical components that perform substantially the same functions as those described above. For example, beam splitter 242 may be replaced with a prism that directs incident light beams 230A and 232A parallel to optical axis 252 through lens 243. Beam combiner 244 may not be used or may be replaced with an optical table that does not affect the path spacing of the reflected light beams. Furthermore, optical components 241-245 may have different sizes and shapes and may be arranged in different configurations or orientations, as desired. For example, optical train 240 of optical assembly 202 may be configured for a compact design.

[0099] Additionally, in alternative embodiments, the parallel light 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 light beams. In alternative embodiments, the focus detector 250 may include two focus detectors positioned side-by-side at fixed, known positions relative to each other. Each focus detector may detect a separate reflected light beam. The relative separation between the reflected light beams may be determined based on the positions of the beam spots associated with the respective focus detectors and the relative positions of the focus detectors relative to each other.

[0100] Although not illustrated in FIGS. 3 and 4 , the optical assembly 202 can also be configured to facilitate collecting output light projected from the object 268. For example, the optical assembly 202 can include an epifluorescence input (EPI) reflector 280 positioned to reflect incident light provided by an excitation light source (not shown). The light can be directed toward a beam splitter 242, which reflects at least a portion of the excitation light and directs it along an optical axis 252 through a lens 243. The lens 243 directs the light toward the object 268, which can provide the output light. The lens 243 then receives the output light (e.g., optical emission) from the object 268 and directs the output light back toward the beam splitter 242. The beam splitter 242 can allow a portion of the output light to propagate therethrough along the optical axis. The output light can then be detected by an object detector (not shown).

[0101] 4, EPI input reflector 280 includes two paths 282 and 284 that allow light beams 230 and 232 to propagate therethrough without being affected by input reflector 280. Thus, beam splitter 242 can reflect incident light beams 230A and 230B and reflected light beams 232A and 232B, as well as reflecting excitation light.

[0102] 5-8 illustrate different projective relationships between reflected light beams 230B and 232B and corresponding beam spots 270 and 272 on detector surface 264. As discussed above, the projective relationship between the reflected light beams is based on where the object is positioned relative to the focal zone. When the object moves relative to the focal zone, the projective relationship between the reflected light beams changes, and as a result, the relative separation between the reflected light beams also changes. FIGS. 5-8 illustrate how the separation distance SD measured between the beam spots can change as the projective relationship between the reflected light beams changes. However, the separation distance SD is merely one method of determining the relative separation between the reflected light beams. Therefore, one skilled in the art will understand that FIGS. 5-8 illustrate merely one method of determining the relative separation, and that other methods for determining the relative separation or projective relationship are possible.

[0103] 5-6 illustrate the projective relationship between reflected light beams 230B and 232B when optical assembly 202 (FIG. 3) is focused on object 268. As shown, incident light beams 230A and 232A propagate parallel to one another through lens 243 and are spaced apart by path spacing PS1. In the illustrated embodiment, incident light beams 230A and 232A propagate parallel to and are equidistant from optical axis 252 of lens 243. In alternative embodiments, incident light beams 230A and 232A may propagate non-parallel to and have a different spacing from optical axis 252. In certain alternative embodiments, one of incident light beams 230A or 232A is coincident with optical axis 252 of lens 243, and the other is spaced apart from optical axis 252.

[0104] Incident light beams 230A and 232A are directed by lens 243 to converge toward focal zone 256. In such embodiments in which the incident light beams are not parallel to the optical axis, the focal zone may have a different location than that shown in FIG. 5 . Incident light beams 230A and 232A are reflected by object 268 to form reflected light beams 230B and 232B. Reflected light beams 230B and 232B return to and propagate through lens 243 and parallel to optical axis 252. Reflected light beams 230B and 232B exit lens 243 parallel to each other and separated by path spacing PS2. When optical assembly 202 is focused, path spacings PS1 and PS2 are equal.

[0105] Thus, when optical assembly 202 is focused, the projective relationship of reflected light beams 230B and 232B exiting lens 243 comprises two parallel light beams. Optical train 240 is configured to maintain the parallel projective relationship. For example, when optical assembly 202 is focused, reflected light beams 230B and 232B are parallel to each other when they exit dual beam generator 241, when they exit beam combiner 244, and when they are reflected by fold mirror 245. While the projective relationship is maintained, path spacing P2 can be rescaled by the beam combiner.

[0106] As shown in FIG. 6, reflected light beams 230B and 232B of FIG. 5 are incident on detector surface 264 and form beam spots 270 and 272. When optical assembly 202 is focused, beam spots 270 and 272 have a separation distance SD2. Separation distance SD2 may be based on (or may be a function of) the dimensions of beam combiner 244, the angle of incidence with respect to the parallel surfaces of beam combiner 244, and the incident reflected light beams 230B and 232B. Separation distance SD2 also depends on the projective relationship of reflected light beams 230B and 232B exiting lens 243. As shown in FIG. 6, detector surface 264 has a center point or central region 266. When all of the optical components 241-245 (FIG. 3) of the optical train 240 are in their 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. Also, as shown, the beam spots 270 and 272 may have a selected shape that correlates with the focusing of the optical assembly 202. For example, the beam spots 270 and 272 may have an imaginary radius that correlates with the focusing of the optical assembly 202.

[0107] 7-8 show the projective relationship between reflected light beams 230B and 232B when optical assembly 202 (FIG. 3) is below the focal point. As explained above, incident light beams 230A and 232A propagate parallel to one another through lens 243 and are separated by path spacing PS1. Incident light beams 230A and 232A intersect at focal zone 256 and are then reflected by object 268 to form reflected light beams 230B and 232B. However, as shown in FIG. 7, when reflected light beams 230B and 232B exit lens 243, they converge slightly toward optical axis 252 and toward one another. Also shown, path spacing PS2 is greater than path spacing PS1.

[0108] Thus, when object 268 is positioned below focal zone 256, the projective relationship of reflected light beams 230B and 232B comprises two light beams converging toward each other. As above, optical train 240 is configured to maintain the converging projective relationship. For example, reflected light beams 230B and 232B converge toward each other when they exit dual beam generator 241, when they exit beam combiner 244, and when they are reflected by fold mirror 245.

[0109] As shown in FIG. 8, when object 268 is positioned below focal zone 256, beam spots 270 and 272 have a separation distance SD3 that is smaller than separation distance SD2 (FIG. 6). Separation distance SD3 is smaller because reflected light beams 130B and 132B converge toward each other across the entire optical track between lens 243 and focus detector 250. Also, as shown in FIG. 8, beam spots 270 and 272 may have a selected shape that correlates with beam spots 270 and 272. The shape of beam spots 270 and 272 when object 268 is positioned below focal zone 256 is different from the shape of beam spots 270 and 272 when object 268 is in focus. Beam spots 270 and 272 may have different imaginary radii that correlate with the object being below focal zone 256.

[0110] 9-10 show the projective relationship between reflected light beams 230B and 232B when optical assembly 202 (FIG. 3) is above the focal point. As explained above, incident light beams 230A and 232A propagate parallel to one another through lens 243 and are separated by path spacing PS1. Before incident light beams 230A and 232A reach focal zone 256, incident light beams 230A and 232A are reflected by object 268 to form reflected light beams 230B and 232B. However, as shown in FIG. 9, as reflected light beams 230B and 232B exit lens 243, reflected light beams 230B and 232B diverge away from optical axis 252 and away from each other. Also shown, path spacing PS2 is less than path spacing PS1.

[0111] Thus, when object 268 is positioned above focal zone 256, the projective relationship of reflected light beams 230B and 232B comprises two light beams that diverge away from each other. Optical train 240 is configured to maintain the divergent projective relationship. For example, reflected light beams 230B and 232B diverge away from each other when they exit dual beam generator 241, when they exit beam combiner 244, and when they are reflected by fold mirror 245.

[0112] As shown in FIG. 10 , when object 268 is positioned above focal zone 256, beam spots 270 and 272 have a separation distance SD4 that is greater than separation distance SD2. Separation distance SD4 is greater because reflected light beams 130B and 132B diverge from each other throughout the optical track between lens 243 and focus detector 250. Also, as shown in FIG. 10 , beam spots 270 and 272 may have selected shapes that correlate with beam spots 270 and 272. The shape of beam spots 270 and 272 when object 268 is positioned above focal zone 256 is different from the shape of beam spots 270 and 272 when object 268 is positioned below focal zone 256. Similarly, beam spots 270 and 272 may have different imaginary radii that correlate with the object being below focal zone 256.

