Dynamic tilt-removal focus tracking

Abstract

Some embodiments of the present disclosure relate to an imaging system including: a sample holder for supporting a sample container having a plurality of sample positions; an optical stage having an assembly with one or more actuators physically coupled to the sample holder for tilting the sample holder relative to the optical stage during imaging of the plurality of sample positions to focus the optical stage on a current sample position; a first light source for projecting a first spot pair 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 spot pair from one or more images taken by an image sensor at one or more of the sample positions.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 300,531, entitled "DYNAMIC DETILT FOCUS TRACKING," filed on January 18, 2022, which is incorporated by reference in its entirety. [Background technology]

[0002] Increasing the numerical aperture (NA) of an optical imaging system improves optical imaging resolution. In sequencing applications, this reduces the sequencing cluster pitch and increases cluster density, 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 NA continue to be used in imaging applications to reduce costs (e.g., to reduce sequencing costs), it becomes more difficult to ensure that the imaged sample remains in focus when translated along the optical axis. For example, as illustrated by FIG. 1A, some current sequencers can tilt the sample out 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 a global adjustment of the sample tilt, local tilt within the sample itself can produce a DoF excursion large enough to cause at least a portion of the sequencing image to be out of focus, which causes 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.

[0005] In one embodiment, the imaging system comprises an assembly comprising 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 to focus the imaging system on a current sample position, a first light source for projecting a first spot pair 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 spot pair from one or more 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 one or more 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 a second direction that is substantially perpendicular to at least 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 specimen tilt is determined along a first direction and a second direction, and the controller is to control the one or more actuators to tilt the specimen holder along the first direction and the second direction based on the specimen 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 a first direction or a 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 image sensor comprises a first image sensor for imaging the first pair of spots and a second image sensor for imaging the second pair of spots.

[0014] 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.

[0015] In some embodiments, the one or more actuators comprise a plurality of linear actuators, the sample holder being a moveable platform mounted on the plurality of linear actuators, the plurality of linear actuators being for tilting the sample holder along a first direction and a second direction.

[0016] In some implementations, each of the linear actuators comprises a carriage that is driven up and down by a screw on a tilt motor.

[0017] In some embodiments, the assembly further comprises a stage, the moveable platform being mounted on the stage, the stage being for laterally moving the sample holder.

[0018] 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.

[0019] In some embodiments, the method further includes tilting the sample in a direction substantially perpendicular to a scanning direction of the imaging system using one or more actuators of the imaging system based on the first tilt of the sample.

[0020] In some embodiments, calculating the first tilt of the sample includes calculating a first tilt of the sample at the first sample position along a scanning direction of the imaging system and a direction substantially perpendicular to the scanning direction based on at least the first sample height and the second sample height.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] In some embodiments, the image sensor comprises a first image sensor and a second image sensor, and the images of the first spot pair and the second spot pair at the first sample position include a first image of the first spot pair at the first sample position acquired by the first image sensor and a second image of the second spot pair at the first sample position acquired by the second image sensor.

[0025] In one embodiment, the imaging system includes an assembly comprising an image sensor, one or more actuators for tilting a sample holder during imaging of a plurality of 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 plurality of sample positions.

[0026] In some embodiments, the imaging system further comprises a 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.

[0027] In one embodiment, the imaging system comprises a light source for projecting a first pair of spots and a second pair of spots onto the sample, a first image sensor for acquiring one or more first images of the first pair of spots and the second pair of spots at one or more sample positions of the sample, a second image sensor for acquiring one or more second images of the sample, and a controller for controlling one or more components of the imaging system such that a relative tilt between the sample and the second image sensor remains substantially the same at the one or more sample positions based on a sample tilt determined from at least the one or more first images.

[0028] In some embodiments, the one or more components include one or more actuators coupled to the second image sensor, and the controller is for controlling the one or more actuators to tilt the second image sensor based on a sample tilt determined from at least the one or more first images, such that a relative tilt between the sample and the second image sensor remains substantially the same at one or more sample positions.

[0029] In some embodiments, the one or more components include one or more actuators coupled to a camera housing a second image sensor, and the controller is for controlling the one or more actuators to tilt the camera based on the sample tilt determined from at least the one or more first images, such that the relative tilt between the sample and the second image sensor remains substantially the same at one or more sample positions.

[0030] In some embodiments, the imaging system further comprises an objective lens in an optical path between the sample and the second image sensor, and the one or more components comprise one or more actuators coupled to the objective lens, and the controller is for controlling the one or more actuators to tilt the objective lens based on the sample tilt determined from the at least one or more first images, such that the relative tilt between the sample and the second image sensor remains substantially the same at one or more sample positions.

[0031] 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 description of the drawings]

[0032] The present disclosure, in accordance with one or more embodiments, will now be described in detail with reference to the following figures, which are provided for illustrative purposes only and merely depict exemplary embodiments. Additionally, it should be noted that for clarity and ease of illustration, elements in the figures have not necessarily been drawn to scale.