[0113] As explained above, when object 268 is below focal zone 256, separation distance SD3 is less than separation distance SD2 when object 268 is within focal zone 256. When object 268 is above focal zone 256, separation distance SD4 is greater than separation distance SD2. Therefore, optical assembly 202 can not only determine that object 268 is not located within focal zone 256, but also determine a direction in which to move object 268 relative to lens 243. Furthermore, the value of separation distance SD3 can be used to determine how far object 268 should be moved relative to lens 243.

[0114] As illustrated by the examples of Figures 6-10, in addition to determining that an object is in focus, measurements of the separation distance on the detector can be used to determine the working distance between the lens and the object being detected through the lens. Additionally, the separation distance on the detector can be used to determine the profile of the object surface.

[0115] As the foregoing examples illustrate, the relative separation (e.g., separation distance) may be a function of the projection relationship (i.e., at what speed reflected light beams 230B and 232B are diverging or converging) and the length of the optical track measured from lens 243 to focus detector 250. As the length of the optical track between lens 243 and focus detector 250 increases, the separation distance may decrease or increase if the object is out of focus. Thus, the length of the optical track may be configured to facilitate distinguishing between separation distances SD3 and SD4. For example, the optical track may be configured so that converging reflected light beams do not cross each other and / or so that diverging light beams do not exceed a predetermined relative separation between each other. To this end, the optical track between the optical components of optical train 240 may be lengthened or shortened as desired.

[0116] Also, as the foregoing examples illustrate, the working distance between the lens and the object being imaged (e.g., WD2 in FIG. 5 , WD3 in FIG. 7 , and WD4 in FIG. 9 ) can be determined based on measurements of the separation distance on the detector. This can then be used to determine the profile of the object surface. For example, the optical system can record the relative separation for a series of data points and associate each data point with a position along the surface. As the working distance decreases (i.e., as the height of the object surface increases), the relative separation can increase, as shown in FIG. 10 . As the working distance increases (i.e., as the height of the object surface decreases), the relative separation can decrease, as shown in FIG. 8 . Thus, the surface profile or topography of the object can be determined based on the relative separation of the reflected beams.

[0117] As discussed above, a sample can have many variations in its topography along the imaging direction (e.g., the scan direction), which cannot be accounted for by performing a single global tilt of the sample before imaging. For example, FIG. 11 shows a series of plots illustrating the surface profile of a flow cell sample scanned using a line-scan system. As depicted, the surface profile of the flow cell can vary significantly (e.g., from about -10 μm to 10 μm) across the scan direction.

[0118] Also, as discussed above, to account for local variations in the sample's topography, the optical imaging system may include a controller configured to dynamically move the sample holder laterally (along the X and / or Y axes extending into the plane of the paper), vertically / elevating along the Z axis, and / or angularly along the X (tip), Y (tilt), and / or Z (torsion) axes during imaging. To this end, it is useful to consider coordinate systems that may be used when designing an assembly that dynamically moves the sample laterally and / or angularly. FIG. 12 depicts one exemplary such coordinate system. In this example, the z-axis coincides with the optical axis of the objective lens and may generally point upward toward the sky. The y-axis is the axis along which the sample may actively move during imaging (e.g., during sequencing using a line scanner). For example, the sample may be a flow cell with a flow cell lane aligned with the Y-axis. Any movement of the sample holder (and thus the sample) in an angular direction can be expressed as a twist of the sample holder about the z-axis (e.g., a change in θz), a tilt of the sample holder about the y-axis (e.g., a change in θy), or a tilt / tilt of the sample holder about the x-axis (e.g., a change in θx).

[0119] 13-15 depict exemplary assemblies that may enable dynamic adjustment of the lateral and angular position of a sample, according to some embodiments of the present disclosure. FIG. 13 shows a sample stage assembly 300 configured to mount a movable platform (FIG. 14) that houses the sample to be imaged. As illustrated, the sample stage assembly 300 includes an XY cradle 301, an XY stage 302, a controller 303, and a tip tilt assembly (TTA) that includes multiple actuators 304. FIG. 14 depicts an exemplary movable platform 400 mounted on the actuators 304 of the TTA. FIG. 15 depicts the movable platform 400 mounted on the sample stage assembly 300. The movable platform 400 includes a sample container 405, which in this example is a flow cell.

[0120] In this example, the XY stage 302 is configured to move the sample holder laterally along the X and Y axes. The TTA is configured to control the angular alignment of the sample holder to position the sample surface within the focal range of the imaging system's optics. The TTA can affect all three axes of rotation, as depicted with respect to FIG. 11 . In some embodiments, the TTA can actively control rotation about all three axes during imaging. In other embodiments, the TTA can actively control rotation about only some axes during imaging. For example, only rotation about the X and Y axes can be actively controlled during the imaging process, and rotation about the Z axis can be manually adjusted as needed.

[0121] The controller 303 may be configured to apply parameters to one or more drive signals 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, 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 smaller voltage, and a smaller duty cycle) will be specified. The control output may be adjusted, for example, by adjusting the current or voltage applied to one or more actuators. Furthermore, in some examples, the time at which the drive signals are applied to one or more actuators may be adjusted based on the amount of translation required for the change in focus. For example, if 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 setting. The parameters of the drive signal and the time the drive signal is applied can be determined based on the actuator type (e.g., piezoelectric vs. voice coil) and drive requirements. Thus, drive signals can be provided to one or more actuators at different power levels to linearly move, tilt, tilt, or otherwise position the sample during imaging.

[0122] In this example, the TTA achieves θX and θY alignment through active manipulation of three linear actuators 304, whereby the sample holder rests on a movable platform 400 that is kinematically mounted to the three linear actuators 304. The actuators 304 may be spaced far enough apart so that relatively large displacements of these actuators can result in small changes in platform tilt. In some embodiments, the three-point kinematic mounting may utilize a "3V coupling," also referred to as a "Maxwell coupling." In other embodiments, a 3-2-1 coupling may be utilized. While this example illustrates angular alignment control through the use of a three-point kinematic mounting, it should be understood that other types or numbers of actuators, or other configurations of actuators, may be utilized to enable angular control to position the sample within the focus.

[0123] 16A-16D show an exploded view, a perspective view, a side view, and another perspective view, respectively, of a specific actuator 304 that may be used in some embodiments of the present disclosure. As depicted, the actuator 304 may include a tilt motor 304-1, an encoder 304-2, a tilt board 304-3, a lead screw 304-4, a proximity sensor reflector 304-5, an encoder scale 304-6, a linear bearing 304-7, a carriage 304-8, a lead screw nut 304-9, and a ball 304-10. In this embodiment, the actuator 304 functions as a linear stage with the carriage 304-8 driven up and down by the 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 carriage 304-8 up and down, while the encoder 304-2 monitors its relative position. The encoder 304-2 may be optically reflective. The scale 304-6 is on the moveable carriage, while the encoder sensor is on the tilt board 304-3. A separate proximity sensor can also be found on the tilt board 304-3. This proximity sensor can help the software verify absolute position and orientation.

[0124] As depicted by Figures 16B-16C, which depict the bottom hard stop position of actuator 304, tracking of actuator 304 can be achieved by driving carriage 304-8 fully down until it contacts the motor support structure at 304-11. Alternatively, a proximity sensor can be left alone to determine global positioning. Actuator 304 can be zeroed at the bottom of carriage travel during instrument initialization, and all actuator commands can be relative to this fully down position.

[0125] 16D, the actuator 304 may incorporate an upper hard stop to prevent the carriage 304-8 from disengaging from the end of the lead screw 304-4. This hard stop may be achieved using 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 over which the carriage 304-8 normally travels during instrument use.

[0126] 17 depicts the underside of the movable platform 400 according to some embodiments of the present disclosure. The underside includes three V-grooves 415 configured to couple with the balls 304-10 of the actuator 304.

[0127] 18 depicts another perspective view of moveable platform 400. To ensure sufficient stability of the system, including under external loads such as wire and tubing disturbances that may upset the kinematic balance of gravity, moveable platform 400 may include tension springs 425 to further increase the contact force at each coupling interface between actuators 304 and moveable platform 400 and make accidental detachment less likely.

[0128] 19 depicts movable platform 400 interfacing with and coupling to actuator 304. As depicted, threaded hole 425 interfaces with hold-down spring fastener and ball 304-10 interfaces with V-groove 415.

[0129] There are different strategies that can potentially be employed to enable dynamic tilting of the sample holder to keep the sample in focus during image scanning. In some embodiments, a feedback tilt elimination mechanism can be employed, whereby tilt is measured in real time during image scanning, and the tilt measurements are fed directly to 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 separation of the projected spot pairs of the focus tracking module can be mapped to a sample height position. The projected spot pairs can be generated using a light source having a wavelength of approximately 620 nm to 700 nm. By projecting two different spot pairs along two different scan positions (i.e., two different Y positions), the sample height at two different positions in the scan direction can be measured and mapped to the change in sample tilt between the two scan positions. For example, as depicted by FIG. 20 showing the scanning of a sample 500 using a line scan 510, the spot beam separation Δx1 can be mapped to a first sample height h1, and the spot beam separation Δx2 can be mapped to a second sample height h2. Separation L between two spot beam pairs y Based on the respective heights h1 and h2, the tilt angle of the sample between the two scan points can be estimated (e.g., by calculating the slope between the two points). The determined tilt angle can be used by the system controller to cause one or more actuators to tilt the sample holder. In some implementations of this embodiment, to ensure that the tilt is measured prior to imaging the sample section, the system can employ a look-ahead focus tracking beam that projects at least one spot pair prior to the line scan reaching the sample section.

[0130] 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 latency in transmitting the real-time measurement to the tilt controller. If the local tilt changes more quickly than the combined latency of the maximum tilt speed and the latency in transmitting the real-time measurement to the tilt controller, any real-time feedback mechanism may experience latency that makes such a method unsuitable.

[0131] Therefore, in some embodiments, to address this latency issue, it may be preferable to employ a technique for generating a "tilt trajectory" prior to tilting the sample during image scanning. An example of this is illustrated by FIGS. 21A-21C. In particular, FIG. 21A is an operational flow diagram illustrating 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 (e.g., sample stage assembly 300) that carries the sample.

[0132] Operation 610 includes determining a tilt map for the sample, the tilt map including multiple entries corresponding to multiple sample positions, each entry indicating the amount to tilt the sample along at least the scan direction for the corresponding sample position. The tilt map may be in the form of a table, a one-dimensional array, a two-dimensional (2D) array, or another suitable data structure. For example, as depicted by FIG. 21B, the tilt map may specify five tilt angles θy1 for five different sample positions. In practice, tilt maps may be generated for more than five positions and may be constructed by estimating a smooth fit between multiple sample tilt measurements. FIG. 21C conceptually illustrates a smooth fit of tilt trajectories in the z direction based on different tilt angles. An initial tilt map is generated prior to scanning the sample. As discussed further below, the tilt map may be generated by performing a “pre-scan” along several positions in the scan direction (e.g., several Y positions along the Y direction) to determine the sample height at various positions. For samples scanned in multiple strips (eg, a flow cell scanned in a swath), the sample height can be determined at multiple Y positions for each strip.

[0133] 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 having one or more actuators tilt the sample holder at least along the scan direction according to the tilt map. Images of the sample may be collected by moving the sample holder at a constant velocity using a motorized stage (e.g., XY stage 302) in a direction perpendicular to the long dimension of the image sensor array (e.g., TDI sensor). In embodiments in which the sample is imaged in strips (e.g., flow cells), after each strip is imaged, the motorized stage may move the sample in the X direction a distance corresponding to the strip width. In such embodiments, a tilt map may be generated and used for each sample strip (e.g., operations 610-640 may be applied to each sample strip).

[0134] Operation 630 includes updating the gradient map for the next imaging cycle. Over time, the sample's topography may change due to thermal expansion (e.g., due to excitation or other light sources increasing the sample's temperature) and / or other changes in the sample. Therefore, to account for potential changes in sample topography, the gradient map may be updated every imaging cycle. In an alternative embodiment, the gradient map may be updated after a predetermined number of imaging cycles. To update the gradient map prior to the next imaging cycle, gradient map measurements for the next imaging cycle may be made during the current imaging cycle. The optical imaging system may generate the updated gradient map using the same mechanism as that used to generate the original gradient map.

[0135] Operation 640 includes scanning the sample on the sample holder during the next imaging cycle and adjusting the tilt of the sample at each of the sample positions during the next imaging cycle by tilting the sample holder with one or more actuators along at least the scan direction according to the updated tilt map. Operations 630-640 may be repeated until all imaging cycles (e.g., sequencing cycles) are completed. In the case of a sequencer, each imaging cycle described above may correspond to a sequencing cycle.

[0136] To illustrate one particular embodiment of a system for generating a "tilt trajectory" prior to tilting the sample during an image scan, it is instructive to consider an exemplary system that utilizes the controller of assembly 300 to perform dynamic correction during the duration of each scan of the 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 tilt removal servo update rate of approximately 10 Hz, a tile θY acquisition rate of approximately 100 Hz, 99 tiles per scan swath, two surfaces (top and bottom) per flow cell, two flow cells per instrument, eight lanes per flow cell surface, and four scans per flow cell lane. In this embodiment, the system is capable of performing 128 scans per sequencing cycle.

[0137] In the preceding example, it is assumed that at least 10 measurements per scan swath are required to characterize the required tilt correction (approximately 1 correction per 10 tiles). Therefore, a set of correction tables with 10 entries per table and one table per scan can be created for the entire sequencing cycle, requiring a total of 128 10-entry tables per imaging cycle. Each correction entry can be based on a centroid calculation performed (e.g., using a dual projection beam, as described above) at each of the 10 measurement points along each scan. These tables can be stored in the TTA controller. In this example, a smooth tilt removal trajectory can be determined to minimize energy input to the instrument structure and maximize the quality of the tilt removal correction. The tilt removal trajectory can be created using smooth curve approximation rather than piecewise linear correction between subsequent entries in the table. For example, if the curve approximation interpolates 9 points between each entry (5 points at the beginning of the scan plus 5 points at the end of the scan), the total number of corrections per scan will be 100, which for a 1 Hz scan rate would require a slope / tilt update rate of 100 Hz. To generate a smooth slope-removed trajectory, a mathematical operation such as a cubic Hermite approximation can be applied to all collected data, which has the advantage of specifying both the position and slope at every target trajectory point.

[0138] In this example, a first set of compensation tables for the first sequencing cycle may be generated by scanning the flow cell before sequencing begins. Subsequent sequencing cycles may use compensation tables that are updated based on the centroid calculations performed during each scan of the previous cycle. One advantage of this exemplary approach is that it may avoid the requirement for a low-latency connection between the centroid calculations and the TTA controller. The controller may have enough time during a time window during the cycle to update the 128 tables for the next sequencing cycle, or the controller may update each table for the next sequencing cycle in the background as it completes each scan in the current sequencing cycle.

[0139] 22 is a block diagram illustrating an exemplary 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 or down) during a sample imaging cycle. The standard resolution tilt actuator(s) 721-2 can adjust the global tilt of the sample holder 720 during an imaging cycle. For example, if the actuators correspond to the actuators 304 of the assembly 300, the actuator 304 shown on the right side of FIG. 13 can be fixed during an image scan, while the actuator 304 shown on the left side of FIG. 13 can dynamically tilt the sample up or down during an image scan.

[0140] 23A-23C illustrate one exemplary implementation for generating a gradient map according to some embodiments of the present disclosure. In particular, FIG. 23A is an operational flow diagram illustrating an exemplary method for determining a gradient map along a scan direction. The method may be implemented, for example, to generate an initial gradient map before beginning imaging of the sample (e.g., operation 610) and generate gradient maps for subsequent imaging cycles during each imaging cycle (e.g., operation 630). In this example, the gradient map is determined using an optical assembly (e.g., optical assembly 202) that projects dual focused beams onto the surface of the sample.

[0141] Operation 810 includes scanning a sample in a scan direction along multiple sample locations of the sample by projecting one beam spot pair onto the surface of each sample location. Operation 820 includes estimating the height of the sample location for each sample location of the multiple sample locations based on the separation distance of at least one beam spot pair. For example, as depicted by FIG. 23B , a focus tracking module or other suitable module may project one spot beam pair onto the surface of the sample across different sample locations in the scan direction, and images may be captured using an image sensor of an optical imaging system. 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 image of the sample, or it may be a different image sensor. As discussed above, the separation distance of the imaged spot beams may be mapped to the distance between the objective lens and the surface of the sample. Thus, the spot beam separation may be mapped to the sample height for a given sample location.