[0033] 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 descriptive 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. [Diagram 2] 1 illustrates a block diagram of an optical imaging system according to some embodiments of the present disclosure. [Diagram 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. [Diagram 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 a 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 a detector surface provided by the reflected light beam shown in FIG. 7; [Figure 9]4 illustrates incident and reflected light beams when the optical assembly shown in FIG. 3 is above focus. [Figure 10] 10 illustrates the beam spot on a detector surface provided by the reflected light beam shown in FIG. [Figure 11] 1 shows a series of plots illustrating the surface profile of a flow cell sample scanned using a line scan 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 moveable platform including a sample container and a corresponding controller, according to some embodiments of the present disclosure. [Figure 14] 1 depicts a moveable 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] FIG. 16B shows a perspective view of the actuator of FIG. 16A. [Figure 16C] FIG. 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] 1 illustrates tilt angles at different sample positions for a tilt map according to some embodiments of the present disclosure. [Figure 21C] 13 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. 2 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 that projects one spot beam pair onto the surface of the sample across different sample positions in the scan direction during the process. [Figure 23C] 13 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] 1 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 that projects 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 a scan direction and 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 the relative orientation of two projected beam spot pairs and one of two scan lines in a two-channel line-scan imaging system, according to some embodiments of the present disclosure. [Figure 25B] 14 illustrates another relative orientation of two projected beam spot pairs and two scan lines in a two-channel line-scan imaging system according to some embodiments of the present disclosure. [Figure 25C] 14 illustrates another relative orientation of two projected beam spot pairs and two scan lines in a two-channel line-scan imaging system according to some embodiments of the present disclosure. [Figure 25D] 14 illustrates another relative orientation of two projected beam spot pairs and two scan lines in a two-channel line-scan imaging system according to some embodiments of the present disclosure. [Figure 25E] 14 illustrates another relative orientation of two projected beam spot pairs and two scan lines in a two-channel line-scan imaging system according to some embodiments of the present disclosure. [Figure 25F] 14 illustrates another relative orientation of two projected beam spot pairs and two scan lines in a two-channel line-scan imaging system according to some embodiments of the present disclosure. [Figure 26] 1 conceptually illustrates the advantages of using multiple tilt actuators, according to some embodiments of the present disclosure. [Figure 27] 1 illustrates 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. [Figure 28] In one exemplary embodiment utilizing the focus tracking system described with reference to FIG. 27, both the object plane and the sensor plane are imaged during scanning of the flow cell surface.

[0034] The figures are not exhaustive and are not intended to limit the disclosure to the precise forms disclosed. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] 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).

[0036] 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.

[0037] 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 an imaging dataset of one imaging cycle.

[0038] As used herein, the term "xy coordinates" is intended to mean information that specifies a position, size, shape, and / or orientation in an xy plane. The information may be, for example, numerical coordinates in a Cartesian coordinate system. The coordinates may be provided relative to one or both of the x and y axes, or may be provided relative to another location in the xy 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.

[0039] As used herein, the term "xy plane" is intended to mean a 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 observation direction between the detector and the object being detected. When used herein to refer to a line scanner, the term "y direction" refers to the direction of scanning.

[0040] As used herein, the term "z coordinate" is intended to mean information that specifies the location of a point, line, or area along an axis orthogonal to the xy plane. In certain embodiments, the z axis is orthogonal to the area of ​​an object observed by a detector. For example, the direction of the focal point of an optical imaging system may be specified along the z axis.

[0041] As used herein, the term "scanning" is intended to mean detecting a two-dimensional cross-section in the xy plane of an object, the cross-section being 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 the emission from that area can be specifically acquired (to the exclusion of other areas) at a given time point of the scan.

[0042] As mentioned above, there is an increasing need to enable dynamic tilting of the sample in optical imaging systems that utilize larger NA and sacrifice DoF to resolve finer optical features. In such systems, even small amounts of twisting of the sample that defocus a portion of the sample in the field of view can cause significant errors. Small amounts of tilt in the scan direction, or in a direction perpendicular to the scan direction, can blur or defocus the region of interest being scanned. Hence, there is a need for dynamic multi-axis tilting of the sample.

[0043] Various embodiments of the present disclosure relate to systems and methods for dynamically tilting a sample holder in one or more directions during a sample scan based on the local topography of the sample to keep the sample in focus. One set of embodiments describes a sample stage assembly including a tilt-tilt assembly for enabling multi-axis tilt of the sample holder during scanning. 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 that is utilized by a controller to direct the operation of an assembly that dynamically tilts the sample holder during a sample scan.

[0044] 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 a 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 paper), vertically / elevationally along a Z-axis, and / or angularly along the X-axis (tilt), Y-axis (tilt), and / or Z-axis (twist). The optical imaging system 100 may also include a system controller or computing system 120 operably coupled to the optical assembly 106 , the stage controller 115 , and / or the object holder 102 .

[0045] In some embodiments, the object 110 is a sample container that contains a biological sample to be imaged using one or more fluorescent dyes. For example, in certain embodiments, the sample container can be implemented as a patterned flow cell that includes a semi-transparent cover plate, a substrate, and a liquid sandwiched therebetween, and the biological sample can be located on the inner surface of the semi-transparent cover plate or the inner surface of the substrate. The flow cell can include a large number (e.g., thousands, millions, or billions) of depressions or regions that are patterned into a defined array (e.g., a hexagonal array, a rectangular array, etc.) within the substrate. Each region can form a cluster (e.g., a monoclonal cluster) of biological samples, such as DNA, RNA, or another genomic material, that can be sequenced using sequencing by synthesis. The flow cell can be divided into several physically separated lanes (e.g., eight lanes), each lane 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.

[0046] Although not shown, the optical imaging system 100 may include one or more subsystems or devices for implementing various test protocols. For example, if the sample includes a flow cell having 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 reagents (e.g., fluorescently labeled nucleotides, buffers, enzymes, cleavage reagents, etc.) to (and through) the sample containers and relief valves. Another subsystem that may be included is a temperature control system, which may have heaters / coolers configured to regulate 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.

[0047] As shown in the figure, the optical assembly 106 is configured to direct input light to the object 110 and to receive and direct output light to one or more detectors. The output light may be input light that has been at least one of reflected and refracted by the object 110 and / or the output light may be light emitted from the object 110. To direct the input light, the optical assembly 106 may include at least one reference light source 112 and at least one excitation light source 114 that direct light, such as a light beam having a predetermined wavelength, through one or more optical components of the optical assembly 106. The optical assembly 106 may include various optical components, including a conjugate lens 118, for directing the input light toward the object 110 and the output light toward the detectors.

[0048] 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 arranged to illuminate a bottom surface of the object 110, such as in TIRF imaging, or may be arranged to illuminate a top surface of the object 110, such as in epifluorescence imaging. As shown in FIG. 2, the conjugate lens 118 directs the input light to a focal zone 122 located in a focal plane FP. The lens 118 has an optical axis 124 and is positioned at a working distance WD1 away 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 an area of ​​interest of the object 100 within the FP by rotating the object holder 102 about the X-, Y-, and / or Z-axes.

[0049] To determine whether the object 110 is in focus (i.e., well within the focal zone 122 or focal plane FP), the optical assembly 106 is configured to direct at least one pair of light beams to the focal zone 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 towards 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 illustrative purposes, 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.

[0050] In the embodiment shown in FIG. 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 the imaging optics, or alternatively, the objective lens illustrated herein can be dedicated to focus determination or distance measurement.

[0051] The reflected light beams 130B and 132B propagate through the lens 118 and may be optionally further directed by other optical components of the optical assembly 106. As shown in the figure, the reflected light beams 130B and 132B are detected by at least one focus detector 144. In the illustrated embodiment, both the reflected light beams 130B and 132B are detected by a single focus detector 144. The reflected light beams may be used to determine a relative separation RS1. For example, the relative separation RS1 may be determined by a distance (i.e., a separation distance) that separates the beam spots from the reflected light beams 130B and 132B incident on the focus detector 144. The relative separation RS1 may be used to determine a 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.

[0052] 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 located above the focal plane FP (or focal zone 122), the object 110 can be relatively moved toward the focal plane FP by a distance ΔZ1, or if the object 110 is located below the focal plane FP (or focal zone 122), the object 110 can be relatively moved toward the focal plane FP by a distance ΔZ2. In some embodiments, the optical imaging system 100 can replace the lens 118 with another lens 118 or other optical components to move the focal zone 122 of the optical assembly 106.

[0053] The examples described above and in Fig. 2 are 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.

[0054] In addition, as described further below, the system may be useful for determining a 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 of the object, the variation in the working distance between the object 110 and the lens 118 along the imaging direction may be determined, which may be mapped to the object height along the imaging direction (i.e., in the z-direction). In a particular embodiment, described further below, the optical assembly 106 is configured to direct multiple light beam pairs (e.g., at least two pairs) along different positions of the scanned object surface. Based on the relative separation of each of the light beam pairs and the distance between the different light beam pairs, the surface profile of the object may be determined in one or more dimensions. Given knowledge of the object's surface profile, the optical imaging system 100 may actively orient an area of ​​interest of the object 100 within the FP by rotating the object holder 102 about the X-axis, the Y-axis, and / or the Z-axis via the stage controller 115.

[0055] 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.

[0056] In one embodiment, during operation, the excitation light source 114 directs input light (not shown) to the object 110 to excite the fluorescently labeled biological or chemical agent. The biological or chemical agent label provides an optical signal 140 (also referred to as an optical emission) having a predetermined wavelength. The optical signal 140 is received by the lens 118 and then directed by other optical components of the optical assembly 106 to at least one object detector 142. Although the illustrated embodiment shows only one object detector 142, the object detector 142 may comprise multiple detectors. For example, the 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. The optical assembly 106 may include a lens / filter assembly that directs different optical signals along different optical paths toward corresponding object detectors.

[0057] 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 communication lines (e.g., the Internet). As an example, the object data may include imaging data that is processed to generate an image of the object 110. The image 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 emissions from biological or chemical materials, 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.

[0058] 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 detect the reflected light beam pair (e.g., reflected light beams 130B and 132B) as well as the optical signal (e.g., optical signal 140).

[0059] The optical imaging system 100 may include a user interface 125 for interacting with a user through the computing system 120. For example, the user interface 125 may include a display (not shown) for presenting and requesting information from the user, and a user input device (not shown) for receiving user input.

[0060] The computing system 120 may include, among other things, an object analysis module 150 and a focus control module 152. The focus control module 152 is configured to receive focus data acquired by the focus detector 144. The focus data may include signals representative of the beam spots incident on the focus detector 144. The data may be processed to determine a relative separation (e.g., a separation distance between the beam spots). A degree of focus of the optical imaging system 100 on the object 110 may then be determined based on the relative separation. In certain embodiments, a working distance WD1 between the object 110 and the lens 118 may be determined. Similarly, the object analysis module 150 may receive the object data acquired by the object detector 142. The object analysis module may process or analyze the object data to generate an image of the object.

[0061] Additionally, computing system 120 may include any processor-based or microprocessor-based system, including systems using microcontrollers, reduced instruction set computers (RISC), application specific integrated circuits (ASICs), field programmable gate arrays (FPGAs), logic circuits, and any other circuits or processors capable of performing the functions described herein. The above examples are merely illustrative and thus are not intended to limit the definition and / or meaning of the term system controller. In one embodiment, computing system 120 executes a set of instructions 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.

[0062] The set of instructions may include various commands to 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 a surface profile of the object 110. The set of instructions may be in the form of a software program.

[0063] As described above, the excitation light source 114 generates excitation light that is directed to 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 once and stop sequentially through the regions in a "step-and-shoot" scanning pattern.

[0064] In some implementations, 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 at a desired aspect ratio. In certain implementations, 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., about 520-565 nm) and the second wavelength may be a "red" wavelength (e.g., about 625-740 nm). Such a line scanning system may be utilized in conjunction with a TDI sensor.

[0065] The object 110 generates an optical signal 140, which may include optical emissions generated in response to illumination of a label within the object 110 and / or light reflected or refracted by an optical substrate of the object 110. Alternatively, the optical signal 140 may be generated in the absence of illumination based overall on the emission characteristics of materials within the object 110 (e.g., radioactive or chemiluminescent moieties within the object).

[0066] 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 and 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.

[0067] 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 in the figures, the optical assembly 202 includes an optical train 240 of optical components 241-245 that directs the 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 of 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.

[0068] 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 of about 620 nm to 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 towards 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 the opposing second surface 262 towards the first surface 260, which forms the incident light beam 232A.

[0069] The dual beam generator 241 directs the parallel incident light beams 230A and 232A towards the beam splitter 242. The beam splitter 242 reflects the incident light beams 230A and 232A towards the conjugate lens 243. In this example, the beam splitter 242 includes a pair of reflectors (e.g., aluminum tabs) positioned to reflect the incident light beams 230A and 232A and the reflected light beams 230B and 232B. The beam splitter 242 is positioned to reflect the incident light beams 230A and 232A so that the incident light beams 230A and 232A propagate parallel to the optical axis 252 of the lens 243. The optical axis 252 extends through the center of the lens 243 and intersects the focal zone 256. The lens 243 can 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.