[0142] Operation 830 includes calculating the tilt of the sample along the scan direction based on the heights of the multiple sample locations. For example, based on the estimated heights of two adjacent sample locations and the separation distance of the two sample locations, the tilt slope and angle can be determined between the adjacent locations.

[0143] Operation 840 includes generating a tilt map including multiple entries corresponding to multiple sample tilt positions based on the tilt of the sample along the scan direction, with each entry indicating the amount of tilt of the sample along the scan direction for the corresponding sample tilt position. In some embodiments, a tilt map entry corresponds to each sample position at which one beam spot pair is projected. For example, each entry may indicate the tilt angle, tilt gradient, or tilt height for each sample position at which one beam spot pair is projected. In some embodiments, to minimize energy input to the instrument structure and maximize the quality of the tilt removal correction, the tilt map may be created to provide a smooth tilt removal trajectory. In such embodiments, a smooth curve fit may be performed between entries corresponding to initial spot beam measurements. As an illustrative diagram, FIG. 23C illustrates a smooth tilt trajectory generated by interpolating between five initial entries. In this illustrative diagram, the black dots may correspond to the five initial entries generated by spot beam separation measurements. The gray dots may indicate additional entries added by interpolation. During sample imaging, each tilt map entry is then read by the TTA's controller to cause an actuator to tilt the sample holder.

[0144] While the preceding examples have been described primarily in the context of determining sample tilt along the scan direction (Y-direction), it may also be important to consider sample tilt in a direction orthogonal to the scan direction (X-direction). To this end, and as further illustrated by Figures 24A-24C, optical imaging systems may utilize focusing systems that generate at least two pairs of beam spots to account for tilt in both directions.

[0145] 24A is an operational flow diagram illustrating an exemplary method for determining a gradient map along a scan direction and a direction orthogonal to the scan direction. The method may be implemented, for example, to generate an initial gradient map before beginning imaging of the sample (e.g., operation 610) and generate gradient maps for subsequent imaging cycles during each imaging cycle (e.g., operation 630). In this example, the gradient map is determined using an optical assembly (e.g., optical assembly 202) that projects at least two focused beam pairs onto the surface of the sample.

[0146] Operation 910 includes scanning a sample in a scan direction along a plurality of sample locations on the sample by projecting two pairs of beam spots onto a surface of each sample location, and operation 920 includes estimating, for each sample location of the plurality of sample locations, based on at least a separation distance of each of the two pairs of beam spots, a height of the sample location, and a tilt of the sample location in a second direction substantially orthogonal to the scan direction.

[0147] As depicted in FIGS. 24B-24C , a focus tracking module or other suitable module can project two spot beam pairs onto the surface of the sample across different sample positions in the scan direction. In contrast to the previous embodiment, in which each spot beam pair was separated along the X-axis, in this embodiment, each spot beam pair is rotated 90 degrees and separated along the scan axis (Y-axis). For example, referring to FIG. 24C , the first spot beam pair is separated along the Y-axis by Δy1, and the second spot beam pair is separated along the Y-axis by Δy2. In this example, the sample height at the center of the field of view of the sample position can be estimated from the beam spot separation Δy1 and the beam spot separation Δy2 as follows: The first beam spot separation can be mapped to a first sample height, and the second beam spot separation can be mapped to a second sample height. An average between the first and second sample heights can be determined. Alternatively, one of the two sample heights can be selected as the sample height for that position. In addition to determining the sample height, the difference between the separation of the two beam spot pairs (Δy1, Δy2) and the distance between the two beam spot pairs (L x ) can be used to determine the slope along the X direction.

[0148] Operation 930 includes calculating the tilt of the sample along the scan direction and the tilt of the sample along a second direction based on the heights of the multiple sample positions. Operation 940 includes generating a tilt map based on the tilt of the sample along the scan direction and the tilt of the sample along the second direction. As described above, the tilt map may include multiple entries corresponding to multiple sample tilt positions. In this case, each entry may indicate, for the corresponding sample tilt position, the amount of sample tilt along both the scan direction and a direction substantially perpendicular to the scan direction. Such a tilt map may also be referred to as a "tilt-tilt map." During sample imaging, each tilt-tilt map entry may subsequently be read by the TTA's controller to cause one or more actuators to tilt and tilt the sample holder along both the X-axis and the Y-axis as needed.

[0149] Although the above examples are described in the context of generating gradient maps in the scan direction or in both the scan direction and a direction orthogonal to the scan direction, it should be understood that the techniques described above may be applied to generate gradient maps only in a direction orthogonal to the scan direction.

[0150] While the foregoing examples for enabling dynamic tilt of the sample have been described primarily in the context of imaging systems utilizing tilt maps that may be updated after each imaging cycle or some multiple of imaging cycles, it should be understood that the foregoing examples may also be used in embodiments utilizing feedback tilt elimination mechanisms that do not rely on tilt maps generated prior to scanning an area. For example, the method of FIG. 23A or FIG. 24A may be adapted such that tilt measurements (e.g., as determined for each sample position) are provided directly to a tilt controller that adjusts one or more tilt actuators in response to receiving the measurements. In such embodiments, the frequency at which the sample holder is tilted may be the same as, greater than, or less than the scan frequency.

[0151] In the foregoing examples, depending on system requirements, the relative positioning between the projected beam spots (e.g., two beam spot pairs) used to capture tilt measurements and the projected excitation light (e.g., scan line) used to capture sample images can vary. For example, while FIG. 24C illustrates two beam spot pairs being projected in front of the scan line in the scan direction, in some embodiments, one of the two beam spot pairs can be projected in front of the scan line and the other of the beam spot pair can be projected behind the scan line. In other embodiments, both pairs of beam spots can be projected behind the scan line. In still other embodiments, one of the beam spot pairs can be projected on the scan line.

[0152] Similarly, in a multi-channel line-scan imaging system that utilizes multiple excitation light sources to image a sample, the relative orientation between the multiple scan lines and the projected beam spot pairs can vary. For example, FIGS. 25A-25F illustrate the relative orientation of two projected beam spot pairs and two scan lines (e.g., laser lines) in a two-channel line-scan imaging system in different configurations. In FIG. 25A, both beam spot pairs are projected in front of both scan lines. In FIG. 25B, one beam spot pair is projected in front of both scan lines and one beam spot pair is projected behind both scan lines. In FIG. 25C, both beam spot pairs are projected behind both scan lines. In FIG. 25D, both beam spot pairs are projected in front of one scan line and behind the other scan line. In FIGS. 25E-25F, one beam spot pair is projected in front of one scan line and the other beam spot pair is projected behind the same scan line.

[0153] FIG. 26 conceptually illustrates the benefits of using multiple tilt actuators, according to some embodiments of the present disclosure. As depicted by the top schematic, when a single tilt actuator is used, the tilt / rotation axis may be off-center from the scanned sample surface. Dynamically changing the tilt according to the level arm L may cause latency-induced errors due to z-displacement, and the x-position of sample features may shift as the sample holder tilts up or down. To overcome this problem, in some embodiments, multiple tilt actuators may be used to change the position of the tilt axis to be closer to or centered on the sample area being scanned. For example, as depicted by the bottom schematic, the rotation axis x can be changed by simultaneously moving z1 and z2, where:

[0154]

number

[0155] While some of the foregoing examples have been described in the context of using spot beam separation measurements to enable dynamic tilting of the sample holder to keep the sample in focus during an image scan, it should be understood that these measurements can also be utilized to move the Z-stage to provide movement of the objective lens relative to the sample container to keep the sample in focus. For example, one or more actuators can be configured to move the objective lens and / or the sample container in the z-direction while maintaining the sample within a focal region of the focal plane of the imaging system. To illustrate, FIG. 27 is a block diagram illustrating some components of an exemplary focus control system for focus tracking, according to some embodiments of the present disclosure. The exemplary focus control system includes a focus tracking circuit 2732 configured to determine a focus setting that is used to generate a drive signal that drives a focus tracking feedback loop in a Z-stage 2734. In the example of FIG. 27 , a command 2752 based on a focus setting difference is provided to the Z-stage 2734. In this example, the Z-stage 2734 is configured to move the objective lens 2746. In other embodiments, the stage holding the sample holder can be moved. The actuator 2744 moves the optical stage, 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 regarding the position of the actuator and its movement. This encoder information 2754 may be fed back to the focus tracking circuit 2732 through the z-stage controller 2736 and may be used in determining an error signal.