[0070] 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 propagate back to and 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).

[0071] As shown in Figures 3 and 4, the reflected light beams 230B and 232B are incident on and directed by the dual beam generator 241 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 that separates 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 given 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, so 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.

[0072] After exiting the lens 243, the reflected light beams 230B and 232B propagate substantially parallel to each other between the optical components. In the illustrated embodiment, the reflected light beams 230B and 232B propagate substantially parallel to each other along the optical track between the lens 243 and the 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 cross 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.

[0073] The optical train 240 may be configured to maintain a projective relationship between the reflected light beams 230B and 232B throughout the optical track so that a degree of focus may be determined. By way of example, when the optical assembly 202 is focused on the object, the reflected light beams 230B and 232B will propagate parallel to one another between each optical component in the optical train 240. When the optical assembly 202 is not focused on the object, the reflected light beams 230B and 232B are coplanar but propagate at a slight angle relative to one another. For example, the reflected light beams 230B and 232B may diverge from one another or converge toward one another as the reflected light beams 230B and 232B travel along the optical track to the focus detector 250.

[0074] 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 such 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 the incident light beams 230B and 232B. Optical components 241 and 244 may also have parallel surfaces that each 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 each other after exiting each optical component. If the reflected light beams 230B and 232B are converging or diverging towards each other at a particular speed, then the reflected light beams 230B and 232B will be converging or diverging towards each other at the same speed after exiting each optical component. Thus, the 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.

[0075] The optical imaging system may include one or more optical assemblies as discussed above for determining the working distance or focus. For example, the optical imaging system may include two optical assemblies of the type shown in Figures 3 and 4 to allow the focus to be determined at two different positions on the object or to provide a determination of the working distance between the optical imaging system and the object at two different positions. For embodiments in which there is more than one optical assembly, the optical assemblies may be discrete and separate, or the optical assemblies may share optical components. The optical assemblies may share optical components such as the reference light source 212, the focus detector 250, the folding mirror 245, the beam combiner 244, the dual beam generator 241, the beam splitter 242, the epi-fluorescent input (EPI) reflector 280, and the range limiter 254. The optical components may be shared by placing the beam splitter in the optical train upstream of the shared components. 3 and 4 are illustrated, 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.

[0076] 4, the reflected light beams 230B and 232B eventually impinge on the detector surface 264 of the 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 the optical assembly 202 has a sufficient degree of focus on the object. The separation distance SD1 on the detector surface 264 may also indicate the working distance between the lens 243 and the object being imaged.

[0077] In other embodiments, optical components 241-245 may be replaced with alternative optical components that perform substantially the same functions as 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. Additionally, 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.

[0078] Further, 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 position of the beam spot with the respective focus detector and the relative positions of the focus detectors with respect to each other.

[0079] Although not illustrated in FIGS. 3 and 4 , the optical assembly 202 may also be configured to facilitate collecting output light projected from the object 268. For example, the optical assembly 202 may include an epifluorescence input (EPI) reflector 280 positioned to reflect incident light provided by an excitation light source (not shown). The light may 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 to the object 268, which may 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 may allow a portion of the output light to propagate therethrough along the optical axis. The output light may then be detected by an object detector (not shown).

[0080] 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, and can also reflect excitation light.

[0081] 5-8 show different projection relationships between the reflected light beams 230B and 232B and the corresponding beam spots 270 and 272 on the detector surface 264. As discussed above, the projection 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 projection 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 projection relationship between the reflected light beams changes. However, the separation distance SD is just one way of determining the relative separation between the reflected light beams. Thus, one skilled in the art will understand that FIGS. 5-8 illustrate just one way of determining the relative separation, and other methods for determining the relative separation or projection relationship are possible.

[0082] 5-6 show the projective relationship between reflected light beams 230B and 232B when the optical assembly 202 (FIG. 3) is focused on the object 268. As shown in the figures, the incident light beams 230A and 232A propagate parallel to one another through the lens 243 and are spaced apart by a path spacing Ps1. In the illustrated embodiment, the incident light beams 230A and 232A propagate parallel to and are equidistant from the optical axis 252 of the lens 243. In alternative embodiments, the incident light beams 230A and 232A may propagate in a non-parallel manner to and have a different spacing from the optical axis 252. In certain alternative embodiments, one of the incident light beams 230A or 232A is aligned with the optical axis 252 of the lens 243 and the other is spaced apart from the optical axis 252.

[0083] The incident light beams 230A and 232A are directed by the lens 243 to converge towards the focal zone 256. In such embodiments where the incident light beams are not parallel to the optical axis, the focal zone may have a different position than that shown in FIG. 5. The incident light beams 230A and 232A are reflected by the object 268 to form the reflected light beams 230B and 232B. The reflected light beams 230B and 232B return to and propagate through the lens 243 and parallel to the optical axis 252. The reflected light beams 230B and 232B exit the lens 243 parallel to each other and spaced apart by a path spacing PS2. When the optical assembly 202 is focused, the path spacings PS1 and PS2 are equal.

[0084] Thus, when the optical assembly 202 is focused, the projective relationship of the reflected light beams 230B and 232B exiting the lens 243 comprises two parallel light beams. The optical train 240 is configured to maintain the parallel projective relationship. For example, when the optical assembly 202 is focused, the reflected light beams 230B and 232B are parallel to each other when they exit the dual beam generator 241, when they exit the beam combiner 244, and when they are reflected by the fold mirror 245. The projective relationship is maintained, but the path spacing PS2 may be rescaled by the beam combiner.

[0085] As shown in FIG. 6, the reflected light beams 230B and 232B of FIG. 5 are incident on the detector surface 264 and form beam spots 270 and 272. When the optical assembly 202 is focused, the beam spots 270 and 272 have a separation distance SD2. The separation distance SD2 may be based on (or may be a function of) the dimensions of the beam combiner 244, the angle of incidence on the parallel surfaces of the beam combiner 244, and the incident reflected light beams 230B and 232B. The separation distance SD2 is also based on the projective relationship of the reflected light beams 230B and 232B exiting the lens 243. As shown in FIG. 6, the detector surface 264 has a center point or central area 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.

[0086] 7-8 show the projective relationship between reflected light beams 230B and 232B when the optical assembly 202 (FIG. 3) is below the focal point. As explained above, the incident light beams 230A and 232A propagate parallel to each other through the lens 243 and are spaced apart by a path spacing PS1. The incident light beams 230A and 232A intersect each other at the focal zone 256 and are then reflected by the object 268 to form the reflected light beams 230B and 232B. However, as shown in FIG. 7, when the reflected light beams 230B and 232B exit the lens 243, the reflected light beams 230B and 232B are slightly convergent toward the optical axis 252 and toward each other. Also, as shown in the figure, the path spacing PS2 is greater than the path spacing PS1.

[0087] Thus, when object 268 is positioned below focal zone 256, the projective relationship of reflected light beams 230B and 232B comprises two light beams that converge towards one another. As above, optical train 240 is configured to maintain a converging projective relationship. For example, reflected light beams 230B and 232B converge towards one another when they exit dual beam generator 241, when they exit beam combiner 244, and when they are reflected by fold mirror 245.

[0088] As shown in FIG. 8, when the object 268 is located below the focal zone 256, the beam spots 270 and 272 have a separation distance SD3 that is smaller than the separation distance SD2 (FIG. 6). The separation distance SD3 is smaller because the reflected light beams 130B and 132B converge toward each other across the optical track between the lens 243 and the focus detector 250. Also, as shown in FIG. 8, the beam spots 270 and 272 may have a selected shape that correlates with the beam spots 270 and 272. The shape of the beam spots 270 and 272 when the object 268 is located below the focal zone 256 is different from the shape of the beam spots 270 and 272 when the object 268 is in focus. The beam spots 270 and 272 may have different imaginary radii that correlate with the object being below the focal zone 256.

[0089] 9-10 show the projective relationship between reflected light beams 230B and 232B when the optical assembly 202 (FIG. 3) is above the focal point. As explained above, the incident light beams 230A and 232A propagate parallel to each other through the lens 243 and are spaced apart by a path spacing PS1. Before the incident light beams 230A and 232A reach the focal zone 256, the incident light beams 230A and 232A are reflected by the object 268 to form the reflected light beams 230B and 232B. However, as shown in FIG. 9, when the reflected light beams 230B and 232B exit the lens 243, the reflected light beams 230B and 232B diverge away from the optical axis 252 and away from each other. Also, as shown in the figure, the path spacing PS2 is less than the path spacing PS1.

[0090] 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 diverging 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.

[0091] As shown in FIG. 10, when the object 268 is positioned above the focal zone 256, the beam spots 270 and 272 have a separation distance SD4 that is greater than the separation distance SD2. The separation distance SD4 is greater because the reflected light beams 130B and 132B diverge from each other throughout the optical track between the lens 243 and the focus detector 250. Also, as shown in FIG. 10, the beam spots 270 and 272 may have a selected shape that correlates with the beam spots 270 and 272. The shape of the beam spots 270 and 272 when the object 268 is positioned above the focal zone 256 is different from the shape of the beam spots 270 and 272 when the object 268 is positioned below the focal zone 256. Similarly, the beam spots 270 and 272 may have different imaginary radii that correlate with the object being below the focal zone 256.

[0092] As explained above, when the object 268 is below the focal zone 256, the separation distance SD3 is less than the separation distance SD2 when the object 268 is within the focal zone 256. When the object 268 is above the focal zone 256, the separation distance SD4 is greater than the separation distance SD2. Thus, the optical assembly 202 can not only determine that the object 268 is not located within the focal zone 256, but also determine a direction in which to move the object 268 relative to the lens 243. Furthermore, the value of the separation distance SD3 can be used to determine how far the object 268 should be moved relative to the lens 243.

[0093] In addition to determining focus, measurements of the separation distance on the detector can be used to determine the working distance between the lens and an object being detected through the lens, as illustrated by the examples of Figures 6-10. Additionally, the separation distance on the detector can be used to determine the profile of the object surface.

[0094] 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 the reflected light beams 230B and 232B are diverging or converging) and the length of the optical track measured from the lens 243 to the focus detector 250. As the length of the optical track between the lens 243 and the focus detector 250 increases, the separation distance may decrease or increase if the object is not in focus. Thus, the length of the optical track may be configured to facilitate distinguishing between the separation distances SD3 and SD4. For example, the optical track may be configured such that the converging reflected light beams do not cross each other and / or the diverging light beams do not exceed a predetermined relative separation between each other. To this end, the optical track between the optical components of the optical train 240 may be lengthened or shortened as desired.

[0095] Also, as the above 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 the measurement of the separation distance on the detector. This can further 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. When the working distance decreases (i.e., when the height of the object surface increases), the relative separation can increase, as shown in FIG. 10. When the working distance increases (i.e., when 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.

[0096] 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 prior to 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.

[0097] Also, as discussed above, to account for local variations in the topography of the sample, the optical imaging system may include a controller configured to dynamically move the sample holder laterally (along the X-axis and / or Y-axis extending into the plane of the paper), vertically / elevationally along the Z-axis, and / or angularly along the X-axis (tip), Y-axis (tilt), and / or Z-axis (twist) during imaging. To this end, it is useful to consider a coordinate system that may be used in designing an assembly that dynamically moves the sample laterally and / or angularly. FIG. 12 depicts one such exemplary coordinate system. In this example, the z-axis is aligned with the optical axis of the objective lens and may generally point upward into the sky. The y-axis is the axis along which the sample may be actively moved 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 of the sample holder about the x-axis (e.g., a change in θx).

[0098] 13-15 depict exemplary assemblies that may enable dynamic adjustment of lateral and angular positions 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 a 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 a number of actuators 304. FIG. 14 depicts an exemplary movable platform 400 mounted on the actuators 304 of the TTA. FIG. 15 depicts a 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.