[0156] FIG. 28 illustrates an example architecture of a Z-stage controller, according to some embodiments of the present disclosure. This example incorporates feedback and feedforward control. During operation, a target focus setting (e.g., target z position 2870 determined using spot separation measurements) is used to command the position of the Z stage. The target z position 2870 is provided to a controller 2888, which determines the drive signal needed to command an actuator 2890 to position the Z stage. The controller 2888 may also include drive circuitry for generating the drive signal. The drive signal determination can be made using, for example, the magnitude of the difference between the target focus setting (e.g., target z position 2870 as determined by the most recent spot separation measurement) and the current focus setting (current z position 2872 as determined by a past spot separation measurement), which may be provided 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 2892. The correction information can be determined, for example, 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 can be stored electronically in a history file in memory so that they can be recalled for later use during a scanning operation. The history file or the information therein can be tagged to identify it as containing focus information for a particular sample container. The correction information can be added to the commanded stage position to adjust the drive signal according to the gradient of the change in focus setting for the scanning operation.

[0157] More generally, the techniques described herein can be implemented by creating a relative tilt between the sample and the image sensor that images the sample by adjusting any component of the optical imaging system along the imaging light path from the sample to the sample image sensor based on the sample tilt measurement. Thus, based on the sample tilt measurement, the system can be configured to alternatively tilt the image sensor that images the sample, the camera carrying the sample image sensor, and / or the sample holder. Other optical components along the sample imaging / light path from the sample to the sample image sensor can 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 can include, for example, an objective lens or one or more mirrors that receive light corresponding to an image of the sample.

[0158] Adjustments to imaging system components other than the sample holder to account for sample tilt (e.g., adjustments to one or more mirrors and / or the image sensor) can be made based on real-time tilt measurements communicated to one or more controllers in real time, as described above with reference to FIGS. 21A-25F, or based on a sample tilt trajectory calculated prior to tilt offset during an image scan. For example, the embodiments of FIGS. 21A, 23A, and 24A can be implemented by creating a tilt map that includes entries corresponding to multiple sample positions, each entry indicating the amount to tilt the image sensor or otherwise adjust some optical component of the imaging system to tilt the sample. Thus, sample tilt offset can be implemented by tilting or otherwise adjusting one or more components of the imaging system along the imaging path from the sample to the sample image sensor based on one or more sample tilt measurements. The tilt or adjustment of one or more optical components can be controlled using one or more system controllers.

[0159] In embodiments where the sample vessel is a flow cell, tilt adjustments can be made after every swath, multiple times per swath, after every tile, or after some other subsection of the sample vessel. Such adjustments can account for sample tilt along one axis (e.g., tilt along the scan direction or a direction substantially perpendicular to the scan direction) or sample tilt along multiple axes (e.g., tilt along both the scan direction and a direction substantially perpendicular to the scan direction). Such embodiments can be implemented with or without a tiltable sample holder.

[0160] As described above, the optical sequencing systems described herein can map the spot beam separation of one or more focus tracking modules to sample height position(s). This mapping can be performed when operating the sequencing system in focus model generation mode, sequencing mode, or both. During focus tracking model generation, the system can determine and store information regarding the focus setting of a given sample container to control the focusing of the system during real-time imaging. For example, using one or more focus tracking modules, a sample container can be scanned at each of multiple sample positions on the sample container, and a focus setting can be measured and determined for each of the multiple sample positions.

[0161] 29 and 30A-30B illustrate a specific example of the design and operation of a focus tracking system utilizing two focus tracking spot pairs per sample surface, according to some embodiments of the present disclosure. The focus tracking system utilizes a light beam architecture that projects two spot pairs onto each of a first and second surface (e.g., top and bottom) of a sample container (e.g., a flow cell lane). In alternative designs, the two spot pairs can be projected onto a single sample surface or more than two sample surfaces.

[0162] As depicted by Figure 29, the two reflected light beams corresponding to each spot pair are incident on the surface of respective sensors 2911, 2912 to form beam spots (e.g., spot pair 2901, 2902 corresponding to light reflected from the top surface of the sample, and spot pair 2903, 2904 corresponding to light reflected from the bottom surface of the sample). For simplicity, this discussion focuses on spot pairs 2901 and 2902 projected onto one surface. Spot pairs 2901, 2902 are detected using respective sensors 2911, 2912 with respective spot separations Δx 1、 Δx2. In this example, L is a "lever arm" and ROI refers to a region of interest that can be dynamically determined and set around the peak maximum. For example, the ROI can be set around the peak maximum with a width twice the full width at half maximum (FWHM). The two sensors can function as a parallel linear sensor array for spot detection. In some implementations, each focus tracking module can be associated with a respective light source that projects two spots and a sensor that detects the two spots. For example, a four-beam system as described herein can be associated with two separate focus tracking modules.

[0163] 30A illustrates pixel intensity as a function of pixel number for sensor 2911 after incidence of light corresponding to spot pair 2901 and 2903. As depicted, the spot separation Δ x_1 can be calculated by determining the pixel corresponding to the peak intensity of each detected spot and determining the difference. Figure 30B illustrates pixel intensity as a function of pixel number for sensor 2912 after incidence of light corresponding to spot pair 2902 and 2904. As depicted, the spot separation Δx2 on sensor 2912 of the left and right spots corresponding to spot pair 2902 can be calculated by determining the pixel corresponding to the peak intensity of each detected spot and determining the difference.

[0164] In some implementations, the following parameters may be defined for a four-beam focus tracking system as described above: The average spot separation Δx of two spot pairs in the sensor plane may be defined by equation (1):

[0165]

number

[0166]

number

[0167]

number

[0168]

number

[0169] FIG. 31 depicts both the object plane and the sensor plane during scanning of the flow cell surface in one exemplary embodiment utilizing the focus tracking system described with reference to FIG. 29. In this case, Δy1 and Δy2 refer to spot separations on the surface of the sample, and L refers to the distance between each pair of spots on the flow cell surface. The focus tracking module spots are shown relative to the two scanning laser lines. For illustrative purposes, sensors 2911 and 2912 with incident light beams are superimposed on the drawing. In one specific embodiment, Δy1 and Δy2 are approximately 60 μm at best focus, and L is approximately 900 μm.

[0170] While the imaging systems described herein can utilize focus-tracking spot separation estimation and keep the sample in focus (e.g., by moving the z-stage in the z-direction and / or tilting the stage holding the sample holder), in some cases, it may not be possible to make an accurate estimate of spot separation due to “bad spots” caused by issues with the sample or sample container (e.g., bubbles, defects, debris on the surface), issues with the sensor (e.g., glitches in sensor readings), or some other issue during imaging that affects the readings of one or both spots on the focus-tracking module's sensor. Such issues can cause the detected spots to be saturated or non-Gaussian in nature, leading to inaccurate estimates of spot separation. These inaccurate estimates of spot separation can result in large Z-step spikes during focus tracking, which can interact with the Z-stage control loop sensitivity and introduce large errors in movement along the Z direction. This issue is illustrated by FIG. 32, which is a plot showing Z-steps (in nm) as a function of time for a Z-stage used with a focus-tracking system that does not implement “spot error” processing. These inaccurate estimates of the spot separation can also introduce errors into the stage movements that tilt the sample holder.

[0171] To address potential issues related to spot error, some embodiments of the present disclosure are directed to correcting spot error, including potentially inaccurate measurements of spot separation, based on at least previous focus-tracking measurements of spot separation. According to some embodiments described further below, statistics of previous spot separation measurements, statistics of previous specimen tilt measurements, and / or error codes tracking the current state of the measured focus-tracking spots can be used to determine which Z-stage and / or tilt adjustments to apply for a given frame, including which spot separation measurements to use for a given frame. By implementing the techniques described herein, significant improvements in focus-tracking performance can be realized. Additionally, the techniques described herein can be implemented to address spot error and / or spot separation glitch spikes in real time (e.g., at an update rate of approximately 3.3 kHz).

[0172] Although the spot error processing techniques described herein are primarily described in the context of an imaging system that utilizes two substantially parallel focus tracking spot pairs detected using two substantially parallel sensors, it should be understood that the techniques described herein may be applied with focus tracking systems that utilize a single focus tracking spot pair detected using one or more sensors, focus tracking systems that use two substantially parallel focus tracking spot pairs detected using one or more sensors, or focus tracking systems that use more than two substantially parallel focus tracking spot pairs detected using one or more sensors.