[0099] 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 optics. The TTA may affect all three rotational axes, as depicted with respect to FIG. 11. In some implementations, the TTA may actively control rotations about all three axes during imaging. In other implementations, the TTA may actively control rotations about only some axes during imaging. For example, only rotations about the X and Y axes may be actively controlled during the imaging process, and rotations about the Z axis may be manually adjusted as needed.

[0100] 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. In general, 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 the one or more actuators. Furthermore, in some examples, the time at which the drive signal is applied to the one or more actuators may be adjusted based on the amount of translation required for the change in focus. For example, if the translation required is larger, the drive signal may be applied earlier. However, in other examples, the drive signal is applied as soon as possible after imaging is completed at the current sample position, regardless of the difference in focus settings. The parameters of the drive signal and the time 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.

[0101] 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 moveable 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 slope. 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.

[0102] 16A-16D show an exploded view, a perspective view, a side view, and another perspective view, respectively, of a particular 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. The actuator 304 functions as a linear stage with a carriage 304-8, which in this embodiment is driven up and down by a lead screw 304-4 that is integrated into a 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 of the light-reflective type. 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 to verify absolute position and orientation.

[0103] 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, the proximity sensor can be left solely 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.

[0104] 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 that the carriage 304-8 normally travels over during instrument use.

[0105] 17 depicts the underside of the moveable platform 400 according to some embodiments of the present disclosure. The underside includes three V-grooves 415 configured to couple to the balls 304-10 of the actuator 304.

[0106] 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.

[0107] 19 depicts the moveable platform 400 interfacing and coupling with the actuator 304. As depicted, the threaded hole 425 interfaces with the hold-down spring fastener and the ball 304-10 interfaces with the V-groove 415.

[0108] 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 explained 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 about 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 a 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 between two spot beam pairs L y , and the respective heights h1 and h2, the sample tilt angle (θ y ) 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 of 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.

[0109] Although feedback-based tilt as described above may 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 may be tilted, and ii) the latency in communicating 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 communicating the real-time measurement to the tilt controller, any real-time feedback mechanism may experience latency that makes such methods unsuitable.

[0110] Therefore, in some embodiments, to accommodate 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-C. In particular, FIG. 21A is an operational flow diagram illustrating an exemplary method for dynamically tilting a sample based on a predefined tilt trajectory. The method may be implemented by an optical imaging system (e.g., optical imaging system 100) that includes a suitable mechanism for dynamically tilting a stage carrying the sample (e.g., sample stage assembly 300).

[0111] Operation 610 includes determining a tilt map for the sample, the tilt map including a plurality of entries corresponding to a plurality of sample positions, each entry indicating an amount to tilt the sample at least along 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 array, or an otherwise 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, the tilt map 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 the smooth fit of the tilt trajectory in the z direction based on different tilt angles. The 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 a sample scanned in multiple strips (eg, a flow cell scanned in a swath), the sample height may be determined at multiple Y positions for each strip.

[0112] 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 an embodiment 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 an embodiment, a tilt map may be generated and used for each sample strip (e.g., operations 610-640 may be applied to each sample strip).

[0113] Operation 630 includes updating the gradient map for the next imaging cycle. Over time, the topography of the sample may change due to thermal expansion (e.g., due to an excitation light source or other light source increasing the temperature of the sample) and / or due to other changes in the sample. Thus, 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, the gradient map measurement for the next imaging cycle may be performed during the current imaging cycle. The optical imaging system may generate the updated gradient map using the same mechanism that was used to generate the original gradient map.

[0114] 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 at least along 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.

[0115] 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 a controller in assembly 300 to perform dynamic correction during the duration of each scan of a flow cell. For example, consider an optical imaging system that images a flow cell and has the following parameters: scan rate of 1 Hz, dynamic tilt removal servo update rate of about 10 Hz, tile θY acquisition rate of about 100 Hz, 99 tiles per scan swath, 2 surfaces (top and bottom) per flow cell, 2 flow cells per instrument, 8 lanes per flow cell surface, and 4 scans per flow cell lane. In this embodiment, the system is capable of performing 128 scans per sequencing cycle.

[0116] In the above 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). Thus, 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's controller. In this example, a smooth tilt removal trajectory can be determined to minimize the energy input to the instrument structure and maximize the quality of the tilt removal correction. The tilt removal trajectory can be created using a 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), then the total number of corrections per scan will be 100, requiring a slope / tilt update rate of 100 Hz for a 1 Hz scan rate. To generate a smooth tilt-removal trajectory, a mathematical operation such as a third order Hermite approximation can be applied to all of the collected data, which has the advantage of specifying both the position and slope at every target trajectory point.

[0117] In this example, a first set of compensation tables for the first sequencing cycle may be created by scanning the flow cell before the start of sequencing. Subsequent sequencing cycles may use compensation tables that are updated based on the centroid calculations made during each of the scans of the previous cycle. One advantage of this exemplary approach is that it may avoid the requirement of a low latency connection between the centroid calculations and the controller of the TTA. 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.

[0118] FIG. 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 721-2 may 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 may be fixed during an image scan, and the actuator 304 shown on the left side of FIG. 13 may dynamically tilt the sample up or down during an image scan.

[0119] 23A-23C illustrate one exemplary embodiment 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 a dual focused beam onto the surface of the sample.

[0120] Operation 810 includes scanning the sample in a scan direction along a plurality of sample locations of the sample by projecting one beam spot pair on the surface of each sample location. Operation 820 includes estimating a height of the sample location for each sample location of the plurality of sample locations based on a 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 on 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 a distance between the objective lens and the surface of the sample. Thus, the spot beam separation may be mapped to a sample height for a given sample location.

[0121] Operation 830 includes calculating a 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 a separation distance of the two sample locations, a tilt slope and angle can be determined between the adjacent locations.

[0122] Operation 840 includes generating a tilt map including a plurality of entries corresponding to a plurality of sample tilt positions based on the tilt of the sample along the scan direction, where each entry indicates an amount of tilting the sample along the scan direction for a corresponding sample tilt position. In some embodiments, a tilt map entry corresponds to each of the sample positions where one beam spot pair is projected. For example, each entry may indicate a tilt angle, a tilt gradient, or a tilt height for each of the sample positions where one beam spot pair is projected. In some embodiments, to minimize the 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 an embodiment, a smooth curve approximation may be performed between the entries corresponding to the 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 the spot beam separation measurement. 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.