[0173] Before describing specific implementations of the spot error handling techniques described herein, it is helpful to consider examples of different problems with the specimen / specimen holder and / or sensor that can cause spot errors, focus tracking performance when spot errors are not considered, and improvement in focus tracking performance when spot errors are considered, according to some implementations of the present disclosure. To that end, they are illustrated in Figures 33A-37C.

[0174] Figure 33A depicts an image of a portion of a flow cell 3300 with a large air bubble 3301. Superimposed on the image are sensors 3321 and 3322. Sensor 3321 is configured to detect the reflection of a first (right) pair of focus-tracking beams, and sensor 3322 is configured to detect the reflection of a second (left) pair of focus-tracking beams. Defect 3301 causes a higher reflection of the right pair of focus-tracking beams. This higher reflection can saturate the signal readout of the two reflected light beams incident on sensor 3321, resulting in a noisy sensor signal without a clear peak intensity spike (e.g., corresponding to the center of one or both spots as illustrated in Figures 30A-30B) that allows for spot separation measurements.

[0175] 33B-33C depict plots showing focus tracking performance, including a summary of defocus error, when focus tracking was performed on flow cell 3300 with spot error processing turned off (FIG. 33B) and with spot error processing turned on (FIG. 33C). In this plot, the x-axis refers to the current focus tracking frame. The y-axis of the bottom plot measures the distance in sensor pixels from the best z focus position for each sensor ("Sensor 1" and "Sensor 2"). As depicted, a defect occurs between focus tracking frames 38690 and 38700 for sensor 3321 (shown as "Sensor 1" in the plot). By accounting for the spot error generated by the defect, aggressive Z stage movements can be avoided when the focus tracking spot for Sensor 1 is over defect 3301. As measured, focus tracking performance improved by approximately 20 percent on average for this flow cell 3300 with defect 3301. Performance in the worst swath improved by approximately 50%.

[0176] Figure 34A depicts an image of a portion of flow cell 3400 having a defect 3401 that causes saturation of the focus tracking spot on sensor 1 over approximately eight frames. Figure 34B depicts a plot showing focus tracking performance, including a summary of defocus error, when focus tracking is performed on flow cell 3400 with spot error processing turned off, and a simulation of performing focus tracking on flow cell 3400 with spot error processing turned on. In this example, the plot with spot error processing turned off is overlaid with the simulation with spot error processing turned on. As depicted with spot error processing on, the curve is smoother and there is no Z step spike commanded to the Z stage when the flow cell defect is encountered.

[0177] Figure 35A depicts an image of a portion of flow cell 3500 with a large air bubble 3501 causing poor measurement of the focus tracking spot on sensor 2 over approximately 50 frames. Figure 35B depicts a plot showing focus tracking performance, including a summary of defocus error, when focus tracking is performed on flow cell 3500 with spot error processing turned off, and a simulation of performing focus tracking on flow cell 3500 with spot error processing turned on. In this example, the plot with spot error processing turned off is overlaid with the simulation with spot error processing turned on. As depicted with spot error processing on, the curve is smoother and there is no Z step spike commanded to the Z stage when the air bubble in the flow cell is encountered.

[0178] Figure 36A depicts an image of a portion of a flow cell 3600 with debris 3601 (appears as a dark dot) on its surface causing an insufficient measurement of the focus tracking spot. For example, the debris is preventing some light from reaching the portion of the flow cell it obscures. Figures 36B-36C depict plots showing focus tracking performance, including a summary of the defocus error, when focus tracking is performed on the flow cell 3600 with spot error processing turned off (Figure 36B) and with spot error processing turned on (Figure 36C). As depicted with spot error processing on, there is no Z step spike commanded to the Z stage when debris is encountered.

[0179] Figure 37 depicts an image of a portion of a flow cell 3700 with residue 3701 (appears as a dark pattern) on its surface that causes insufficient measurement of the focus tracking spot. For example, the residue 3701 causes unpredictable reflections of the incident focus tracking light that distort the measured spot. Figures 37B-37C depict plots showing focus tracking performance, including a summary of the defocus error, when focus tracking is performed on the flow cell 3700 with spot error processing turned off (Figure 37B) and with spot error processing turned on (Figure 37C). As depicted with spot error processing on, there is no Z-step spike commanded to the Z stage when the residue is encountered.

[0180] In some cases, glitches in the system electronics (e.g., focus tracking sensors) and / or during data collection can lead to missing and / or inaccurate measurements. Figure 38A depicts a plot showing focus tracking performed on a flow cell using a four-beam, two-sensor focus tracking system without spot error processing. In this example, a spot separation glitch on one sensor occurred and was not considered. The fourth plot shows that error codes were not used to track the current state of the measured focus tracking spot. Figure 38B depicts a plot showing focus tracking performed on the flow cell of Figure 38A using a four-beam, two-sensor focus tracking system with spot error processing. In contrast to the example shown by Figure 38A, smoother Z movement of the Z stage is commanded despite an unusual spot separation glitch on one or both sensors. The fourth plot shows that error codes were used to track the current state of the measured focus tracking spot.

[0181] FIG. 39 depicts a flow diagram illustrating an example method 3900 for implementing spot error processing, according to some embodiments of the present disclosure.

[0182] Operation 3901 includes capturing an image of a spot pair at a sample location. For example, using one or more light sources of an imaging system, a first spot pair can be projected onto the sample location in the sample container, and an image sensor of the imaging system can capture an image of the first spot pair. A focus tracking system, such as described above, can be used to capture the image.

[0183] At decision 3902, a determination is made as to whether the image of the spot pair is valid. In some embodiments, this determination can be made based on the intensity distribution of pixels in the first image. For example, as described above with reference to FIGS. 30A-30B, when detecting spot pairs on an image sensor, a valid image is expected to result in two intensity peaks from a sample with one surface, or four intensity peaks from a sample with two surfaces, each with a substantially Gaussian distribution. If this is not the case (e.g., there are no intensity peaks, one intensity peak, three intensity peaks, and / or a non-Gaussian distribution of intensities around the peaks, and / or asymmetric pixel locations of the peaks), this can indicate that a spot error has occurred. Thus, in some embodiments, one or more of the following parameters can be examined by the system to determine whether the image is valid: the intensity distribution of the first spot, the intensity distribution of the second spot, the intensity distribution of the third spot, the intensity distribution of the fourth spot, the pixel location of the peak intensity, the number of intensity peaks, whether any saturated pixels are present, etc.

[0184] In some embodiments, if an image is determined to be invalid, an identifier such as an error code can be stored in system memory, indicating why the image is invalid. In some embodiments, the identifier can be a number or other alphanumeric indicator associated with the type of error / problem. For example, the system can associate different identifiers with problems such as one or more spots with saturated pixels, non-Gaussian spots, an incorrect number of valid peaks, incorrect symmetry, other errors, or unidentifiable errors. In some embodiments, the system can also associate an identifier with images determined to be valid. Thus, an identifier indicating whether an image is valid or invalid (and, optionally, the reason it is invalid) can be stored for each frame. By implementing and storing the identifier in memory, the system can preserve a history of errors across different frames, which can be used to implement spot error processing. In some cases, the identifier can also be used to troubleshoot the imaging system (e.g., the sensor of the focus tracking system that collects the images) or the sample container during sample pre-scanning, sample scanning, and / or after sample scanning. For example, an identifier for saturated pixels can identify the presence of an air bubble. As another example, an identifier for a non-Gaussian spot can identify the presence of debris or scratches on the sample container. In some embodiments, the identifiers can be stored in a circular buffer that maintains a history of recent identifiers (e.g., during the current swath scan or other sample scan). In embodiments in which multiple spot pairs are detected using multiple respective sensors, the system can store a history of identifiers associated with each sensor.

[0185] If the image is determined to be valid, a current separation distance measurement (e.g., Δx1 as described above) for the spot pair is obtained based on the image in operation 3903. As described herein, separation distance measurements can be made in pixels, as depicted by FIGS.

[0186] At decision 3904, a determination is made as to whether the current separation distance measurement is within a threshold range determined from previous separation distance measurements. For example, to prevent large Z steps that may indicate measurement errors and / or be detrimental to imaging, the system may maintain a recent history of previous separation distance measurements (e.g., in a circular buffer or other fixed-size data structure). The threshold range may be based on an average / running average of the most recent measurements, or the change between adjacent frames from the most recent measurement, or the frequency content of the most recent measurement. For example, the threshold range may be set as a number of standard deviations above / below averaged past valid measurements (e.g., 1, 1.5, 2, 2.5, 3, 3.5, 4, etc.), or an averaged change above / below the last valid measurement. If the most recent measurement is outside this threshold range, it may indicate an abnormally large Z step that should not be taken. In some implementations, the threshold range can be set so that the most recent measurements in a fixed data structure are weighted more heavily (e.g., an average of the measurements or an average of the change in measurements between neighboring frames weights the most recent measurements in the data structure more heavily than the last measurement in the data store). In some implementations, the threshold range can be set based on the most recent valid separation distance measurement used during Z-stage adjustment.

[0187] If the separation distance measurement is within the threshold range, the Z stage may be controlled to focus the imaging system on the sample location based on at least the current separation distance measurement in operation 3905. If the spot pair image 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 on the sample location based on an earlier separation distance measurement in operation 3906. For example, the Z stage may be controlled based on the most recent of the previous separation distance measurements determined to be valid, or a combination of previous separation distance measurements (e.g., a moving average weighted toward more recent measurements).

[0188] During operation, the Z stage can be controlled based on a Z step commanded based on one or more separation distance measurements. The Z step can move the stage holding the objective lens and / or sample container of the imaging system up or down in the Z direction. As discussed above, the Z step can be calculated as a function of one or more separation distance measurements (e.g., the current separation distance measurement) and a gain value associated with the optics of the focus tracking module. For example, the Z step can be calculated by taking the current separation distance measurement in pixels and multiplying it by a gain value in nm / pixel.

[0189] In an alternative embodiment, method 3900 can be implemented without decision 3904 (e.g., after the image is determined to be valid and a current separation distance measurement is obtained, it is used to control the Z stage).

[0190] While method 3900 has been described in the context of spot error processing for an imaging / focus tracking system that generates one imaged spot pair, it should be understood that method 3900 can be extended to an imaging / focus tracking system that generates multiple pairs of imaged spots using one or more sensors (e.g., two pairs of substantially parallel pairs of spots imaged by two sensors). In such a system, operations 3901-3904 as described above can be implemented for each pair of detected spots (e.g., for each sensor in the system).

[0191] To that end, Figure 40 is a flow diagram depicting a method 4000 of spot error processing in a system that performs focus tracking using at least two pairs of substantially parallel spots, according to some embodiments 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 spot pairs are substantially parallel and may be captured by a single sensor or multiple sensors (e.g., two substantially parallel linear sensors as described above).

[0192] Operation 4002 includes determining that a first image of a first spot pair is invalid or that a current separation distance measurement (e.g., Δx1) for the first spot pair obtained from the first image is not within a threshold range. For example, operation 4002 can be performed as described above with reference to operations 3902-3904 of method 3900.

[0193] At decision 4003, a determination is made as to whether the second image of the second spot pair is valid. For example, this determination may be made based on the intensity distribution of pixels in the second image according to the techniques described above with reference to decision 3902 of method 3900. If the second image is determined to be valid, then at operation 4004, a current separation distance measurement (e.g., Δx2 as described above) for the second spot pair is obtained based on the second image.

[0194] At decision 4005, a determination is made as to whether the current separation distance measurement of the second spot pair is within a threshold range determined from the previous separation distance measurement of the second spot pair. In an alternative embodiment, the comparison of the obtained separation distance measurement to the threshold may be skipped. In such an embodiment, operation 4005 may be skipped and operation 4002 may only include determining that the first image of the first spot pair is invalid.

[0195] If the first image is invalid but the second image is valid, operation 4006 can be performed, which includes controlling the Z stage to focus the imaging system on the sample location based on the current separation distance measurement of the second spot pair and, optionally, one or more of the previous separation distance measurements of the first spot pair. Thus, by using two spot pairs, even if there is a detected error in one of the spot pairs, the separation distance measurement of the other spot pair can be used to determine the Z step. In some implementations, the current separation distance measurement of the second spot pair can be averaged with a separation distance measurement determined from one or more of the previous separation distance measurements of the first spot pair (e.g., the most recently valid one, or some type of average of previous measurements). This average can then be used to command the Z stage.

[0196] If both the first image and the second image are invalid, operation 4007 can be performed, which includes controlling the Z stage to focus the imaging system on the sample position based on one or more previous separation distance measurements of the first spot pair and / or one or more previous separation distance measurements of the second spot pair. For example, the Z step can be determined using an average of the most recently valid separation distance measurement of the first spot pair and the most recently valid separation distance measurement of the second spot pair.

[0197] While the preceding 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 in a tilt stage configured to tilt a sample container in a direction orthogonal to the scan direction.

[0198] To this end, Figure 41 is a flow diagram depicting a method 4100 of detecting gradient spikes in a system that performs focus tracking using at least two pairs of substantially parallel spots projected onto at least two locations on a sample, according to some embodiments of the present disclosure. Prior to performing method 4100, separation distance measurements can be obtained for the two spot pairs (e.g., by following the techniques described above).

[0199] Operation 4101 includes obtaining a current specimen tilt measurement based on a current separation distance measurement of a first pair of spots (e.g., Δx1) and a current separation distance measurement of a second pair of spots (e.g., Δx2). For example, the measurement may be obtained based on the difference between the two separation distance measurements (e.g., equation (3) above).

[0200] 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 described above, each tilt measurement may be obtained by taking the difference between two separation distance measurements of two pairs of substantially parallel spots. The threshold range may be based on the average / moving average of the most recent measurements, or the change between adjacent frames from the most recent measurement, or the frequency content of the most recent measurement. For example, the threshold range may be set as the number of standard deviations above / below averaged previous valid measurements (e.g., 1, 1.5, 2, 2.5, 3, 3.5, 4, etc.), or the averaged change above / below the last valid measurement. If the most recent tilt measurement is outside this threshold range, it may indicate a tilt spike that should be avoided. In some embodiments, the threshold range can be set such that the most recent measurements in a fixed data structure are weighted more heavily (e.g., an average of measurements weights the most recent measurements in the data structure more heavily than the last measurement in the data store). In some embodiments, the threshold range can be set based on the most recent valid specimen tilt measurement used during tilt stage adjustment.

[0201] If the current sample tilt measurement is within the threshold range, operation 4103 includes controlling the tilt stage to focus the sample at the desired position based on the current sample tilt measurement. If the current sample tilt measurement is not within the threshold range, optional operation 4104 includes identifying the sensor(s) having the spot separation glitch. For example, in an embodiment where two parallel sensors are used to detect two corresponding spot pairs, a determination can be made as to whether the current separation distance measurement of one sensor has deviated significantly or whether the current separation distance measurements of both sensors have deviated significantly. In some embodiments, the system can store an error code when it is detected that the current sample tilt measurement is not within the threshold range.

[0202] In this document, the terms "machine-readable medium," "computer-readable medium," and similar terms are used generally to refer to a non-transitory medium, either volatile or non-volatile, that stores data and / or instructions that cause a machine to operate in a specific fashion. Common forms of machine-readable medium include, for example, a hard disk, a solid-state drive, a magnetic tape, or any other magnetic data storage medium, an optical disk, or any other optical data storage medium, any physical medium with a pattern of holes, RAM, PROM, EPROM, FLASH-EPROM, NVRAM, any other memory chip or cartridge, and networked versions thereof.

[0203] These and various other forms of computer-readable media may be involved in carrying one or more sequences of one or more instructions to a processing device for execution. Such instructions embodied on the medium are generally referred to as "instructions" or "code." The instructions may be grouped in the form of a computer program or other grouping. Such instructions, when executed, may enable the processing device to perform the features or functions of the present application as discussed herein.

[0204] In this document, a "processing device" may be implemented as a single processor that performs processing operations, or as a combination of special purpose 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 circuitry.

[0205] As used throughout this disclosure, including the claims, the terms "substantially" and "about" are used to describe and account for small variations, such as those due to variations in processing, etc. For example, small variations can refer to ±5% or less, such as ±2% or less, such as ±1% or less, such as ±0.5% or less, such as ±0.2% or less, such as ±0.1% or less, such as ±0.05% or less.

[0206] Where applicable, the terms "first," "second," "third," etc., used herein are used merely to indicate each object described by those terms as a separate entity, and no chronological order is implied unless otherwise expressly stated herein.