[0123] Although the above 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-C, the optical imaging system may utilize a focusing system that generates at least two beam spot pairs to account for tilt in both directions.

[0124] In particular, Figure 24A 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. 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.

[0125] Operation 910 includes scanning the sample in a scan direction along a plurality of sample locations of 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.

[0126] As depicted in FIG. 24B-C, the focus tracking module or other suitable module may 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 where 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 height of the sample at the center of the field of view of the sample position may be estimated from the beam spot separation Δy1 and the beam spot separation Δy2 as follows: The first beam spot separation may be mapped to a first sample height, and the second beam spot separation may be mapped to a second sample height. The average between the first sample height and the second sample height may be determined. Alternatively, one of the two sample heights may 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 ) may be used to determine the slope (also referred to herein as "tilt") along the X direction.

[0127] 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 an amount to tilt the sample along both the scan direction and a direction substantially orthogonal to the scan direction for a corresponding sample tilt position. Such a tilt map may also be referred to as a "tilt-tilt map." During sample imaging, each tilt-tilt map entry may then 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 necessary.

[0128] 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 for a direction orthogonal to the scan direction.

[0129] Although the above 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 above examples may also be used in embodiments utilizing feedback tilt elimination mechanisms that do not rely on tilt maps being 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 an implementation, the frequency at which the sample holder is tilted may be the same as, greater than, or less than the scan frequency.

[0130] In the above 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.

[0131] Similarly, in a multi-channel line-scan imaging system that utilizes multiple excitation light sources to image a sample, the relative orientation between multiple scan lines and projected beam spot pairs may vary. For example, Figures 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 Figure 25A, both beam spot pairs are projected in front of both scan lines. In Figure 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 Figure 25C, both beam spot pairs are projected behind both scan lines. In Figure 25D, both beam spot pairs are projected in front of one scan line and behind the other scan line. In Figures 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.

[0132] FIG. 26 conceptually illustrates the advantages 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 sample surface being scanned. Dynamically changing the tilt according to the level arm L may cause latency-induced errors due to z-displacement, and the x-position of the sample features may be shifted as the sample holder tilts up and down. To overcome this issue, 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 may be changed by simultaneously moving z1 and z2, where:

[0133]

number

[0134] Although some embodiments have been described thus far in the context of tilting a sample / sample holder based on one or more separation measurements of one or more spot pairs from an image captured by an image sensor, the techniques described herein may be implemented generally by creating a relative tilt between a sample and an image sensor that images the sample. Thus, based on a sample tilt measurement, the system may be configured to instead 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 may be tilted and / or otherwise adjusted to create a relative tilt between the sample and the image sensor that images the sample. Such optical components may include, for example, an objective lens or a mirror that receives light corresponding to an image of the sample.

[0135] For example, the embodiments of Figures 21A, 23A, and 24A can be implemented by creating a tilt map that includes entries corresponding to multiple sample positions, where each entry indicates an amount to tilt the image sensor, or some other optical component of the imaging system. Thus, sample tilt removal 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 the one or more optical components can be controlled using one or more system controllers.

[0136] 27 illustrates 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 vessel (e.g., a flow cell lane). In alternative designs, the two spot pairs can be projected onto a single sample surface, or onto more than two sample surfaces.

[0137] As depicted by Fig. 27, the two reflected light beams corresponding to each spot pair are incident on the surface of the 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 the 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 of 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.

[0138] In some implementations, the following parameters can 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 can be defined by equation (1):

[0139]

number

[0140]

number

[0141]

number

[0142]

number

[0143] FIG. 28 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. 27. In this case, Δy1 and Δy2 refer to the spot separation on the surface of the sample, and L refers to the distance between each spot pair on the flow cell surface. The focus tracking module spots are shown relative to the two scanning laser lines. For illustration purposes, the sensors 2911 and 2912 with the incident light beams are superimposed on the drawing. In one particular embodiment, Δy1 and Δy2 are about 60 μm at best focus, and L is about 900 μm.

[0144] 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 manner. Common forms of machine-readable media 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.

[0145] 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 a processing device to perform the features or functionality of the present application as discussed herein.

[0146] In this document, a "processing device" may be implemented as a single processor that performs processing operations, or 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.

[0147] 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.

[0148] To the extent applicable, the terms "first," "second," "third," etc., used herein are used merely to indicate each object described by these terms as separate entities, and no chronological sense is implied unless otherwise specified herein.

[0149] Terms and phrases used in this document, and variations thereof, unless otherwise expressly stated, should be construed as open ended as opposed to limiting. As an example of the foregoing, the term "including" should be read to mean "including but not limited to" and the like. The term "example" is used to provide representative instances of the item in the 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, and adjectives such as "conventional," "traditional," "usual," "standard," "known," and terms of similar meaning 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, ordinary, or standard technology that may be available or known at any time now or in the future. Similarly, when this document refers to technology that may be apparent or known to one of ordinary skill in the art, such technology encompasses those technologies that are apparent or known to one of ordinary skill in the art at any time now or in the future.

[0150] 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.

[0151] Moreover, 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 their various alternatives 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.

[0152] 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.

[0153] To the extent applicable, the terms "first," "second," "third," etc., used herein are used merely to indicate each object described by these terms as separate entities, and no chronological sense is implied unless otherwise specified herein.

[0154] Although 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 by way of limitation. Similarly, various figures may depict example architectures or other configurations for the present disclosure, which 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 may be implemented using various alternative architectures and configurations. Indeed, it will be apparent to one of ordinary skill in the art how alternative functional, logical, or physical partitionings and configurations may be implemented to implement the desired features of the present disclosure. Also, many different configuration 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 mandate that the various embodiments be implemented to perform the recited functions in the same order, unless otherwise dictated by context.

[0155] Although the present disclosure has been described above with reference to 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 their applicability to the particular embodiment 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.