[0207] Terms and phrases used in this document, and variations thereof, unless expressly stated otherwise, should be construed as open-ended as opposed to limiting. As in the preceding examples, the term "including" should be read to mean "including, but not limited to," and the like. The term "example" is used to provide a representative instance of the item in discussion, rather than an exhaustive or limiting list thereof. The terms "a" or "an" should be read to mean "at least one," "one or more," or the like. Adjectives such as "conventional," "traditional," "usual," "standard," "known," and similar terms should not be construed as limiting the described items to items available in a given period or at a given time, but rather should be read to encompass conventional, traditional, usual, or standard technology that may be available or known now or at any time in the future. Similarly, when this document refers to technology that would be apparent or known to those of ordinary skill in the art, such technology encompasses those technologies that are apparent or known to those of ordinary skill in the art now or at any time in the future.

[0208] In some cases, the presence of broader words and phrases such as "one or more," "at least," "including but not limited to," or other similar phrases should not be read to imply that a narrower case is intended or required in the absence of such broader phrase.

[0209] Furthermore, various embodiments described herein are described with reference to exemplary block diagrams, flow charts, and other illustrative diagrams. As will become apparent to one of ordinary skill in the art after reading this document, the illustrated embodiments and various alternatives thereof can be implemented without being limited to the illustrated examples. For example, block diagrams and their accompanying description should not be construed as mandating a particular architecture or configuration.

[0210] As used throughout this disclosure, including the claims, the terms "substantially" and "about" are used to describe and account for small variations, such as those due to variations in processing, etc. For example, small variations can refer to ±5% or less, such as ±2% or less, such as ±1% or less, such as ±0.5% or less, such as ±0.2% or less, such as ±0.1% or less, such as ±0.05% or less.

[0211] Where applicable, the terms "first," "second," "third," etc., used herein are used merely to indicate each object described by those terms as a separate entity, and no chronological order is implied unless otherwise expressly stated herein.

[0212] While various embodiments of the present disclosure have been described above, it should be understood that they are presented by way of example only, and not limitation. Similarly, various diagrams may depict example architectures or other configurations for the present disclosure, and this is done to aid in understanding the features and functionality that may be included in the present disclosure. The present disclosure is not limited to the illustrated example architectures or configurations, but the desired features can be implemented using a variety of alternative architectures and configurations. Indeed, it will be apparent to one skilled in the art how alternative functional, logical, or physical partitionings and configurations can be implemented to implement the desired features of the present disclosure. Also, many different component module names other than those depicted herein may be applied to the various partitions. Furthermore, with respect to flow diagrams, operational descriptions, and method claims, the order in which steps are presented herein does not obligate various embodiments to be implemented to perform the recited functions in the same order, unless the context dictates otherwise.

[0213] While the present disclosure has been described above in terms of various exemplary embodiments and implementations, it should be understood that various features, aspects, and functions described in one or more of the individual embodiments are not limited in applicability to the particular embodiments for which they are described, but instead may be applied alone or in various combinations to one or more of the other embodiments of the present application, regardless of whether such an embodiment is described and whether such features are presented as being part of the described embodiment. Thus, the breadth and scope of the present disclosure should not be limited by any of the exemplary embodiments described above.

[0214] It is to be understood that all combinations of the foregoing concepts (provided that such concepts are not mutually inconsistent) are intended to be part of the inventive subject matter disclosed herein. In particular, all combinations of claimed subject matter appearing in this disclosure are contemplated to be part of the inventive subject matter disclosed herein.

Claims

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

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

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

4. Determining whether the first image of the first spot pair is valid includes determining that the first image is not valid; and in response to determining that the first image is not valid, storing in a memory of the imaging system an identifier indicating why the first image is not valid.

5. 2. The non-transitory computer-readable medium of claim 1, wherein the operations further include, before controlling the Z-stage, determining that the current separation distance measurement of the first spot pair is within a threshold range determined from multiple previous separation distance measurements of the first spot pair associated with other sample positions of the sample.

6. 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 previous separation distance measurements, the number being greater than 0 and less than 4.

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

8. 2. The non-transitory computer-readable medium of claim 1, wherein the operations further include, if 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 spot pair associated with one or more other sample positions of the sample.

9. the one or more previous separation distance measurements are a plurality of previous separation distance measurements stored in a memory of the imaging system; controlling the Z-stage to focus on the sample at the first sample position based on at least the one or more previous separation distance measurements; retrieving a most recent separation distance measurement from the plurality of previous separation distance measurements stored in the memory; and and controlling the Z-stage to focus on the sample at the first sample position based on the most recent separation distance measurement of the plurality of previous separation distance measurements.

10. the one or more previous separation distance measurements are a plurality of previous separation distance measurements stored in a memory of the imaging system; 9. The non-transitory computer-readable medium of claim 8, wherein controlling the Z stage to focus on the sample at the first sample position based on at least the one or more previous separation distance measurements comprises 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 operation is capturing a second image of a second pair of spots projected substantially parallel to the first pair of spots at the first sample position using the one or more image sensors of the imaging system; determining whether the second image of the second spot pair is valid; and 10. The non-transitory computer-readable medium of claim 1, further comprising: if the second image is determined to be valid, obtaining a current separation distance measurement of the second spot pair based on the second image.

12. 12. The non-transitory computer-readable medium of claim 11, wherein the operations further include, if 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 on the current separation distance measurement of at least the second spot pair.

13. 12. The non-transitory computer-readable medium of claim 11, wherein the operations further include, if 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 on at least the current separation distance measurement of the second spot pair and one or more previous separation distance measurements of the first spot pair associated with one or more other sample positions of the sample.

14. 12. The non-transitory computer-readable medium of claim 11, wherein the operations further include, if 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 on at least the current separation distance measurement of the first spot pair and the current separation distance measurement of the second spot pair.

15. controlling the Z stage of the imaging system to focus the imaging system onto the first sample position based on at least the current separation distance measurement of the first spot pair and the current separation distance measurement of the second spot pair; determining an average of the current separation distance measurement of the first pair of spots and the current separation distance measurement of the second pair of spots; and controlling the Z-stage of the imaging system to focus the imaging system onto the first sample position based at least on the average.

16. 12. The non-transitory computer-readable medium of claim 11, wherein the operations further include, if 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 spot pair and the current separation distance measurement of the second spot pair.

17. 17. The non-transitory computer-readable medium of claim 16, wherein the operations further comprise determining whether the current specimen tilt measurement is within a threshold range determined from a previous specimen tilt measurement.

18. one or more light sources for projecting a first pair of spots onto a first sample location on the sample; one or more image sensors for capturing first images of the first spot pair projected onto the first sample location; and one or more processors that perform operations, said operations comprising: determining whether the first image of the first spot pair is valid; If the first image is determined to be valid, obtaining a current separation distance measurement for the first spot pair based on the first image; and controlling a Z-stage to focus at the first sample position based on at least the current separation distance measurement.

19. 20. The system of claim 18, wherein determining whether the first image of the first spot pair is valid comprises determining whether the first image of the first spot pair is valid based on an intensity distribution of pixels in the first image.

20. 20. The system of claim 18, further comprising a memory for storing one or more previous separation distance measurements associated with one or more other sample positions of the sample, and wherein the operations further include, if the first image is determined to be invalid, controlling the Z stage to focus on the sample at the first sample position based on at least the one or more previous separation distance measurements.

21. the one or more light sources for projecting a second pair of spots at the first sample position substantially parallel to the first pair of spots; 20. The system of claim 18, wherein the one or more image sensors capture a second image of the second spot pair projected onto the first sample location.

22. the one or more light sources include a first light source for projecting the first pair of spots onto the first sample location and a second light source for projecting the second pair of spots onto the first sample location; 22. The system of claim 21, wherein the one or more image sensors include a first image sensor for capturing the first image and a second image sensor for capturing the second image.

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

24. The operation is determining whether the second image of the second spot pair is valid; and 22. The system of claim 21, further comprising: if the second image is determined to be valid, obtaining a current separation distance measurement of the second spot pair based on the second image.

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

26. the system further comprising a tilt assembly including one or more actuators for tilting a sample holder of the sample during imaging of the sample; The operation is if 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 spot pair and the current separation distance measurement of the second spot pair; 25. The system of claim 24, further comprising: controlling the tilt assembly 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, the Z stage including an objective lens.

Images