[0156] 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. 1. An imaging system, comprising: a sample holder having a surface for supporting a sample vessel, the sample vessel having a plurality of sample positions; An objective lens; an assembly comprising 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 to focus the imaging system on a 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 spot pair from one or more images taken by an image sensor at one or more of the plurality of sample positions.

2. the first light source for projecting the first pair of spots and the second pair of spots onto the sample container; 2. The imaging system of claim 1, wherein the sample tilt is determined from the first separation measurement from the one or more images taken by the image sensor at the one or more sample positions and a second separation measurement of the second spot pair.

3. the sample tilt is determined at least along the second direction substantially perpendicular to the first direction of imaging; The imaging system of claim 2 , wherein the controller is for controlling the one or more actuators to tilt the sample holder along the second direction based on the sample tilt.

4. the specimen tilt is determined along the first direction and along the second direction; 4. The imaging system of claim 3, wherein 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.

5. The sample tilt along the second direction is determined by the imaging system: determining a first sample height based on at least the first separation measurement; determining a second sample height based on at least the second separation measurement; and 3. The imaging system of claim 2, wherein the sample tilt is determined by: calculating the sample tilt along the second direction based on at least the first sample height and the second sample height.

6. the one or more actuators comprise a plurality of actuators; The imaging system of claim 2 , wherein the controller is for controlling the plurality of actuators to center a tilt axis of the sample holder along the first direction of imaging.

7. 2. The imaging system of claim 1, wherein 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.

8. 3. The imaging system of claim 2, wherein the controller is for controlling the one or more actuators to tilt the sample stage along the first direction and the second direction based on a tilt map determined from a plurality of first separation measurements of the first spot pair and a plurality of second separation measurements of the second spot pair from a plurality of images taken by the image sensor at a plurality of sample positions.

9. The imaging system of claim 2 , wherein the image sensor comprises a first image sensor for imaging the first pair of spots and a second image sensor for imaging the second pair of spots.

10. Further comprising a second light source; the second light source comprises a line scanner for scanning the sample during the imaging of the sample container; The imaging system of claim 1 , wherein the direction of imaging is a direction in which the line scanner scans the sample container.

11. Further comprising a movable platform; the one or more actuators comprising a plurality of linear actuators; the movable platform is mounted on the plurality of linear actuators; The imaging system of claim 1 , wherein the plurality of linear actuators are for tilting the sample holder along the first direction and the second direction.

12. The imaging system of claim 11 , wherein each of the linear actuators comprises a carriage that is driven up and down by a screw of a tilt motor.

13. The assembly further comprises a stage; the movable platform is mounted on the stage; The imaging system of claim 11 , wherein the stage is for laterally moving the sample holder.

14. 1. A method comprising: projecting a first pair of spots and a second pair of spots at a first sample location on the sample using one or more light sources of an imaging system; capturing images of the first and second spot pairs at the first sample location using an image sensor of the imaging system; and 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 image; 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.

15. 15. The method of claim 14, further comprising: tilting the sample in the 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.

16. 15. The method of claim 14, wherein calculating the first tilt of the specimen comprises calculating the first tilt of the specimen at the first sample position along the scanning direction of the imaging system and the direction substantially perpendicular to the scanning direction based on at least the first sample height and the second sample height.

17. 17. The method of claim 16, further comprising tilting the sample in the scanning direction and in the direction substantially perpendicular to the scanning direction using one or more actuators of the imaging system based on the first tilt of the sample.

18. 15. The method of claim 14, further comprising generating a tilt map comprising a plurality of entries corresponding to a plurality of sample tilt positions based on the first tilt of the specimen at the first sample position and additional tilts of the specimen determined at additional sample positions of the specimen, each entry indicating an amount to tilt the specimen along the scan direction or a direction substantially perpendicular to the scan direction for a corresponding sample tilt position.

19. 20. The method of claim 18, further comprising tilting the sample in the direction substantially perpendicular to the scanning direction at each of the plurality of sample tilt positions using one or more actuators of the imaging system according to the tilt map.

20. the image sensor comprises a first image sensor and a second image sensor; 15. The method of claim 14, wherein the images of the first and second spot pairs at the first sample position include a first image of the first spot pair at the first sample position acquired by the first image sensor and a second image of the second spot pair at the first sample position acquired by the second image sensor.

21. 1. An imaging system, comprising: An image sensor; an assembly comprising one or more actuators for tilting a sample holder during imaging of a plurality of sample positions so as 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 and second spot pairs captured by the image sensor at one or more of the plurality of sample positions.

22. The line generating device further comprises: 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; 22. The imaging system of claim 21, comprising one or more line forming optics for shaping the first light beam into a first line and for shaping the second light beam into a second line.

23. 1. An imaging system, comprising: a light source for projecting a first pair of spots and a second pair of spots onto the sample; a first image sensor for acquiring one or more first images of the first pair of spots and the second pair of spots at one or more sample locations of the sample; a second image sensor for acquiring one or more second images of the sample; and and a controller for controlling one or more components of the imaging system such that a relative tilt between the sample and the second image sensor remains substantially the same at the one or more sample positions based on a sample tilt determined from at least the one or more first images.

24. the one or more components comprising one or more actuators coupled to the second image sensor; 24. The imaging system of claim 23, wherein the controller is for controlling the one or more actuators to tilt the second image sensor such that the relative tilt between the sample and the second image sensor remains substantially the same at the one or more sample positions based on the sample tilt determined from at least the one or more first images.

25. the one or more components include one or more actuators coupled to a camera housing the second image sensor; 24. The imaging system of claim 23, wherein the controller is for controlling the one or more actuators to tilt the camera such that the relative tilt between the sample and the second image sensor remains substantially the same at the one or more sample positions based on the sample tilt determined from at least the one or more first images.

26. the imaging system further comprising an objective lens in an optical path between the sample and the second image sensor; the one or more components include one or more actuators coupled to the objective lens; 24. The imaging system of claim 23, wherein the controller is for controlling the one or more actuators to tilt the objective lens such that the relative tilt between the sample and the second image sensor remains substantially the same at the one or more sample positions based on the sample tilt determined from at least the one or more first images.

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